RAF DIMERS AND USES THEREOF

Abstract

Disclosed herein are mutated RAF and KSR nucleic acids and polypeptides. Also disclosed are methods of using the mutated RAF and KSR to inhibit the dimerization of RAF/RAF and RAF/KSR. Also disclosed are methods of using the mutated RAF and KSR to screen for inhibitors of dimerization.

Description

RELATED APPLICATIONS

[0001] This application is a continuation of International Application No. PCT/CA2010/001164, which designated the United States and was filed on Jul. 23, 2010, published in English, which claims the benefit of U.S. Provisional Application No. 61/228,273, filed on Jul. 24, 2009. The entire teachings of the above application(s) are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present generally concerns mutated RAF and KSR isoforms, and more particularly to their inhibition of RAF/RAF and RAF/KSR dimer formation.

BACKGROUND

[0003] The ERK (extracellular signal-regulated kinase) pathway is an evolutionarily conserved signal transduction module that controls cellular growth, differentiation and survival (Wellbrock, C., Karasarides, M. & Marais, R. The RAF proteins take centre stage, Nat Rev Mol Cell Biol., 5, 875-85 (2004)). Activation of receptor tyrosine kinases (RTKs) by the binding of growth factors initiates GTP loading of RAS, which triggers the initial steps in the activation of the ERK pathway by modulating RAF family kinase function. Once activated, RAF participates in a sequential cascade of phosphorylation events that activate MEK, and in turn ERK. Unbridled signaling through the ERK pathway caused by activating mutations in RTKs, RAS or RAF, have been linked to a multitude of human cancers (Roberts, P. J. & Der, C. J., Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer, Oncogene, 26, 3291-310 (2007)). Of note, one member of the RAF family, B-RAF, is the most frequently mutated oncogene within the kinase superfamily (Greenman, C. et al., Patterns of somatic mutation in human cancer genomes, Nature, 446, 153-8 (2007)).

[0004] Not surprisingly, there has been a colossal effort to understand the underlying regulation of this family of kinases. Despite intense scrutiny, the mechanisms governing RAF activation remain only partially understood. In particular, the process by which its kinase domain becomes catalytically activated towards its substrate MEK remains elusive.

[0005] A greater understanding of the mechanisms that govern RAF activation would be useful as a means to identify novel therapeutic intervention strategies for disease such as cancer.

BRIEF SUMMARY

[0006] The following addresses the shortcomings of the above.

[0007] In one aspect, there is provided a composition comprising: an aqueous solution of RAF/RAF homodimer. The composition includes equimolar amounts of RAF monomers. Each RAF monomer includes a RAF kinase domain having a dimerization interface. The RAF/RAF homodimer is a side-to-side dimer having a 2 fold axis of symmetry.

[0008] In one aspect, there is provided a composition comprising: an aqueous solution of RAF/KSR heterodimer. The composition includes equimolar amounts of KSR and RAF monomers. The KSR and the RAF monomer each include a kinase domain having a dimerization interface. The heterodimer has a 2-fold axis of symmetry.

[0009] In one aspect, there is provided a substantially pure nucleic acid encoding a mutated RAF polypeptide. The nucleic acid is DNA which contains the RAF gene. The DNA is genomic DNA or cDNA. The mutated RAF polypeptide is mutated A-RAF, mutated B-RAF or mutated C-RAF. The mutated RAF polypeptide includes at least one mutated residue located in a dimerization interface. The mutated residue is selected from the group consisting of: H449, G450, R481H, L487, F488, M489, Y538, A541 and K542. The mutation is selected from the group consisting of: H449E, G450W, R481H, L487R, F488A, F488L, M489W, Y538F, A541E and K542E. The mutated RAF polypeptide comprises a sequence of SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 15. The nucleic acid comprises a sequence of SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 16. The nucleic acid is DNA which is operably linked to regulatory sequences for expression of a mutated RAF polypeptide and wherein the regulatory sequences comprise a promoter. The promoter is a constitutive promoter, is inducible by one or more external agents, or is cell-type specific.

[0010] In another aspect, there is provided a method of producing a mutated RAF polypeptide, the method comprising: [0011] a) providing a cell transfected with a nucleic acid sequence encoding a mutated RAF polypeptide positioned for expression in the cell; [0012] b) culturing the transfected cells under conditions for expressing the nucleic acid; and [0013] c) producing the mutated RAF polypeptide.

[0014] In another aspect, there is provided a substantially pure mammalian mutated RAF polypeptide, or fragment thereof, the polypeptide being encoded by the nucleic acid, as described above. The polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9. The polypeptide includes at least one mutant selected from the group consisting of: H449E, G450W, R481H, L487R, F488A, F488L, M489W, Y538F, A541E and K542E.

[0015] In one aspect, there is provided a substantially pure nucleic acid encoding a mutated KSR polypeptide. The nucleic acid is DNA which contains the KSR gene. The DNA is genomic DNA or cDNA. The mutated KSR polypeptide is mutated KSR-1 or KSR-2. The mutated KSR polypeptide includes at least one mutated residue located in a dimerization interface. The mutated residue is selected from the group consisting of: H699, G700, R732, L738, F739, M740, Y790, A793 and R794. The mutation is selected from the group consisitng of: H699E, G700W, R732H, L738R, F739A, F739L, M740W, Y790F, A793E and R794E. The mutated KSR polypeptide comprises a sequence of SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 17. The nucleic acid comprises a sequence of SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 18. The nucleic acid is DNA which is operably linked to regulatory sequences for expression of the polypeptide and wherein the regulatory sequences comprise a promoter. The promoter is a constitutive promoter, is inducible by one or more external agents, or is cell-type specific.

[0016] In another aspect, there is provided a method of producing a mutated KSR polypeptide, the method comprising: [0017] a) providing a cell transfected with a nucleic acid sequence encoding a mutated KSR polypeptide positioned for expression in the cell; [0018] b) culturing said transfected cells under conditions for expressing the nucleic acid; and [0019] c) producing the mutated KSR polypeptide.

[0020] In another aspect, there is provided a substantially pure mammalian mutated KSR polypeptide, or fragment thereof, the polypeptide being encoded by the nucleic acid, as described above. The polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 17. The polypeptide includes at least one mutant selected from the group consisting of: H699E, G700W, R732H, L738R, F739A, F739L, M740W, Y790F, A793E and R794E.

[0021] The polypeptide or the nucleic acid, as described above is mammalian. The mammal is murine or human.

[0022] The polypeptide or the nucleic acid, as described above, is non-mammalian. The non-mammal is Drosophila melanogaster.

[0023] In another aspect, there is provided a vector comprising the nucleic acid, as described above, the vector being capable of directing expression of the polypeptide encoded by the nucleic acid in a vector-containing cell.

[0024] In another aspect, there is provided a cell that contains the nucleic acid, as described above.

[0025] In another aspect, there is provided a transgenic cell that contains the nucleic acid, as described above, wherein the nucleic acid is expressed in the transgenic cell.

[0026] In another aspect, there is provided a transgenic non-human mammal generated from the cell, as described above, wherein the nucleic acid is expressed in the transgenic mammal. The transgenic non-human mammal is a mouse.

[0027] The cell, as described above, is a mammalian cell, a yeast cell, or a bacterial cell.

[0028] In one aspect, there is provided a method of detecting the presence of a mutation in a RAF kinase domain, the method comprising: [0029] a) providing a WT RAF kinase domain and a suspected mutant RAF kinase domain, each domain having a cysteine residue located at its N-terminus; [0030] b) incubating the WT RAF kinase domain and the suspected mutant RAF kinase domain with different cross-linking detectable labels; [0031] c) incubating together equimolar amounts of the labeled WT RAF kinase domain and detecting a signal from the detectable label so as to provide a dimerization reference signal; and [0032] d) incubating equimolar amounts of the labeled suspected mutant B-RAF kinase domain and detecting a signal from the detectable labels, an absent signal or a reduce signal compared to that of the dimerization reference signal being an indication that a mutant B-RAF kinase domain is present.

[0033] In one aspect, there is provided a method of monitoring the formation of RAF/RAF or RAF/KSR kinase domain dimers to detect mutations inhibiting dimerization or drug-like molecules interfering with dimerization, the method comprising: [0034] a) fusing either (i) a RAF kinase domain or (ii) a KSR kinase domain at either of their N- or C-termini to a BRET donor or a BRET acceptor to produce donor labeled and acceptor labeled fusion proteins; [0035] b) expressing the fusion proteins to identify combinations that provide specific BRET signals; [0036] c) introducing dimer interface mutations into either of the labeled fusion proteins; [0037] d) expressing the labeled mutated fusion proteins with WT RAF or KSR kinase domains; [0038] e) measuring the BRET signals, a loss or significant reduction of the BRET signal using dimer interface mutations as opposed to mutations remote from the interface, being an indication that a specific BRET signal which depends on the RAF/RAF or RAF/KSR dimerization interface has been obtained.

[0039] The BRET donor is renilla luciferase variant II or rlucII. The BRET acceptor is GFP10. The acceptor label is Yellow Fluorescent Protein (YFP). The donor labeled fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 42 and SEQ ID NO: 48. The acceptor labeled fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 40 and SEQ ID NO: 54. The donor labeled mutated fusion proteins comprise sequences SEQ ID NO: 36 and SEQ ID NO: 50. The acceptor labeled mutated fusion proteins comprises a sequence of SEQ ID NO: 32.

[0040] In another aspect, there is provided a method of identifying a potential inhibitor of RAF/RAF homodimerization, the method comprising. [0041] a) fusing a RAF kinase domain at either of its N- or C-termini to a BRET donor or a BRET acceptor to produce donor labeled and acceptor labeled fusion proteins; [0042] b) expressing the fusion proteins to identify combinations that provide specific BRET signals; [0043] c) introducing dimer interface mutations into either of the labeled fusion proteins; [0044] d) expressing the labeled mutated fusion proteins with WT RAF kinase domains; [0045] e) contacting the interface with the potential inhibitor; and [0046] f) measuring the BRET signals, a loss or significant reduction of the BRET signal for the wild-type RAF/RAF BRET pair being an indication that the inhibitor is specifically bound to the interface.

[0047] In another aspect, there is provided a method of identifying a potential inhibitor of RAF/RAF homodimerization, the method comprising: [0048] a) detectably labeling at least one of the dimerization interface residues to generate a detectably labeled RAF monomer; [0049] b) incubating the detectably labeled RAF monomer with the potential inhibitor and a non-labeled RAF monomer; [0050] c) measuring a signal from the detectable label; [0051] d) contacting the RAF dimerization interface with the inhibitor to determine the ability of the potential inhibitor to inhibit RAF/RAF homodimerization.

[0052] The interface residues include H449, G450, R481, L487, F488, M489, Y538, A541 or K542.

[0053] In one aspect, there is provided a method of identifying a potential inhibitor of RAF/RAF homodimerization, the method comprising. [0054] a) fusing a RAF kinase domain at either of its N- or C-termini to a BRET donor or a BRET acceptor to produce donor labeled and acceptor labeled fusion proteins; [0055] b) expressing the fusion proteins to identify combinations that provide specific BRET signals; [0056] c) introducing dimer interface mutations into either of the labeled fusion proteins; [0057] d) expressing the labeled mutated fusion proteins with WT RAF kinase domains; [0058] e) contacting the interface with the potential inhibitor; and [0059] f) measuring the BRET signals, a loss or significant reduction of the BRET signal for the wild-type RAF/RAF BRET pair being an indication that the inhibitor is specifically bound to the interface.

[0060] In another aspect, there is provided a method of identifying compounds that bind to a RAF or a KSR dimerization interface, the method comprising: [0061] a) contacting the interface with a probe to form a probe: interface complex, the probe being displaceable by a test compound; [0062] b) measuring a signal from the probe so as to establish a reference level; [0063] c) incubating the probe:interface complex with the test compound; [0064] d) measuring the signal from the probe; [0065] e) comparing the signal from step d) with the reference level, a modulation of the signal being an indication that the test compound binds to the BIR domain, wherein the probe is a compound labeled with a detectable label or an affinity label.

[0066] In another aspect, there is provided a method of identifying a potential inhibitor of RAF/RAF homodimerization, the method comprising: [0067] a) using the atomic coordinates of at least one of the interface residues to generate a three dimensional structure of a RAF dimerization interface; [0068] b) using the three-dimensional structure to design or select the potential inhibitor; [0069] c) synthesizing the inhibitor; and [0070] d) contacting the RAF dimierization interface with the inhibitor to determine the ability of the potential inhibitor to inhibit RAF/RAF homodimerization.

[0071] The interface residues are H449, G450, R481, L487, F488, M489, Y538, A541 or K542.

[0072] In one aspect, there is provided a method of identifying a potential inhibitor of RAF/KSR heterodimerization, the method comprising: [0073] a) using the atomic coordinates of at least one of interface residues to generate a three dimensional structure of a KSR dimerization interface; [0074] b) using the three-dimensional structure to design or select the potential inhibitor; [0075] c) synthesizing the inhibitor; and [0076] d) contacting the KSR dimerization interface with the inhibitor to determine the ability of the potential inhibitor to inhibit RAF/KSR heterodimerization.

[0077] The interface residues are H699, G700, R732, L738, F739, M740, Y790, A793 or R794.

[0078] In another aspect, there is provided a method of detecting in a subject the susceptibility to develop a condition or an increased likelihood of developing a condition characterized by impaired regulation of protein RAF or KSR dimerization, the method comprising: [0079] a) obtaining from said subject a biological sample having DNA; [0080] b) sequencing predetermined regions of said DNA encoding a RAF or KSR polypeptide; and [0081] c) comparing the sequence obtained at (b) with a corresponding sequence from a non-susceptible control subject for identifying a RAF or KSR mutation known to be indicative of the susceptibility.

[0082] In another aspect, there is provided a human RAF or KSR polypeptide which comprises a mutation compared to wild type RAF or KSR, wherein said mutation produces a mutant version of human RAF or KSR polypeptide that includes at least one mutant H449, G450, R481, L487, F488, M489, Y538, A541 or K542 residue, and wherein the mutant version prevents the formation of a RAF/RAF homodimer.

[0083] In another aspect, there is provided a human RAF kinase domain which comprises a mutated dimerization interface having at least one mutant H449, G450, R481H, L487, F488, M489, Y538, A541 or K542 residue.

[0084] In another aspect, there is provided a human KSR kinase domain which comprises a mutated dimerization interface having at least one mutant H699, G700, R732, L738, F739, M740, Y790, A793 or R794 residue.

[0085] In another aspect, there is provided a substantially pure nucleic having the sequence of full length RAF and encoding the polypeptide sequences of SEQ ID NO: 7 and SEQ ID NO: 9.

[0086] In another aspect, there is provided a substantially pure nucleic acid having about 50% or greater nucleotide sequence identity to the sequences, as described above.

[0087] In another aspect, there is provided a substantially pure nucleic having the sequence of full length KSR and encoding the polypeptide sequences of SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 17.

[0088] In another aspect, there is provided a substantially pure nucleic acid having about 50% or greater nucleotide sequence identity to the sequences of SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 18.

[0089] In another aspect, there is provided a cell in vitro expressing a recombinant nucleic acid comprising a nucleic acid sequence encoding a mutated RAF polypeptide, as described above. In one example, the cell is a mammalian cell, a yeast cell, or a bacterial cell.

[0090] In one aspect, there is provided a method of producing a drug which inhibits RAF/RAF homodimerization, the method comprising: identifying a drug or designing a drug which interacts with at least one of the H449, G450, R481, L487, F488, M489, Y538, A541 and K542 residues; and synthesizing the drug.

[0091] In another aspect, there is provided a method of producing a drug which inhibits RAF/KSR heterodimerization, the method comprising: identifying a drug or designing a drug which interacts with at least one of the H699, G700, R732, L738, F739, M740, Y790, A793 and R794 residues; and synthesizing the drug.

[0092] In another aspect, there is provided a composition comprising: an inhibitor adapted to inhibit the formation of a RAF/RAF homodimer or a RAF/KSR heterodimer, in which the inhibitor binds to at least one of the H449, G450, R481, L487, F488, M489, Y538, A541 and K542 residues in a RAF monomer or at least one of the H699, G700, R732, L738, F739, M740, Y790, A793 and R794 residues in a KSR monomer.

[0093] In another aspect, there is provided a method of treating or preventing a disease in a subject, the disease being characterized by RAF/RAF homodimerization or RAF/KSR heterodimerization, the method comprising: administering to the subject in need thereof, an expression vector encoding mutated RAF or KSR polypeptide, the mutated RAF or KSR polypeptide being positioned in the vector for expression in a cell of the subject in which RAF/RAF homodimerization or RAF/KSR heterodimerization is taking place, so as to treat or prevent the disease.

[0094] In another aspect, there is provided a dominant negative mutant polypeptide of mammalian RAF or KSR, wherein the mutant polypeptide comprises a kinase domain having a dimerization interface and does not bind to a WT mammalian RAF or KSR dimerization interface.

[0095] In another aspect, there is provided a purified antibody which specifically binds to a mammalian mutated RAF or KSR polypeptide. The mammal is a human. The mammal is a mouse. The mutated RAF polypeptide is B-RAF. The KSR polypeptide is KSR-1. The antibody is a polyclonal antibody. The antibody is a monoclonal antibody.

BRIEF DESCRIPTION OF THE FIGURES

[0096] In order that the herein described may be readily understood, certain embodiments are illustrated by way of example in the accompanying drawings.

[0097] FIG. 1--KSR possesses intrinsic RAF activating potential. A) Co-overexpression of KSR, RAF and its substrate MEK as indicated in S2 cells leads to activation of RAF in a KSR concentration-dependent manner in the presence or absence of RNAi-mediated knockdown of RAS or co-overexpression of a constitutively active RAS^V12 variant. RAF activation was assessed by immunoblotting for phospho-MEK. The catalytically-inactive RAF K455M (KM) mutant served as a negative control.B and C) The RAF activation potential of overexpressed KSR is not affected by RNAi-mediated knockdown of CNK, HYP or CK2α or by mutation of the proposed CK2 α regulatory sites in KSR (T399A/K402A) and RAF (S416A/S417A). Assessment of the RNAi-mediated knock-downs for endogenous RAS, CNK, HYP and CK2 α is provided in FIG. 13.

[0098] FIG. 2--KSR_R732H mutation abolishes its inherent RAF activating potential. Wild type KSR but not the KSR_R732H mutant is able to drive RAF activation in an S2 cell overexpression system. Experiments were performed as in FIG. 1.

[0099] FIG. 3--A side-to-side dimer configuration of RAF underlies an allosteric mode of regulation. A) Projection of highly conserved residues across both KSR and RAF orthologues onto the crystal structure (PDB ID=1UWH) (Wan, P. T. et al., Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)) of the B-RAF kinase domain (top panel) highlights common side-to-side dimer contact surfaces visualized originally in crystal structures of B-RAF (bottom panel). B) Crystal structure of B-RAF highlighting the position of Arg481 (equivalent to Arg732 in KSR) at the center of the side-to-side dimer interface (PDB ID=1UWH) (Wan, P. T. et al., Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)). Residue numbering scheme corresponds to Drosophila RAF. One protomer is displayed as a surface representation in orange and the other is shown as a ribbons representation in violet. Inset displays a close-up view of hydrogen bonding interactions involving Arg481, an ordered solute molecule, and main-chain carbonyl groups in the linker joining helix α C to strand β4.C) Analytical ultracentrifugation analysis reveals that mutation of Arg481 (R481H) in B-RAF transitions the protein from a dimer (left panel) to a monomer (right panel) in solution. The red line denotes a fitted curve to the self-association model. The residuals for the fit are shown in the upper panels.

[0100] FIG. 4--Side-to-side dimer interface residues are conserved in all KSR and RAF proteins. Sequence alignment of the kinase domains of KSR and RAF from divergent organisms highlighting conserved residues. For comparison, the sequence of the kinase domains of LCK and PKA are co-aligned demonstrating that the side-to-side dimer contact residues are unique to the KSR/RAF family. The sequence of the kinase domain N-lobe is boxed in red and the secondary structural elements are indicated above the sequence. Aligned sequences correspond to those from Drosophila (d), human (h), mouse (m), zebrafish (z), and chicken (c). Only the B-RAF sequence is shown for species where multiple RAF isoforms exist.

[0101] FIG. 5--Perturbing the side-to-side dimer interface on RAF and KSR impairs RAF activation. A) Left panel: Model of a side-to-side heterodimer between KSR and RAF kinase domains. RAF is displayed as a surface representation in purple while KSR is shown in ribbons representations in green. Highlighted in red stick representation are the positions of residues selected for mutational analysis in KSR (G700W, R732H, F739A, M740W and Y790F). Position of analogous mutated sites in RAF (G450W, R481H, F488A, M489W and Y538F, respectively) are denoted by yellow surface; residue numbering scheme corresponds to Drosophila RAF. Right panel: The individual effect of KSR and RAF mutations on RAF activation was assessed by monitoring the levels of phosphorylated MEK in S2 cells as performed in FIG. 1. Control mutations outside the side-to-side dimer interface correspond to K460A, E601A and M640A in RAF, and D710A, E859A and V898A in KSR. B) Left panel: Schematic for induced side-to-side dimer formation using FRB/FKBP fusions to the kinase domains of KSR and RAF. Right panel: The RAF activation potential of the FRB/FKBP fused kinase domains of KSR and RAF were assessed by monitoring the levels of phosphorylated MEK in the presence or absence of rapamycin in S2 cells as performed in FIG. 1. C) Left panel: Schematic for induced side-to-side homodimer formation of RAF kinase domains. The catalytically-inactive (K455S) FRB-RAF fusion is indicated by an `X` within the N-lobe. Right panel: Activation potential of FRB/FKBP RAF homodimers was assessed as in FIG. 3B.

[0102] FIG. 6--The kinase domain of RAF adopts a side-to-side dimeric configuration in the crystal structure. A) The side-to-side dimer configuration of the kinase domain of B-RAF is shown viewed perpendicular to the 2-fold axis of symmetry (PDB ID=1UWH) (Wellbrock, C., Karasarides, M. & Marais, R., The RAF proteins take centre stage, Nat Rev Mol Cell Biol, 5, 875-85 (2004)). The N-lobes of the two kinase domains, which compose the majority of the dimer interaction surfaces, are colored in darker tint. B) Superposition of the six reported B-RAF kinase domain structures reveal an identical mode of side-to-side dimerization (PDB IDs: 1UWH (Wan, P. T. et al., Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)), 1UWJ (Wan, P. T. et al., Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)), 2FB8 (King, A. J. et al., Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885, Cancer Res, 66, 11100-5 (2006)), 3C4C (Tsai, J. et al., Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity, Proc Natl Acad Sci, USA, 105, 3041-6 (2008)), 3C4D (Tsai, J. et al., Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc Natl Acad Sci., USA, 105, 3041-6 (2008)) and 3D4Q (Hansen, J. D. et al., Potent and selective pyrazole-based inhibitors of B-Raf kinase, Bioorg Med Chem Lett, 18, 4692-5 (2008)). C) Comparison of the B-RAF side-to-side mode of dimerization with the specific mode of dimerization of PKR (PDB ID=2A19 (Dar, A. C., Dever, T. E. & Sicheri, F., Higher-order substrate recognition of eIF2alpha by the RNA-dependent protein kinase PKR, Cell, 122, 887-900 (2005)) and EGFR (PDB ID=2GS2 (Zhang, X., Gureasko, J., Shen, K., Cole, P. A. & Kuriyan, J., An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor, Cell, 125, 1137-49 (2006)) kinase domains. Helix αC is a participant in all three modes of dimerization.

[0103] FIG. 7--An engineered KSR/RAF chimera can functionally mimic wildtype KSR. A) Schematics of wild type and chimeric KSR/RAF constructs involving either a full RAF kinase domain swap into KSR (Chimera-A) or just a RAF N-lobe swap into KSR (Chimera-B). B) The ability of wild type RAF, KSR, and KSR/RAF chimeric constructs to drive RAF activation in S2 cells was assessed by the levels of phosphorylated MEK.

[0104] FIG. 8--Perturbing the side-to-side dimer interface on RAF and KSR impairs RAF activation. A) Model of a side-to-side heterodimer between KSR and RAF kinase domains. RAF is displayed as a surface representation in purple while KSR is shown in ribbons representations in green. Highlighted in red stick representation are the positions of residues selected for mutational analysis in KSR (H699E, L738R, F739L, A793E, and R794E). Position of analogous mutated sites in RAF (H449E, L487R, F488L, A541E and K542E, respectively) are denoted by yellow surface; residue numbering scheme corresponds to Drosophila RAF. B and C) The RAF activation potential of the FRB/FKBP fused kinase domains of KSR and RAF were assessed by monitoring the levels of phosphorylated MEK in the presence or absence of rapamycin in S2 cells as illustrated in the schematic.

[0105] FIG. 9--KSR contains a putative 14-3-3 binding site C-terminal to its kinase domain. A) Sequence alignment of the C-terminus of KSR reveals a highly conserved 14-3-3 recognition site common to that found in RAF (Drosophila RAF residue numbering is indicated above the alignment). Aligned sequences correspond to those from Drosophila (d), human (h), mouse (m), zebrafish (z), and chicken (c). B) S2 cell overexpression assay for RAF activation showing the effects of RNAi-mediated knockdown of 14-3-3 isoforms or mutation of putative 14-3-3 binding sites in KSR (R950A/S951A) and RAF (S701A). The effect of RNAi on endogenous 14-3-3 protein levels is shown in FIG. 13.

[0106] FIG. 10--Binding of 14-3-3 to KSR and RAF may promote the formation of hetero- and/or homotypic dimers by the kinase domain. Structural model showing that the geometry of the KSR/RAF (or RAF/RAF) side-to-side dimer is compatible with the spatial requirements for binding to dimeric 14-3-3 proteins. Surface representation of 14-3-3 bound to phospho-peptides is based on PDB ID 1YWT (Wilker, E. W., Grant, R. A., Artim, S. C. & Yaffe, M. B., A structural basis for 14-3-3sigma functional specificity, J Biol Chem, 280, 18891-8 (2005)).

[0107] FIG. 11--Side-to-side dimer formation underlies the aberrant signaling potential of oncogenic RAF mutants. A) RAF activation assay using overexpressed full-length RAF and MEK proteins in S2 cells. The dimer interface mutation (RAF_R481H) abrogates the pronounced activation potential of the activation segment mutation (analogous to oncogenic B-RAF mutation) RAF_T571E/T574D (denoted RAF-AL^ED). B) Glu558 locates to the side-to-side dimer interface in RAF and is mutated to Lys in human cancers (E558K mutation; residue numbering scheme corresponds to Drosophila RAF). The longer Lys residue could potentially engage in hydrogen bonding interactions with Ser561 on the opposite protomer. C) Left panel: Schematic for induced side-to-side homodimer formation of RAF kinase domains as in FIG. 3C. Right panel: Catalytically inactive FRB-RAF harboring the E558K mutation was assessed for its activation potential towards FKBP-RAF in trans as in FIG. 3C.

[0108] FIG. 12--Oncogenic B-RAF E558K mutation promotes kinase domain dimerization. Analytical ultracentrifugation analysis reveals that the oncogenic E558K mutation in B-RAF transitions the B-RAF_L487R dimer mutant from weak monomer-dimer equilibrium (left panel) to a dimer (right panel) in solution; residue numbering scheme corresponds to Drosophila RAF. The red line denotes a fitted curve to the self-association model. The residuals for the fit are shown in the upper panels.

[0109] FIG. 13--Depletion of specific endogenous targets by RNAi. To ensure that dsRNAs directed against RAS, CNK, HYP, CK2α, 14-3-3ε or 14-3-3ζ worked as expected, we separately incubated S2 cells with specific dsRNAs (15 μg/ml) against these intended targets and monitored their respective protein or mRNA levels using either specific antibodies or qPCR. GFP dsRNA was used as negative control. In panel A, the effect of RAS depletion was also monitored by assessing phospho-MAPK (pMAPK) levels induced by the activated Sevenless (SEV^S11) RTK expressed under the control of the hsp70 promoter (Laberge, G., Douziech, M., & Therrien, M. Src42 binding activity regulates Drosophila RAF by a novel CNK-dependent derepression mechanism, EMBO J, 24, 487-98 (2005)).

[0110] FIG. 14A shows polypeptide and polynucleotide sequences (SEQ ID NO: 7 and 8) of homo sapiens v-raf murine sarcoma viral oncogene homolog B1 (BRAF) showing mutated residues as underlined and highlighted.

[0111] FIG. 14B shows polypeptide and polynucleotide sequences (SEQ ID NO: 9 and 10) of mus musculus Braf transforming gene (Braf) showing mutated residues as underlined and highlighted.

[0112] FIG. 14C shows polypeptide and polynucleotide sequences (SEQ ID NO: 11 and 12) of homo sapiens kinase suppressor of ras 1 (KSR 1) showing mutated residues as underlined and highlighted.

[0113] FIG. 14D shows polypeptide and polynucleotide sequences (SEQ ID NO: 13 and 14) of mus musculus kinase suppressor of ras 1 (Ksr 1) showing mutated residues as underlined and highlighted.

[0114] FIG. 14E shows polypeptide and polynucleotide sequences (SEQ ID NO: 15 and 16) of Drosophila melanogaster pole hole (phi) transcript variant A showing mutated residues as underlined and highlighted.

[0115] FIG. 14F shows polypeptide and polynucleotide sequences (SEQ ID NO: 17 and 18) of Drosophila melanogaster kinase suppressor of ras (ksr) showing mutated residues as underlined and highlighted.

[0116] FIG. 15--Development of a Bioluminescence Resonance Energy Transfer (BRET) assay to monitor RAF/RAF homodimerization. (A) Structure of the human BRAF kinase. RBD, CRD and Ser/Thr stand for Ras-Binding Domain, Cysteine-Rich Domain and Ser/Thr-rich domains respectively. The Kinase domain (KD) and its C-terminal extension (dashed box) were used in all BRET constructs described here. (B) Structure of the BRAF-KD donor (rlucII) and acceptor (GFP10) expression constructs used in the BRET assay. (C) Saturation curve of the BRAF-KD-wt and BRAF-KD-R481H alleles showing a significant reduction in the BRET_max and BRET₅₀ when a dimer interface mutation (R481H) is introduced in the BRAF-KD. (D) Parameters derived from fit of our data with a hyperbolic function. R² denotes the goodness of fit of our data to a hyperbolic function. BRET_max and BRET₅₀ were interpolated using the hyperbolic function.

[0117] FIG. 16 shows CAAX-box and BRET donor and acceptor polypeptide sequences (human KRAS CAAX-box CDS: SEQ ID NO: 19; human KRAS CAAX-box: SEQ ID NO: 20; GFP10 CDS: SEQ ID NO: 21; GFP10: SEQ ID NO: 22; rlucII CDS: SEQ ID NO: 23; and rlucII: SEQ ID NO: 24).

[0118] FIG. 17 shows human BRAF (hBRAF) polypeptide sequences (hBRAF-KD-wt CDS: SEQ ID NO: 25; hBRAF-KD-wt: SEQ ID NO: 26; hBRAF-KD-R481H CDS: SEQ ID NO: 27; hBRAF-KD-R481H: SEQ ID NO: 28). The bolded residues indicate linker and restriction sites. Mutated residues are shaded in black.

[0119] FIG. 18 shows human BRAF-KD clones between the NheI and XbaI sites in pCDNA3.1-zeo (GFP10-hBRAF-KD-wt-CAAX CDS: SEQ ID NO: 29; GFP10-hBRAF-KD-wt-CAAX: SEQ ID NO:30; GFP10-hBRAF-KD-R481H-CAAX CDS: SEQ ID NO: 31; GFP10-hBRAF-KD-R481H-CAAX: SEQ ID NO: 32; rlucII-hBRAF-KD-wt-CAAX CDS: SEQ ID NO: 33: rlucII-hBRAF-KD-wt CAAX: SEQ ID NO: 34; rlucII-hBRAF-KD-R481H-CAAX CDS: SEQ ID NO: 35; and rlucII-hBRAF-KD-R481H-CAAX: SEQ ID NO: 36). The bolded residues indicate linker and restriction sites. Mutated residues are shaded in black.

[0120] FIG. 19 shows human CRAF (hCRAF) polypeptide sequences (hCRAF-KD-wt CDS: SEQ ID NO: 37: hCRAF-KD-wt: SEQ ID NO: 38).

[0121] FIG. 20 shows hCRAF-KD fusions that are cloned between NheI and XbaI in pCDNA3.1-zeo (GFP10-hCRAF-KD-wt-CAAX CDA: SEQ ID NO: 39; GFP10-hCRAF-KD-wt-CAAX: SEQ ID NO: 40; rlucII-hCRAF-KD-wt-CAAX CDS: SEQ ID NO: 41; and rlucII-hCRAF-KD-wt-CAAX: SEQ ID NO: 42). The bolded residues indicate linker and restriction sites.

[0122] FIG. 21 shows human KSR1 (hKSR1) sequences (hKSR1-KD-wt-CDS: SEQ ID NO: 43; hKSR1-KD-wt: SEQ ID NO: 44; hKSR1-KD-C922Y CDS: SEQ ID NO: 45; and hKSR1-KD-C922Y: SEQ ID NO: 46). The bolded residues indicate linker and restriction sites. Mutated residues are shaded in black.

[0123] FIG. 22 shows human KSR1-KD-rlucII fusions cloned between KpnI and PmeI in pCDNA3.1-zeo (hKSR1-KD-wt-rlucII CDS: SEQ ID NO: 47; hKSR1-KD-wt-rlucII: SEQ ID NO: 48; hKSR1-KD-C922Y-rlucII CDS: SEQ ID NO: 49; and hKSR1-KD-C922Y-rlucII: SEQ ID NO: 50). The bolded residues indicate linker and restriction sites.

[0124] FIG. 23 shows human MEK1 (hMEK1) sequences (hMEK1 CDS: SEQ ID NO: 51; and hMEK1: SEQ ID NO: 52).

[0125] FIG. 24 shows human GFP10-MEK1 full length fusion cloned between NheI and XbaI in pCDNA3.1-zeo (GFP10-hMEK1 CDS: SEQ ID NO: 53; and GFP10-hMEK1: SEQ ID NO: 54). The bolded residues indicate linker and restriction sites.

[0126] FIG. 25 shows the sequences of cloning oligonucleotides. Human BRAF (OL5_hBRAF_KD_+start_F: SEQ ID NO: 55; OL3_hBRAF_KD_+CAAX_+stop_R: SEQ ID NO: 56); human CRAF (OL5_hCRAF_KD_+start_F: SEQ ID NO: 57; OL5_hCRAF_KD_+CAAX_+stop_R: SEQ ID NO: 58); human KSR1 (OL5_hKSR1_KpnI_cloning_BRET: SEQ ID NO: 59; OL3_hKSR_XbaI_cloning_BRET: SEQ ID NO:60); and human MEK (OL5_hMEK1_KpnI_cloning_BRET: SEQ ID NO: 61; OL3_hMEK_XbaI_cloning_BRET: SEQ ID NO: 62). The bolded residues indicate linker and restriction sites.

[0127] FIG. 26 shows sequencing oligonucleotides (OL5_hBRAF_seq1_F: SEQ ID NO: 63; OL5_hBRAF_seq2_F: SEQ ID NO: 64; OL5_hCRAF_seq1_F: SEQ ID NO: 65; and OL5_hCRAF_seq2_F: SEQ ID NO: 66).

[0128] FIG. 27 shows mutagenesis oligonucleotides. The following primer pair were used to generate the side-to-side dimer interface mutant R481H in BRAF: OL5_hBRAF_R481H_F (SEQ ID NO: 67); and OL3_hBRAF_R481H_R (SEQ ID NO: 68). The following primer pair was used to introduce the C922Y hMEK1 interaction mutant in hKSR1: OL5_hKSR1_C922Y_F (SEQ ID NO: 69) and OL3_hKSR1_C922Y_R (SEQ ID NO: 70). The bolded residues indicate linker and restriction sites.

DETAILED DESCRIPTION

[0129] In the following description of the embodiments, references to the accompanying Figures are by way of illustration of an example by which the embodiments described herein may be practiced. It will be understood that other embodiments may be made without departing from the scope of that disclosed herein.

[0130] Definitions:

[0131] Unless otherwise specified, the following definitions apply throughout:

[0132] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a mutation" includes one or more of such mutations and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0133] As used herein, the term "comprising" is intended to mean that the list of elements following the word "comprising" are required or mandatory but that other elements are optional and may or may not be present.

[0134] As used herein, the term "consisting of" is intended to mean including and limited to whatever follows the phrase "consisting of". Thus the phrase "consisting of" indicates that the listed elements are required or mandatory and that no other elements may be present.

[0135] As used herein, the term "RAF" is intended to refer to a protein, a polypeptide or fragment thereof, encoded by a RAF gene. Examples of Wild-type (WT) human RAF proteins include the RAF protein isoforms known as A-RAF, B-RAF and C-RAF (e.g., genbank accession numbers P10398 for Homo sapiens A-RAF; P15056 for Homo sapiens B-RAF; and PO4049 for Homo sapiens C-RAF). Examples of RAF xenologues are (e.g. genbank accession number P11346 for Drosophila melanogaster pole hole (phl; RAF); P04627 for Mus musculus A-RAF; P28028 for Mus musculus B-RAF; and Q99N57 for Mus musculus C-RAF. Included in this definition are any functional RAF fragment, or any fusion of functional RAF fragments. Examples of these fragments include those that consist of, consist essentially of, or comprise the RAF kinase domain. Furthermore, the term also encompasses any fusion of full length RAF, or a functional fragment thereof, with another polypeptide. These fusions include, but are not limited to, GST-RAF, HA tagged RAF, or Flag tagged RAF. These additional polypeptides may be linked to the N-terminus and/or C-terminus of RAF. Chimeric RAF protein, including a protein comprising a fusion of a RAF domain or domains with a portion of another protein, wherein the chimeric RAF retains the properties of human RAF, are also included. Examples of chimeric RAF proteins include the fusion of any of the above RAF domains, or fragments thereof, to any domain or fragment of the following proteins such as, for example, GST, luciferase or GFP derivatives. RAF also includes any protein with at least 70% sequence identity with mammalian or non-mammalian RAF. The term also includes any conservative substitutions of amino-acid residues in RAF. The term "conservative substitution" refers to replacement of an amino acid residue by a chemically similar residue, e.g., a hydrophobic residue for a separate hydrophobic residue, a charged residue for a separate charged residue, etc. Examples of conserved substitutions for non-polar R groups are alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. Examples of substitutions for polar, but uncharged R groups are glycine, serine, threonine, cysteine, asparagine, or glutamine. Examples of substitutions for negatively charged R groups are aspartic acid or glutamic acid. Examples of substitutions for positively charged R groups are lysine, arginine, or histidine. Furthermore, the term RAF includes conservative substitutions with non-natural amino-acids.

[0136] The following are Accession numbers for RAF cDNA and protein sequences for various species:

[0137] Accession numbers for RAF cDNA sequences

[0138] NM_--080308: Drosophila melanogaster pole hole (phl; RAF)

[0139] NM_--009703: Mus musculus A-RAF

[0140] NM_--139294: Mus musculus B-RAF

[0141] AB057663: Mus musculus C-RAF

[0142] X04790: Homo sapiens A-RAF

[0143] NM_--004333: Homo sapiens B-RAF

[0144] NM_--002880: Homo sapiens C-RAF

[0145] Accession numbers for RAF protein sequences

[0146] P11346: Drosophila melanogaster pole hole (phl; RAF)

[0147] P04627: Mus musculus A-RAF

[0148] P28028: Mus musculus B-RAF

[0149] Q99N57: Mus musculus C-RAF

[0150] P10398: Homo sapiens A-RAF

[0151] P15056: Homo sapiens B-RAF

[0152] P04049: Homo sapiens C-RAF

[0153] As used herein, the terms "mutated RAF protein" and "mutated RAF polypeptide" are used interchangeably throughout and are intended to mean a WT RAF protein in which one or more amino acid residues have been changed. In certain examples described herein, the mutations include H449E, G450W, R481H, L487R, F488A, F488L, M489W, Y538F, A541E and K542E, which are located in the dimerization interface. Unless otherwise stated, amino acid residue positions in RAF proteins refer to those of the Drosophila. melanogaster sequences.

[0154] As used herein, the term "KSR" is intended to refer to a Kinase Suppressor of Ras protein, a polypeptide or fragment thereof, encoded by a KSR gene. Examples of Wild type (WT) human KSR proteins include the KSR protein isoforms known as KSR1 and KSR2 (e.g. genbank accession number A8MY87 for Homo sapiens kinase suppressor of ras 1 (KSR1) and Q6VAB6: for Homo sapiens kinase suppressor of ras 2 (KSR2). Examples of KSR xenologues are (e.g. genbank accession numbers Q24171 for Drosophila melanogaster kinase suppressor of ras (KSR); Q61097 for Mus musculus kinase suppressor of ras 1 (KSR1); and Q3UVC0 for Mus musculus kinase suppressor of ras 2 (KSR2). The term "KSR" also means any functional KSR fragment, or any fusion of functional KSR fragments. Examples of these fragments include those that consist of, consist essentially of, or comprise the KSR kinase domain. Included in this definition are fusion of full length KSR, or a functional fragment thereof, with another polypeptide. These fusions include, but are not limited to, GST-KSR, HA tagged KSR, or Flag tagged KSR. These additional polypeptides may be linked to the N-terminus and/or C-terminus of KSR. Any chimeric KSR protein including a protein comprising a fusion of a KSR domain or domains with a portion of another protein, wherein the chimeric KSR retains the properties of human KSR, are also included. Examples of chimeric KSR proteins include the fusion of any of the above KSR domains, or fragments thereof, to any domain or fragment of the following proteins such as, for example, GST, luciferase or GFP derivatives. KSR also includes any protein with at least 70% sequence identity with mammalian or non-mammalian KSR. The term also includes any conservative substitutions of amino-acid residues in KSR. The term "conservative substitution" refers to replacement of an amino acid residue by a chemically similar residue, e.g., a hydrophobic residue for a separate hydrophobic residue, a charged residue for a separate charged residue, etc. Examples of conserved substitutions for non-polar R groups are alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. Examples of substitutions for polar, but uncharged R groups are glycine, serine, threonine, cysteine, asparagine, or glutamine. Examples of substitutions for negatively charged R groups are aspartic acid or glutamic acid. Examples of substitutions for positively charged R groups are lysine, arginine, or histidine. Furthermore, the term KSR includes conservative substitutions with non-natural amino-acids.

[0155] The following are Accession numbers for KSR cDNA and protein sequences for various species:

[0156] Accession numbers for KSR cDNA sequences

[0157] NM_--079512: Drosophila melanogaster kinase suppressor of ras (KSR)

[0158] NM_--013571: Mus musculus kinase suppressor of ras 1 (KSR1)

[0159] DQ531035: Mus musculus kinase suppressor of ras 2 (KSR2)

[0160] NM_--014238: Homo sapiens kinase suppressor of ras 1 (KSR1)

[0161] NM_--173598: Homo sapiens kinase suppressor of ras 2 (KSR2)

[0162] Accession numbers for KSR protein sequences

[0163] Q24171: Drosophila melanogaster kinase suppressor of ras (KSR)

[0164] Q61097: Mus musculus kinase suppressor of ras 1 (KSR1)

[0165] Q3UVC0: Mus musculus kinase suppressor of ras 2 (KSR2)

[0166] Q8IVT5: Homo sapiens kinase suppressor of ras 1 (KSR1)

[0167] Q6VAB6: Homo sapiens kinase suppressor of ras 2 (KSR2)

[0168] As used herein, the terms "mutated KSR protein" and "mutated KSR polypeptide" are used interchangeably throughout and are intended to mean a WT KSR protein in which one or more amino acid residues have been changed. In certain examples described herein, the mutations include H699E, G700W, R732H, L738R, F739A, F739L, M740W, Y790F, A793E and R794E,

[0169] which are located in the dimerization interface. Unless otherwise stated, amino acid residue positions in KSR proteins refer to those of the Drosophila. melanogaster sequences.

[0170] As used herein, the term "mutation" is intended to mean any alteration in a gene which alters function or expression of the gene products, such as mRNA and the encoded for protein. This includes, but is not limited to, altering mutation, point mutation, truncation mutation, deletion mutation, frameshift mutation, and null mutation.

[0171] As used herein, the term "RAF gene" is intended to mean a gene encoding a RAF polypeptide having a dimerization interface. The RAF gene is a gene having about 50% or greater nucleotide sequence identity to at least one of human RAF isoforms (e.g. genbank accession numbers X04790 for Homo sapiens A-RAF; NM_--004333 for Homo sapiens B-RAF; and NM_--002880 for Homo sapiens C-RAF. Examples of RAF xenologues are (e.g. genbank accession numbers NM_--080308 for Drosophila melanogaster pole hole (phl; RAF); NM_--009703 for Mus musculus A-RAF; NM_--139294 for Mus musculus B-RAF; and AB057663 for Mus musculus C-RAF).

[0172] As used herein, the term "KSR gene" is intended to mean a gene encoding a KSR polypeptide having a dimerization interface. The KSR gene is a gene having about 50% or greater nucleotide sequence identity to at least one of human KSR isoforms (e.g. genbank accession numbers NM_--014238 for Homo sapiens kinase suppressor of ras 1 (KSR1); and NM_--173598 for Homo sapiens kinase suppressor of ras 2 (KSR2)). Examples of KSR xenologues are (e.g. genbank accession numbers NM_--079512 for Drosophila melanogaster kinase suppressor of ras (KSR); NM_--013571 for Mus musculus kinase suppressor of ras 1 (KSR1); and DQ531035 for Mus musculus kinase suppressor of ras 2 (KSR2).

[0173] As used herein, the term "gene" refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences. The nucleic acid may also optionally include non-coding sequences such as promoter or enhancer sequences. The term "intron" refers to a DNA sequence present in a given gene that is not translated into protein and is generally found between exons.

[0174] As used herein, the term "dimer interface" is intended to mean a site in the WT RAF or KSR polypeptide sequence or the mutated RAF or KSR polypeptide sequence, which reacts with a RAF or KSR substrate.

[0175] As used herein, the terms "RAF kinase domain" or "KSR kinase domain" are intended to mean the portion of the RAF or KSR proteins that are related in sequence to a generic protein kinase domain.

[0176] As used herein, the term "detectable label" is intended to mean a compound that may be linked to a RAF or KSR kinase domain, such that when the compound is associated with the domain, the label allows either direct or indirect recognition of the compound so that it may be detected, measured and quantified.

[0177] As used herein, the term "affinity tag" is intended to mean a ligand or group, which is linked to a RAF or KSR kinase domain to allow another compound to be extracted from a solution to which the ligand or group is attached.

[0178] As used herein, the term "nucleic acid" or a "nucleic acid molecule" as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction. With reference to nucleic acids described herein, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. Whenever applicable, the term "isolated nucleic acid" may also refer to a RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e. in cells or tissues). An "isolated nucleic acid" (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

[0179] As used herein, the term "vector" is intended to mean a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.

[0180] As used herein, the terms "percent similarity", "percent identity" and "percent homology" when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program.

[0181] As used herein, the term "substantially pure" is intended to refer to a preparation comprising at least 50-60% by weight of a given material (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-95% by weight of the given compound. Purity is measured by methods appropriate for the given compound (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like). Described herein are substantially pure mutated RAF or KSR isoforms (e.g., nucleic acids, oligonucleotides, proteins, fragments, mutants, etc.).

[0182] As used herein, the term "oligonucleotide" is intended to sequences, primers and probes as described herein, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.

[0183] As used herein, the term "primer" is intended to refer to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as appropriate temperature and pH, the primer may be extended at its 3' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically about 20-40, or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.

[0184] As used herein, the term "probe" is intended to refer to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains about 20-40 or more nucleotides in length, although it may contain fewer nucleotides. The probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize" or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.

[0185] With respect to single-stranded nucleic acids, particularly oligonucleotides, the term "specifically hybridizing" refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA molecule as described herein, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence. Appropriate conditions enabling specific hybridization of single-stranded nucleic acid molecules of varying complementarity are well known in the art. For instance, one common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is set forth below (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press):

T_m=81.5° C.+16.6 Log [Na+]+0.41 (% G+C)-0.63 (% formamide)-600/#bp in duplex

[0186] As an illustration of the above formula, using [Na+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T_m is 57° C. The T_m of a DNA duplex decreases by 1-1.5 with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42° C.

[0187] The stringency of the hybridization and wash depends primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25° C. below the calculated T_m of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12-20° C. below the T_m of the hybrid. With regard to the nucleic acids as described herein, a moderate stringency hybridization is defined as hybridization in 6×SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C. and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A high stringency hybridization is defined as hybridization in 6×SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. A very high stringency hybridization is defined as hybridization in 6×SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15 minutes.

[0188] Alternatively, as used herein, the term "probe" is intended to mean a compound which is labeled with either a detectable label or an affinity tag, and which is capable of binding, either covalently or non-covalently, to a RAF or KSR kinase domain. When, for example, the probe is non-covalently bound, it may be displaced by a test compound. When, for example, the probe is bound covalently, it may be used to form cross-linked adducts, which may be quantified and inhibited by a test compound.

[0189] As used herein, the term "isolated protein" or "isolated and purified protein" is intended to refer to a protein produced by expression of an isolated nucleic acid molecule as described herein. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure" form. "Isolated" is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.

[0190] As used herein, the term "amino acid" is intended to mean a radical derived from the corresponding α-amino acid by eliminating the hydroxyl of the carboxy group and one hydrogen of the α-amino group. For example, the terms Gln, Ala, Gly, Ile, Arg, Asp, Phe, Ser, Leu, Cys, Asn, and Tyr represent the residues of L-glutamine, L-alanine, glycine, L-isoleucine, L-arginine, L-aspartic acid, L-phenylalanine, L-serine, L-leucine, L-cysteine, L-asparagine, and L-tyrosine, respectively. Amino Acid residues are provided below:

[0191] Three and single letter abbreviations for α-amino acids used throughout are as follows:

TABLE-US-00001 Amino acid. Abbreviation Abbreviation Alanine Ala A Arginine Arg R Aspartic acid Asp D Asparagine Asn N Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Isoleucine Ile I Histidine His H Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0192] As used herein, the term "subject" is intended to mean humans and non-human mammals such as primates, cats, dogs, swine, cattle, sheep, goats, horses, rabbits, rats, mice and the like.

[0193] As used herein, the term "solid support" refers to any solid or stationary material to which reagents such as antibodies, antigens, and other test components can be attached. Examples of solid supports include, without limitation, microtiter plates (or dish), microscope (e.g. glass) slides, coverslips, beads, cell culture flasks, chips (for example, silica-based, glass, or gold chip), membranes, particles (typically solid; for example, agarose, sepharose, polystyrene or magnetic beads), columns (or column materials), and test tubes. Typically, the solid supports are water insoluble.

[0194] As used herein, the term "instructional material" or a "user manual" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of reagents for performing a method as described herein.

[0195] As used herein, the term "biological sample" is intended to refer to a subset of the tissues of a biological organism, its cells or component parts (e.g. body fluids, including but not limited to, blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).

[0196] We have discovered, using a combination of structural analysis, site-directed mutagenesis and functional studies in vivo, a dimerization interface in RAF and KSR. We have identified a number of residues within the RAF and KSR kinase domains which, when mutated, prevent the formation of oncogenic dimers. Through this, we have discovered that RAF catalytic function is regulated in response to a specific mode of dimerization of its kinase domain (which we term the side-to-side dimer). Furthermore, we have discovered that the RAF-related pseudo-kinase KSR also participates in forming side-to-side heterodimers with RAF and thereby can trigger RAF activation. This mechanism provides an elegant explanation for the longstanding conundrum regarding RAF catalytic activation and provides an explanation for the capacity of KSR, despite lacking catalytic function, to directly mediate RAF activation. We have also demonstrated that RAF side-to-side dimer formation is essential for aberrant signaling by oncogenic B-RAF mutants and we have identified an oncogenic mutation that acts specifically by promoting side-to-side dimer formation. These discoveries allow us to identify the side-to-side dimer interface of RAF as a potential therapeutic target for intervention in B-RAF-dependent tumourigenesis.

[0197] I: Nucleic Acid Molecules, Vectors, Cells, Transgenes and Transgenic Non-Human Mammals

[0198] Described herein are mutated isoforms of RAF and KSR proteins. Furthermore, we have discovered that single point mutations in the dimerization interface of RAF or KSR kinase domains prevents the formation of side-to-side dimers, when compared to wild type RAF or KSR. The single point mutations in the RAF kinase domain are at residues H449, G450, R481H, L487, F488, M489, Y538, A541 and K542 with the mutations being H449E, G450W, R481H, L487R, F488A, F488L, M489W, Y538F, A541E and K542E. The single point mutations in the KSR kinase domain are at residues H699, G700, R732, L738, F739, M740, Y790, A793 and R794 with the mutations being H699E, G700W, R732H, L738R, F739A, F739L, M740W, Y790F, A793E and R794E. We have also discovered that the isolated kinase domain of RAF forms homodimers in aqueous solution. Similar behavior is expected for the isolated KSR kinase domain as well as heterodimers should form in aqueous solution upon mixing equimolar amounts of RAF and KSR kinase domains.

[0199] Thus, a substantially pure DNA molecule, such as genomic, cDNA, or a synthetic DNA molecule, encodes one of the mammalian or non-mammalian RAF or KSR isoforms in which one or more nucleotide substitutions has/have been incorporated into the dimerization interface.

[0200] In certain embodiments, DNA sequences are substantially identical to the DNA sequences, or a fragment thereof, as illustrated in FIGS. 14A through 14F (SEQ ID NO's: 8, 10, 12, 14, 16, and 18). Another aspect features RNA, which is encoded by the DNA described herein. In one example, the RNA is mRNA. In another example, the RNA is antisense RNA.

[0201] Also contemplated are oligonucleotide probes, which specifically hybridize with the nucleic acid molecules as described herein. In certain examples, the probe specifically hybridizes with mutated RAF or KSR nucleic acid molecules (e.g. a nucleic acid having a sequence encoding a mutated RAF or KSR protein) while not hybridizing with the wild type or "normal" sequence under high or very high stringency conditions. Primers capable of specifically amplifying mutated RAF or KSR encoding nucleic acids described herein are also contemplated herein. As mentioned previously, such oligonucleotides are useful as probes and primers for detecting, isolating or amplifying mutated RAF or KSR genes.

[0202] Nucleic acid molecules encoding the mutated RAF or KSR proteins, as described herein, can be prepared by known general methods or isolated from appropriate biological sources using methods known in the art. Additionally, cDNA or genomic clones having homology with human and other known mammalian RAF or KSR, for example, mouse, rat, and the like, or non-mammalian RAF or KSR, such as Drosophila, may be isolated from other species using oligonucleotide probes corresponding to predetermined sequences within the human RAF or KSR encoding nucleic acids.

[0203] Nucleic acids described herein may be maintained as DNA in any convenient vector. Accordingly, vectors comprising a nucleic acid molecule as described herein and more particularly a plasmid expression vector are encompassed. Also encompassed are host cells transformed with such vectors and transgenic animals comprising such a nucleic acid molecule as described herein. Those cells and animals could serve as models of disease in order to study the mechanism of the function of the RAF or KSR gene and also allow for the screening of therapeutics.

[0204] In some embodiments, the vector, host cell or transgenic animal comprise a nucleic acid molecule (a transgene) encoding a mutated RAF or KSR protein that is expressed or delivered to tissues. The host cell is a transformed and stable cell line constitutively expressing the mutant RAF or KSR isoform.

[0205] Methods for producing host cells and transgenic animals are known in the art. Host cells include, but are not limited to mammalian, yeast or bacterial cells Transgenic animals can be selected from non-human mammals such as farm animals (such as pigs, goats, sheep, cows, horses, rabbits, and the like), rodents (such as rats, guinea pigs, mice, and the like), non-human primates (such as baboon, monkeys, chimpanzees, and the like), and domestic animals (such as dogs, cats, and the like) and wild and domestic (such as swans, ducks, fowl and the like). A transgenic animal is an animal having cells that contain a transgene which was introduced into the animal or an ancestor of the animal at a prenatal (embryonic) stage. The cells and transgenic animals can be useful to identify mutated RAF or KSR proteins specific to each organ, and monitoring dimerization of the RAF and KSR in response to therapeutic treatment.

[0206] II: Mutated RAF or KSR Polypeptides

[0207] A mutated RAF or KSR polypeptide sequence may have 80% homology or more with any of the amino acid sequences disclosed herein. A mutated RAF or KSR polypeptide sequence as described herein may also comprise at least 50 or more contiguous amino acids of any of sequences disclosed herein.

[0208] Mutated dimer interface residues in Drosophila RAF or Drosophila KSR and their equivalent positions in mammalian B-RAF or KSR1 are provided in the Tables below:

TABLE-US-00002 Hsap Mmus Dmel RAF BRAF BRAF (Acc. # (Acc. # (Acc. # P11346) P15056) P28028) H449E H477 H514 G450W G478 G515 R481H R509 R546 L487R L515 L552 F488A F516 F553 F488L F516 F553 M489W M517 M554 Y538F Y566 Y603 A541E A569 A606 K542E K570 K607

TABLE-US-00003 Dmel KSR Hsap Mmus (Acc. # KSR1 (Acc. KSR1 (Acc. Q24171) # Q8IVT5) # Q61097) H699E H631 H583 G700W G632 G584 R732H R663 R615 L738R L669 L621 F739A F670 F622 F739L F670 F622 M740W M671 M623 Y790F Y721 Y673 A793E A724 A676 R794E K725 K677

[0209] Other dimer interface residues in Drosophila RAF or Drosophila KSR and their equivalent positions in mammalian B-RAF or KSR1, and which are mutatable include those in the following Tables:

TABLE-US-00004 Hsap Mmus Dmel RAF BRAF BRAF (Acc. # (Acc. # (Acc. # P11346) P15056) P28028) E420 D448 D485 W422 W450 W487 W448 W476 W513 K478 R506 R543 K479 K507 K544 T480 T508 T545 H482 H510 H547 C483 V511 V548 Q502 Q530 Q567 D537 D565 D602 L560 L588 L625 S561 T589 T626 E687 E715 E752

TABLE-US-00005 Dmel KSR Hsap Mmus (Acc. # KSR1 (Acc. KSR1 (Acc. Q24171) # Q8IVT5) # Q61097) K670 Q602 Q554 W672 W604 W556 W698 W630 W582 K729 R660 R612 N730 Q661 Q613 T731 T662 T614 H733 H664 H616 E734 E665 E617 S754 S685 S637 G789 G720 G672 K812 K743 K695 V813 V744 V696 E941 E876 E828

[0210] SwissProt Accession Numbers are Provided for Reference

[0211] Referring to FIGS. 14A through 14F, specifically SEQ ID NO's: 7, 9, 11, 13, 15 and 17, the amino acid positions for the experimentally verified dimer interface residues are shaded in the protein sequences presented. In these Figures, predicted additional dimer interface residues are underlined.

[0212] In some embodiments, the mutated RAF or KSR polypeptide is an isolated mutated protein in which the mutations are located in the RAF or KSR kinase domain, specifically in the dimerization interface. In certain examples, the mutated RAF polypeptide comprises one or more mutations selected from H449E, G450W, R481H, L487R, F488A, F488L, M489W, Y538F, A541E and K542E. In certain examples, the mutated KSR polypeptide comprises one or more mutations selected from H699E, G700W, R732H, L738R, F739A, F739L, M740W, Y790F, A793E and R794E.

[0213] Mutated RAF or KSR proteins or polypeptides as described herein may be prepared in a variety of ways, according to known methods. The proteins may be purified from appropriate sources, e.g., transformed bacterial or animal cultured cells or tissues, by immunoaffinity purification. The availability of nucleic acid molecules encoding mutated RAF or KSR protein enables production of the protein using in vitro expression methods and cell-free expression systems known in the art. In vitro transcription and translation systems are commercially available, e.g., from Promega or Invitrogen.

[0214] Alternatively, larger quantities of mutated RAF or KSR proteins or polypeptides may be produced by expression in a suitable prokaryotic or eukaryotic system. For example, part or all of a DNA molecule encoding for mutated RAF or KSR may be inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli. Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences. Mutated RAF or KSR proteins or polypeptides produced by gene expression in a recombinant prokaryotic or eukaryotic system may be purified according to methods known in the art.

[0215] Thus, another embodiment includes a method of producing a mammalian mutated RAF or KSR polypeptide includes providing a cell transformed with a nucleic acid sequence encoding a mammalian mutated RAF or KSR polypeptide positioned for expression in the cell. The mutated RAF or KSR polypeptide has an amino acid change at one of the positions depicted in FIGS. 14A through 14F (SEQ ID NO's: 7, 9, 11, 13, 15 and 17) that correspond to specific dimerization interface residues. The transformed cell is cultured under conditions for expressing the nucleic acid; which then produces the mammalian mutated RAF or KSR polypeptide.

[0216] A dominant-negative protein is a protein that antagonizes the action of its normal counterpart. A dominant-negative RAF would be a mutant RAF that prevents endogenous RAF from performing its natural (or oncogenic) function. Such a dominant-negative RAF (or KSR) protein could do so by sequestering away key proteins that normally act in concert with endogenous RAF (or KSR). For example, overexpression of a kinase-defective RAF construct is known to act as a dominant-negative in part by its ability to out-compete for endogenous RAS, which is normally critical for RAF activation.

[0217] Thus a dominant negative mutant polypeptide of mammalian RAF or KSR, wherein the mutant polypeptide comprises a kinase domain having a disabled dimerization interface and therefore does not associate to a WT mammalian RAF or KSR dimerization interface.

[0218] The use of a dominant-negative polypeptide could be treating or preventing a disease in a subject, in which the disease being characterized by RAF/RAF homodimerization or RAF/KSR heterodimerization. This method comprises administering to the subject in need thereof, an expression vector encoding mutated RAF or KSR polypeptide, the mutated RAF or KSR polypeptide being positioned in the vector for expression in a cell of the subject in which RAF/RAF homodimerization or RAF/KSR heterodimerization is taking place, so as to treat or prevent the disease.

[0219] III: Detection Methods

[0220] Recombinant WT and mutated RAF or KSR polypeptides can be used during in vitro RAF or KSR dimerization experiments to follow the dimerization of RAF or KSR protomers. RAF or KSR polypeptides mutants can also be co-transfected in mammalian cells with target protein substrates, such as WT RAF or KSR.

[0221] Changes in WT and mutated RAF or KSR polypeptide dimerization in response to a potential therapeutic agent, and across cell phenotypes, can be monitored by measuring the variation of the levels of phosphorylated MEK in the presence or absence of rapamycin in animal cells such as S2 cells.

[0222] The RAF or KSR dimerization appears to be involved in many aspects of cancer from initiation to metastasis. One additional aspect includes a method of detecting in a subject susceptibility to express mutant RAF or KSR polypeptide. The method includes taking a biological sample from the subject that contains a sufficient amount of a nucleic acid, for example, DNA, and sequencing predetermined regions of the DNA, which encodes a RAF or KSR mutated polypeptide. By comparing this sequence with a corresponding sequence from a non-susceptible control subject, a RAF or KSR mutation known to be indicative of the susceptibility can be identified.

[0223] Thus, a method of detecting the presence of a mutation in a RAF kinase domain or a KSR kinase domain, comprises a) providing a WT RAF kinase domain or a WT KSR kinase domain and a suspected mutant RAF kinase domain or a mutant KSR kinase domain, each domain having a cysteine residue located at its N-terminus; b) incubating the WT RAF kinase domain or the WT KSR kinase domain and the suspected mutant RAF kinase domain or the suspected mutant KSR kinase domain with different cross-linking detectable labels; c) incubating together equimolar amounts of the labelled WT RAF kinase domain or the labeled WT KSR kinase domain and detecting a signal from the detectable label so as to provide a dimerization reference signal; and d) incubating equimolar amounts of the labeled suspected mutant B-RAF kinase domain or the suspected mutant KSR kinase domain and detecting a signal from the detectable labels, an absent signal or a reduce signal compared to that of the dimerization reference signal being an indication that a mutant B-RAF kinase domain or a mutatent KSR kinase domain is present.

[0224] Also included is a bioluminescence resonance energy transfer (BRET) fusion molecule, and method of use. The fusion molecule comprises three components: a bioluminescent donor protein (donor) and a fluorescent acceptor molecule (acceptor), wherein the acceptor can accept energy from the donor-generated luminescence when these components are in an appropriate spatial relationship and in the presence of an appropriate substrate. A modulator (a drug-like compound for example) can either influence the proximity/orientation of the donor and the acceptor and thereby the energy transfer between these components, or it can play a different role in affecting the energy transfer between the donor-generated activated product and the acceptor.

[0225] Thus, there is provided a method of monitoring the formation of RAF/RAF or RAF/KSR kinase domain dimers to detect mutations inhibiting dimerization or drug-like molecules interfering with dimerization. This method comprises a) fusing either (i) a RAF kinase domain or (ii) a KSR kinase domain at either of their N- or C-termini to a BRET donor or a BRET acceptor to produce donor labeled and acceptor labeled fusion proteins; b) expressing the fusion proteins to identify combinations that provide specific BRET signals; c) introducing dimer interface mutations into either of the labeled fusion proteins; d) expressing the labeled mutated fusion proteins with WT RAF or KSR kinase domains; e) measuring the BRET signals, a loss or significant reduction of the BRET signal using dimer interface mutations as opposed to mutations remote from the interface, being an indication that a specific BRET signal which depends on the RAF/RAF or RAF/KSR dimerization interface has been obtained.

[0226] In one example, the BRET donor is renilla luciferase variant II or rlucII and the BRET acceptor is GFP10. The acceptor label is Yellow Fluorescent Protein (YFP). The donor labeled fusion protein is SEQ ID NO's: 24, 34, 42 and 48, whereas the acceptor labeled fusion protein is SEQ ID NO's: 22, 30, 40 and 54. The donor labeled mutated fusion proteins are SEQ ID NO's: 36, 50 and the acceptor labeled mutated fusion proteins are SEQ ID NO: 32.

[0227] IV: Antibodies and Kits

[0228] Also provided are antibodies capable of immunospecifically binding to mutated RAF or KSR proteins and polypeptides as described herein. Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Such antibodies may be may be used for immunoaffinity enrichment of the mutated RAF or KSR or they may be used in a kit for detecting in a subject the susceptibility to develop a condition or an increased likelihood of developing a condition characterized by dimerization of RAF and/or KSR.

[0229] Polyclonal antibodies directed toward mutated RAF or KSR protein, polypeptides or fragments thereof may be prepared according to standard methods. In one example, monoclonal antibodies are prepared, such that antibodies react immunospecifically with predetermined epitopes of the mutated RAF or KSR protein. In one example, the antibodies are immunogically specific to mutated RAF or KSR proteins and polypeptides. Monoclonal antibodies may be prepared according to general methods known in the art. Polyclonal or monoclonal antibodies that immunospecifically interact with mutant RAF or KSR proteins can be utilized for identifying and purifying such proteins. For example, antibodies may be utilized for affinity separation of proteins with which they immunospecifically interact. Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules.

[0230] One advantageous use of antibodies as described herein is in the use of a kit for monitoring the RAF or KSR dimerization activity of a cell or the binding of RAF or KSR to specific protein substrates such as the 14-3-3 proteins. This information may be used for purposes of diagnosis, prognosis or for predicting the response to treatment. Examples of diseases include cancer. The kit comprises a substantially pure antibody that specifically binds to a mammalian mutated RAF or KSR polypeptide and a means for detecting the binding of the antibody to the mammalian RAF or KSR polypeptide.

[0231] V: Screening Methods

[0232] Because we have identified the amino acid residues involved in RAF/RAF homodimerization and RAF/KSR heterodimerization, we can use this knowledge to screen for potential therapeutic agents which interact, either covalently or non-covalently, with the WT amino residue counterparts. Thus, one additional aspect includes methods of identifying biological agents or small molecules that modulate or prevent RAF or KSR dimerization activity in the cell or modification of the regulation of protein RAF or KSR dimerization. This could also be exploited for example to screen for inhibitors, activators or modulators of RAF or KSR dimerization. The identified agents or molecules could be exploited as research reagents or for therapeutic purposes. The method could be used for in vitro screening assays using purified RAF or KSR WT polypeptides.

[0233] Generally speaking, there is provided a method of identifying inhibitors of RAF/RAF or RAF/KSR dimerization that bind to a RAF or KSR kinase domain, the RAF or KSR full protein or the kinase domain is bound to a support, and a potential inhibitor is added to the assay. Alternatively, the potential inhibitor may be bound to the support and the RAF or KSR full protein or the kinase domain is added.

[0234] Additionally, the above described BRET assay can be used as a method of identifying a potential inhibitor of RAF/RAF homodimerization. This method comprises a) fusing a RAF kinase domain at either of its N- or C-termini to a BRET donor or a BRET acceptor to produce donor labeled and acceptor labeled fusion proteins; b) expressing the fusion proteins to identify combinations that provide specific BRET signals; c) introducing dimer interface mutations into either of the labeled fusion proteins; d) expressing the labeled mutated fusion proteins with WT RAF kinase domains; e) contacting the interface with the potential inhibitor; and f) measuring the BRET signals, a loss or significant reduction of the BRET signal for the wild-type RAF/RAF BRET pair being an indication that the inhibitor is specifically bound to the interface.

[0235] There are a number of ways in which to determine the binding of a potential inhibitor to the RAF or KSR kinase domain. In one way, the potential inhibitor, for example, may be fluorescently or radioactively labeled and binding determined directly. For example, this may be done by attaching the RAF or KSR full protein or the kinase domain to a solid support, adding a detectably labeled potential inhibitor, washing off excess reagent, and determining whether the amount of the detectable label is present on the solid support. Numerous blocking and washing steps may be used, which are known to those skilled in the art.

[0236] In some cases, only one of the components is labeled. For example, specific residues, such as those identified as described herein, in the RAF or KSR kinase domain may be labeled. Alternatively, more than one component may be labeled with different labels; for example, using I¹²⁵ for the RAF or KSR domain, and a fluorescent label for the potential inhibitor.

[0237] As used herein, the terms "drug candidate", "test compounds" or "potential inhibitor" are used interchangeably and describe any molecule, for example, protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, and the like, to be tested for bioactivity. The compounds may be capable of inhibiting the formation of RAF/RAF homodimers or RAF/KSR heterodimers.

[0238] Drug candidates can include various chemical classes, although typically they are small organic molecules having a molecular weight of more than 100 and less than about 2,500 Daltons. Candidate agents typically include functional groups necessary for structural interaction with proteins, for example, hydrogen bonding and lipophilic binding, and typically include at least an amine, carbonyl, hydroxyl, ether, or carboxyl group. The drug candidates often include cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more functional groups.

[0239] Drug candidates can be obtained from any number of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means.

[0240] Competitive screening assays may be done by combining a RAF or KSR kinase domain and a labeled probe to form a probe:RAF or KSR kinase domain complex in a first sample followed by adding a potential inhibitor from a second sample. The binding of the potential inhibitor is determined, and a change or difference in binding between the two samples indicates the presence of a test compound capable of binding to the RAF or KSR kinase domain and potentially modulating the RAF or KSR's dimerizing ability.

[0241] In one case, the binding of the potential inhibitor is determined through the use of competitive binding assays. In this embodiment, the probe is labeled with a fluorescent label. Under certain circumstances, there may be competitive binding between the potential inhibitor and the probe. Potential inhibitors which displace the probe, resulting in a change in fluorescence as compared to control, are considered to bind to the RAF or KSR kinase domain.

[0242] In one case, the potential inhibitor may be labeled. The potential inhibitor is added first to the RAF or KSR domain for a time sufficient to allow binding to form a complex.

[0243] Formation of the probe:RAF or KSR domain complex typically require incubations of between 4° C. and 40° C., for between 10 minutes to about 1 hour to allow for high-throughput screening. Any excess of reagents are generally removed or washed away. The potential inhibitor is then added, and the presence or absence of the labeled component is followed, to indicate binding to the RAF or KSR kinase domain.

[0244] In one case, the probe is added first, followed by the potential inhibitor. Displacement of the probe is an indication the potential inhibitor is binding to the RAF or KSR domain and thus is capable of binding to, and potentially modulating or inhibiting the dimerization of RAF and KSR. Either component can be labeled. For example, the presence of probe in the wash solution indicates displacement by the potential inhibitor. Alternatively, if the potential inhibitor is labeled, the presence of the probe on the support indicates displacement.

[0245] In one case, the potential inhibitor may be added first, with incubation and washing, followed by the probe. The absence of binding by the probe may indicate the potential inhibitor is bound to the RAF or KSR domain with a higher affinity. Thus, if the probe is detected on the support, coupled with a lack of potential inhibitor binding, may indicate the potential inhibitor is capable of binding to the RAF or KSR kinase domain.

[0246] Modulation is tested by screening for a potential inhibitor's ability to modulate the activity of RAF or KSR and includes combining a potential inhibitor with a RAF or KSR kinase domain, as described above, and determining an alteration in the biological activity of RAF or KSR. Therefore in this case, the potential inhibitor should both bind to the RAF or KSR kinase domain (although this may not be necessary), and alter its biological activity as defined herein.

[0247] Positive controls and negative controls may be used in the assays. All control and test samples are performed multiple times to obtain statistically significant results. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound probe determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound potential inhibitor.

[0248] Typically, the signals that are detected in the assay may include fluorescence, resonance energy transfer, time resolved fluorescence, radioactivity, fluorescence polarization, plasma resonance, or chemiluminescence and the like, depending on the nature of the label. Detectable labels useful in performing screening assays as described herein include a fluorescent label such as Fluorescein, Oregon green, dansyl, rhodamine, tetramethyl rhodamine, texas red, Eu³+; a chemiluminescent label such as luciferase; calorimetric labels; enzymatic markers; or radioisotopes such as tritium, I¹²⁵ and the like

[0249] Affinity tags, which may be useful in performing the screening assays as described herein include biotin, polyhistidine and the like.

EXAMPLES

[0250] 1. S2 Expression Plasmids

[0251] Copper-inducible pMet vectors were used for functional assays conducted in S2 cells as previously described (Douziech, M., Sahmi, M., Laberge, G. & Therrien, M. A KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila. Genes Dev 20, 807-19 (2006)), Roy, F., Laberge, G., Douziech, M., Ferland-McCollough, D. & Therrien, M. KSR is a scaffold required for activation of the ERK/MAPK module. Genes Dev 16, 427-38 (2002)). The FRB-RAF.sup.K455S fusion construct was assembled by inserting an AseI/NotI PCR fragment encompassing residues 328-738 of RAF into the AseI/NotI site of FRB-KSR (Roy, F., Laberge, G., Douziech, M., Ferland-McCollough, D. & Therrien, M. KSR is a scaffold required for activation of the ERK/MAPK module. Genes Dev 16, 427-38 (2002)). The KSR-RAF chimera-A corresponds to KSR^1-665 fused to RAF^417-739, whereas chimera-B replaced the N-lobe of KSR (a.a. positions 666-757) with the one of RAF (a.a. positions 417-505). In both cases, the RAF N-lobe contained a K455M change to catalytically impair its kinase activity and thereby mimicked kinase-inert KSR. Variant full length Drosophila KSR, RAF or FRB/FKBP fusion mutants were generated by QuickChange mutagenesis (Stratagene). Mutagenized cDNAs were fully sequenced to verify that only the desired mutations had been introduced.

[0252] 2. S2 Cell Assays

[0253] S2 cells were maintained in serum-free insect cell medium (Sigma) at 27° C. Cells were seeded at a density of 1.75×10⁶ cells/ml 24 h prior to transfection. Between 10 to 300 ng (or up to 900 ng for KSR_R732H) of DNA was transfected per construct using Effectene (Qiagen). dsRNAs were produced and used in RNAi experiments as described (Roy, F., Laberge, G., Douziech, M., Ferland-McCollough, D. & Therrien, M. KSR is a scaffold required for activation of the ERK/MAPK module. Genes Dev 16, 427-38 (2002)). Protein expression was induced by adding CuSO₄ (0.7 mM) 36 h before harvesting the cells. For FRB/FKBP-mediated dimer formation, rapamycin (Sigma) was added (1 M) to the medium 2 h prior to harvesting the cells. Lysates, immunoprecipitations, western blot procedures and antibodies were essentially as previously described (Douziech, M., Sahmi, M., Laberge, G. & Therrien, M. A, KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila, Genes Dev, 20, 807-19 (2006)).

[0254] 3. Bacterial Protein Expression and Purification

[0255] B-RAF (residues 448-723) and mutant series (R481H, L487R and L487R/E558K) were recombinantly expressed from pProEx (Invitrogen) plasmid in E. coli BL21 cells as TEV protease-cleavable 6× His-tagged fusions. To increase the level of soluble protein expression in E. coli, 16 specific mutations (remote from the side-to-side dimer interface) were introduced in B-RAF as described Tsai, J. et al., Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity, Proc Natl Acad Sci, USA, 105, 3041-6 (2008). Expressed proteins were bound to Ni--NTA and eluted with imidazole and subjected to TEV protease treatment. Further purification was performed by subtractive Ni--NTA and size exclusion (Superdex 200) chromatography.

[0256] 4. Homology Modeling

[0257] A multiple sequence alignment of KSR and RAF kinase domains was used to build a structural model of the kinase domain of Drosophila KSR (residues 670-945) in SWISS-MODEL (Schwede, T., Kopp, J., Guex, N. & Peitsch, M. C. SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res 31, 3381-5 (2003)). An initial model with a total energy of -7474.3 KJ/mol was generated using the structure of the kinase domain of B-RAF as a template (chain A of PDB entry 1UWH) (Wan, P. T. et al., Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)). This model was manually edited in COOT (Emsley, P. & Cowtan, K., Coot: model-building tools for molecular graphics, Acta Crystallogr D Biol Crystallogr, 60, 2126-32 (2004)) and a poorly modelled loop spanning residues 821-838 was removed. To generate the KSR/RAF side-to-side heterodimer, the modelled structure of KSR was superimposed onto chain A of PDB entry 1UWH.

[0258] 5. Analytical Ultracentrifugation

[0259] Equilibrium sedimentation was performed with a Beckman Optima XL-A ultracentrifuge and An60Ti rotor. B-RAF samples were prepared in 20 mM Tris (pH 7.5), 200 mM NaCl, 5% glycerol and 1.5 mM TCEP for analysis. Data was collected at 4° C. for three protein concentrations (25 μM, 12.5 μM, and 6.25 μM) at three rotor speeds (13,000 rpm, 18,000 rpm and 23,000 rpm for B-RAF_wt and B-RAF_R481H or 12,000 rpm, 17,000 rpm and 25,000 rpm for B-RAF_L487R and B-RAF-L487R/E558K). Model analysis of the data was performed simultaneously in a global curve-fitting procedure (Origin software, Beckman). For this, data collected at 13,000 rpm and 18,000 rpm for B-RAF_wt was analyzed at all three protein concentrations; data collected at 18,000 rpm and 23,000 rpm for B-RAF_R481H was analyzed at all three protein concentrations; data collected at 17,000 rpm and 25,000 rpm for B-RAF_L487R was analyzed at all three protein concentrations; data collected at 17,000 rpm and 25,000 rpm for B-RAF_L487R/E558K at 25 μM and 12.5 μM was analyzed. The term "global" refers to fits across all rotor speeds for a given concentration.

[0260] The global self association fit yielded an average molecular weight (MW) of 57,978 Da for B-RAF_wt. The ratio of the observed average MW to the theoretical MW of the monomer is 1.9:1 suggesting that the sample contains mostly dimers. A single-species dimer model (shown by the red line; FIG. 3C) best fit the observed data (blue circles; FIG. 3C), indicated by the random distribution of the residuals--a measure of goodness of fit (the residual is the difference between the observed value and the predicted value). For B-RAF_R481H, the global self association fit yielded an average MW of 34,544 Da. The ratio of the observed average MW to the theoretical MW of the monomer is 1.1:1 suggesting that the sample contains mostly monomers. A single-species monomer model (red line; FIG. 3C) best fit the observed data (blue circles; FIG. 3C). For B-RAF_L487R, the global self association fit yielded an average MW of 48,636 Da. The ratio of the observed average MW to the theoretical MW of the monomer is 1.5:1 suggesting that the sample contains a mixture of monomers and dimers. Consistent with this, a monomer-dimer model (red line; FIG. 12) resulted in the best fit to the observed data (blue circles; FIG. 12) with a dissociation constant (Kd) of 2 M (Note: In order to reliably estimate Kd values from an AUC experiment, both species in a monomer-dimer equilibrium need to be sufficiently represented in solution; in our AUC analyses, we observed such a monomer-dimer equilibrium only for the B-RAF_L487R dimer mutant). For B-RAF_L487R/E558K, the global self association fit yielded an average MW of 55,472 Da. The ratio of the observed average MW to the theoretical MW of the monomer is 1.8:1 suggesting that the sample contains mostly dimers and the data (blue circles; FIG. 12) was best fit to a single-species dimer model (red line; FIG. 12).

[0261] 6. RNA Preparation and Quantitative Real-Time PCR (qPCR)

[0262] S2 cells were treated with specific RNAi for four days and total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. qPCR analyses were performed as follows. A total of 2 μg of RNA was reverse transcribed using the High Capacity cDNA Archive Kit with random primers (Applied Biosystems, Foster City, Calif.) as described by the manufacturer.

[0263] Primer and probe sets from Universal ProbeLibrary were used for quantitative real-time PCR (https://www.roche-applied-science.com/sis/rtper/upl/index.jsp). Primers were chosen so that the amplified regions did not overlap with the areas targeted by dsRNAs used for RNAi. PCR reactions for 384-well plate formats were performed using 2 μl of cDNA, 5 μl of the TaqMan fast Universal PCR Master Mix (Applied Biosystems, CA), 2 μM of each primer and 1 μM of the Universal TaqMan probe in a total volume of 10 μl. The ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems) was used to detect the amplification level. The relative quantification of target genes was determined by using the CT method. Briefly, the Ct (threshold cycle) values of target genes were normalized to an endogenous control gene (Rp149) (CT=Ct target-Ct Rp149) and compared with a calibrator (wild type): CT=Ct.sub.Sample-Ct.sub.Calibrator. Relative expression (RQ) or fold change was calculated using the Sequence Detection System (SDS) 2.2.2 software (Applied Biosystems) and the formula RQ=2^-CT.

TABLE-US-00006 dsRNA primers: GFP amplicon (SEQ ID NO: 71) top 5'- CGTAAACGGCCACAAGTTCAG (SEQ ID NO: 72) bottom 5'- ACGAACTCCAGCAGGACCATG RAS amplicon (SEQ ID NO: 73) top 5'- AATACAAACTGGTCGTCGTTG (SEQ ID NO: 74) bottom 5'- AATCTACGATTCGGCTTGTTC CNK amplicon (SEQ ID NO: 75) top 5'- TTTGGACAGATCTATATGCAG (SEQ ID NO: 76) bottom 5'- TCGGTTCAAAGGTCTCCAG HYP amplicon (SEQ ID NO: 77) top 5'- CCGATTGTGTCACCCCTAAT (SEQ ID NO: 78) bottom 5'- CCACTTGAGCACATCGCTAA CK2α amplicon (SEQ ID NO: 79) top 5'- GACACTTCCTAGTGCGGCTCGCGTG (SEQ ID NO: 80) bottom 5'- GTAATCATACATCTGGTAATCTACC 14-3-3ε amplicon (SEQ ID NO: 81) top 5'- TGACTGAGCGCGAGAACAATG (SEQ ID NO: 82) bottom 5'- TCTTCTGCCTGCATATCGGAC 14-3-3ζ amplicon (SEQ ID NO: 83) top 5'- GACAGTCGATAAGGAAGAGCTGG (SEQ ID NO: 84) bottom 5'- TCGTTCAGTGTGTCCAGCTC

[0264] 7. Screening Assays

[0265] Two independent assays were developed to monitor RAF dimerization. The first is a FRET (fluorescence resonance energy transfer) assay. It is based on the observation that bacterially-expressed human B-RAF kinase domain form dimers in solution.

[0266] The second assay exploits the BRET (bioluminescence resonance energy transfer) technology to assess for RAF homodimerization or RAF-KSR heterodimerization using a cell-based system.

[0267] A. FRET Assay

[0268] A single cysteine residue is engineered at the N-terminus of the wild-type or mutant B-RAF kinase domain for cross-linking fluorescent probes. Following bacterial expression and purification, independent batch of proteins are labeled either with Alexa 555 (donor) or Alexa 647 (acceptor). Labeled proteins are re-purified and then combined in equimolar ratios. FRET detection is carried out using a Luminescence Spectrometer. Various controls are conducted in parallel. For instance, no FRET signal is detected when labeled proteins are tested alone. Similarly, no FRET signal or a significantly reduced one is detected when dimer interface mutants (e.g. R481H-like) are tested in combination with wild-type B-RAF.

[0269] B. BRET Assay

[0270] The BRET assay can use either BRET1 or BRET2 as a means of measuring BRET signals. We used BRET2 donor (renilla luciferase variant II or rlucII) and acceptor (GFP10) fusions rather than BRET1 fusions (rluc and YFP) as well as the addition of a CAAX-box to target RAF to the plasma membrane, since the BRET2 system is more sensitive and has a higher signal to noise ratio (Kocan, See et al., 2008), to independently fuse RAF and KSR kinase domains at either their N- or C-terminus. The addition of the CAAX-box is frequently used to generate BRAF gain of function alleles and is reported in the literature (Leevers, Paterson et al., 1994). We focused on the human BRAF kinase domain (BRAF-KD) expressed from the pCDNA3.1 plasmid backbone (Invitrogen) in a HEK293 transfection setup.

[0271] The assay included co-transfection of rlucII fused to the N-terminus of the human BRAF-KD (referred to below as the donor) and of N-terminally GFP10-tagged hBRAF-KD (referred to below as the acceptor) both targeted to the plasma membrane with a CAAX-box. Transfections were performed in HEK293T cells with PEI as a transfection reagent in a 6-well format with varying molar ratios of pCDNA3.1 donor:acceptor constructs (0:1, 0.25:1, 0.5:1, 1:1, 2:1, 5:1, 10:1 and 20:1). 48 hours post transfection, the cells were washed and resuspended in tyrode buffer and transferred to opaque microtiter plates. GFP10 raw signal was read on a FlexStation 3 plate reader (Molecular Devices) and BRET signals were read using a Mithras LB 940 plate reader following the addition of DeepBlue C (DBC) at a concentration of 10 μM. The data was then analysed using the GraphPad Prism software package.

[0272] The assay was highly reproducible and the BRET ratio obtained for the rlucII-BRAF-KD-CAAX versus GFP10-BRAF-KD-CAAX pair at saturating concentration of the acceptor construct was consistently between 4.7 and 4.9.

[0273] We also generated the dimer interface mutation R481H of the BRAF-KD and measured its impact on the affinity of the BRAF-BRAF interaction. The BRAF wt-wt pair yielded a significantly higher BRET ratio than when the R481H mutant was introduced as the donor or acceptor construct (FIG. 15). This reduction was reproducible and significant in terms of both the BRET_max and BRET₅₀ ratios (FIG. 15). This was indicative of a significant decrease in BRAF-BRAF affinity when the dimer interface is perturbed.

[0274] Altogether, the BRET assay is specific for the BRAF-KD vs BRAF-KD interaction and is highly sensitive to the genetic alteration of the now well-characterized dimer interface (Hatzivassiliou, Song et al.; Poulikakos, Zhang et al.; Rajakulendran, Sahmi et al. 2009).

[0275] All BRET assays were developed with the human B-RAF (hBRAF), C-RAF (hCRAF), KSR1 (hKSR1) and MEK1 (hMEK1) isoforms (see FIGS. 16 through 27 and SEQ ID NO's: 19 through 70). In FIGS. 16 through 27, CDS stands for coding sequences. And KD stands for kinase domain.

[0276] The mutagenised residues are labeled according to their position in the Drosophila orthologous protein sequence. Thus, the R481H mutation of Drosophila RAF corresponds to the R509H mutation of human B-RAF, and the C922Y mutation of Drosophila KSR corresponds to the C722Y mutation of human KSR1.

[0277] Results and Discussion

[0278] In Drosophila, RAF activation is regulated by a core complex that notably includes the proteins RAS, CNK, HYP and KSR amongst others (Claperon, A. & Therrien, M. KSR and CNK: two scaffolds regulating RAS-mediated RAF activation, Oncogene, 26, 3143-58 (2007)). Of these proteins, the function of KSR (Kinase Suppressor of Ras) in RAF activation remains controversial. KSR contains a kinase domain of closest sequence similarity to RAF (Manning, G., Whyte, D. B., Martinez, R., Hunter, T. & Sudarsanam, S., The protein kinase complement of the human genome, Science, 298, 1912-34 (2002)) and was initially thought to drive RAF activation by virtue of its kinase activity. However, subsequent studies have been inconclusive in demonstrating this point and thus relegated KSR as a pseudo-kinase (Boudeau, J., Miranda-Saavedra, D., Barton, G. J. & Alessi, D. R., Emerging roles of pseudokinases, Trends Cell Biol, 16, 443-52 (2006)). Because of its capacity to bring MEK to RAF, the function of KSR is currently considered to be that of an organizing centre (or scaffold) in the ERK pathway (Kolch, W., Coordinating ERK/MAPK signalling through scaffolds and inhibitors, Nat Rev Mol Cell Biol, 6, 827-37 (2005)).

[0279] We previously showed in Drosophila S2 cells that co-overexpression of KSR with RAF and MEK stimulated RAF-dependent MEK phosphorylation (Douziech, M., Sahmi, M., Laberge, G. & Therrien, M., A KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila,. Genes Dev, 20, 807-19 (2006)). If KSR was solely acting as a scaffold, we reasoned that overexpression of KSR without co-overexpression of its scaffold partners would perturb the optimal stoichiometry of KSR containing complexes with the net effect of decreasing RAF activation. Since we observed increased RAF activation, this suggested that KSR might possess an inherent RAF activating capacity that becomes apparent upon overexpression. To investigate whether KSR can stimulate increasing RAF activation in a concentration dependent manner (as would be the case if KSR possessed an intrinsic RAF activating capacity), we titrated in increasing amounts of KSR in S2 cells and monitored the effect by assessing RAF-dependent MEK phosphorylation (Roy, F., Laberge, G., Douziech, M., Ferland-McCollough, D. & Therrien, M., KSR is a scaffold required for activation of the ERK/MAPK module, Genes Dev, 16, 427-38 (2002)). As shown in FIG. 1A, increasing levels of KSR correspondingly increased MEK phosphorylation. Surprisingly, this KSR-dependent RAF activation was unperturbed by RNAi-mediated knockdown of RAS (FIG. 1A; see FIG. 13A for a demonstration of the activity and specificity of the RAS dsRNA). Moreover, co-overexpression of a constitutively active RAS (RAS^V12) under these conditions did not considerably augment MEK phosphorylation, suggesting that KSR can drive RAF activation independently of RAS activity when overexpressed in S2 cells (FIG. 1A). These results suggest a role for KSR in RAF activation beyond a scaffold-only function.

[0280] To more rigorously rule out a scaffolding function as the origin of the observed stimulatory effect of overexpressed KSR on RAF, we used RNAi to knockdown a subset of known scaffold partners of KSR. Interestingly, in this context RAF activation was also unperturbed by RNAi-mediated knockdown of CNK or HYP (also known as AVE), which are normally required under physiological conditions (Douziech, M., Sahmi, M., Laberge, G. & Therrien, M., A KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila, Genes Dev, 20, 807-19 (2006)), Douziech, M. et al., Bimodal regulation of RAF by CNK in Drosophila, Embo J, 22, 5068-78 (2003)) and Roignant, J. Y., Hamel, S., Janody, F. & Treisman, J. E., The novel SAM domain protein Aveugle is required for Raf activation in the Drosophila EGF receptor signaling pathway, Genes Dev, 20, 795-806 (2006) (FIG. 1B). Studies with mammalian cells recently suggested that CK2 (bound to KSR1) phosphorylates and activates B-RAF/C-RAF (Ritt, D. A. et al., CK2 is a component of the KSR1 scaffold complex that contributes to Raf kinase activation, Curr Biol, 17, 179-84 (2007)). We found that KSR not only potently stimulated RAF activation in the presence of RNAi-mediated knockdown of CK2 (FIG. 1C), but that mutating the proposed CK2 binding site on KSR or the sites of CK2-mediated phosphorylation on RAF (Ritt, D. A. et al., CK2 Is a component of the KSR1 scaffold complex that contributes to Raf kinase activation, Curr Biol, 17, 179-84 (2007)) had no impact on the ability of KSR to drive RAF activation under our overexpression conditions (FIG. 1C). Taken together, these results suggest that the overexpression of KSR in S2 cells unmasks an inherent activation potential on RAF beyond its well established role as a scaffold.

[0281] We previously showed that the capacity of KSR to bind MEK was required for the ability of RAF to phosphorylate MEK (Roy, F., Laberge, G., Douziech, M., Ferland-McCollough, D. & Therrien, M., KSR is a scaffold required for activation of the ERK/MAPK module, Genes Dev, 16, 427-38 (2002)). This result supported a scaffolding role for KSR in RAF activation. More recently, we identified a mutation in KSR (R732H) within its kinase domain that completely abolished its RAF activating capacity yet fully retained its ability to bind MEK and RAF Douziech, M., Sahmi, M., Laberge, G. & Therrien, M, A KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila, Genes Dev, 20, 807-19 (2006) (FIG. 2). This mutant provided a starting point for unraveling the mechanism by which KSR directly activates the catalytic function of RAF.

[0282] Since the kinase domain of KSR is most similar to that of RAF (Manning, G., Whyte, D. B., Martinez, R., Hunter, T. & Sudarsanam, S., The protein kinase complement of the human genome, Science, 298, 1912-34 (2002), we hypothesized that the previously determined crystal structure of the kinase domain of human B-RAF (the human orthologue of Drosophila RAF) might provide a good model to discern the mechanism of action of the KSR R732H mutation. Indeed, Arg732 is not only invariant across all KSR proteins, it is invariant across the larger RAF/KSR family (but not in other closely related kinases; FIG. 4). Intriguingly, while the structure of the kinase domain of B-RAF was reported as a monomer (Wan, P. T. et al., Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)), the asymmetric unit of the crystal in fact contains two RAF kinase domains that interact in a unique side-to-side fashion involving the N-lobe of their kinase domains (FIG. 6A). This mode of dimerization, which was not appreciated to date, was observed in a total of five subsequent RAF structure analyses (Wan, P. T. et al., Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)), (Hansen, J. D. et al., Potent and selective pyrazole-based inhibitors of B-Raf kinase, Bioorg Med Chem Lett, 18, 4692-5 (2008)) (in distinct crystal lattices), suggesting that the mode of dimerization/oligomerization is functionally relevant rather than an artifact of crystal packing (FIG. 6B). Side-to-side dimerization of the RAF kinase domain buries a large surface area (˜1280 Ų) and provocatively involves helix αC, a key structural element whose conformation serves a regulatory function in numerous protein kinases (Huse, M. & Kuriyan, J. The conformational plasticity of protein kinases. Cell 109, 275-82 (2002)) (FIG. 6A). Most notably, a specific mode of dimerization involving helix αC underlies an allosteric mechanism for kinase activation for both PKR (Dar, A. C., Dever, T. E. & Sicheri, F. Higher-order substrate recognition of eIF2alpha by the RNA-dependent protein kinase PKR. Cell 122, 887-900 (2005)) and EGFR (Zhang, X., Gureasko, J., Shen, K., Cole, P. A. & Kuriyan, J. An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125, 1137-49 (2006)) kinase domains (FIG. 6C). As the structure of the RAF kinase domain adopts a productive conformation in the dimeric crystal configuration, we reasoned that side-to-side dimerization itself might directly modulate the attainment of an active kinase conformation of RAF.

[0283] Projection of KSR/RAF conserved residues onto the RAF crystal structure revealed that nearly the entire side-to-side dimer contact surface of RAF, but no other surfaces, are conserved across the larger KSR/RAF family (FIG. 3A; FIG. 4). This suggested that KSR might form an analogous dimer structure. Moreover, the position of Arg481 (the equivalent of Arg732 in KSR; FIG. 4) at the center of the side-to-side dimer interface of the B-RAF crystal structure (FIG. 3B) hinted at the basis by which the mutation of Arg732 in KSR might exert a functional effect by perturbing dimerization (for simplicity, we used the Drosophila RAF numbering scheme for discussion of human B-RAF positions; see the Table below for list of residue equivalence between B-RAF and Drosophila RAF).

TABLE-US-00007 Drosophila RAF Human B-RAF Trp422 Trp450 Trp448 Trp476 His449 His477 Gly450 Gly478 Lys478 Arg506 Lys479 Lys507 Thr480 Thr508 Arg481 Arg509 His482 His510 Cys483 Val511 Leu487 Leu515 Phe488 Phe516 Met489 Met517 Gln502 Gln530 Asp537 Asp565 Tyr538 Tyr566 Ala541 Ala569 Lys542 Lys570 Glu558 Glu586 Leu560 Leu588 Ser561 Thr589 Glu687 Glu715

[0284] In order to investigate the potential of the RAF kinase domain to form dimers in solution, we performed analytical ultracentrifugation experiments (FIG. 3C). Equilibrium sedimentation analysis confirmed that RAF can form dimers under the conditions tested (i.e., micromolar concentrations). Consistent with the mode of dimerization seen in the crystal structure, mutation of Arg481 in B-RAF converted it to a predominant monomer in solution. This result shows that the side-to-side dimer configuration of RAF visualized in the crystal environments is also sampled in solution. Based on these findings, we reasoned that the R732H mutation in KSR most likely perturbs KSR's ability to form an analogous side-to-side homodimer or to form a side-to-side heterodimer with RAF. This in turn could explain the mechanism by which the KSR_R732H mutation abolishes RAF activation.

[0285] If KSR mediates RAF activation by a mechanism involving the formation of a specific side-to-side homodimer with itself (i.e. KSR/KSR side-to-side homodimer) or a heterodimer with the kinase domain of RAF (i.e. KSR/RAF side-to-side heterodimer), then mutation of other dimer interface residues on KSR in close vicinity to Arg732 might also impair RAF activation. Using our minimal KSR/RAF/MEK co-overexpression activation assay, we found this to be the case. Specifically, individual mutation of four additional residues (G700W, F739A, M740W and Y790F) on KSR severely impeded its ability to induce RAF activation (FIG. 5A). If KSR mediates RAF activation by forming a specific side-to-side heterodimer with the kinase domain of RAF, then mutations of the corresponding positions (residues) on RAF should also impair RAF activation. As shown in FIG. 5A, we also found this to be the case. In contrast, control mutations remote from the side-to-side dimer interface on the kinase domains of both KSR and RAF showed no significant effect on RAF activation (FIG. 5A). We note that none of the dimer interface mutations in KSR detectably affected the KSR/MEK interaction, indicating that the mutations did not simply destroy protein fold (data not shown). These results confirm that the integrity of the side-to-side dimer interface on KSR and on RAF is essential for RAF activation.

[0286] While our results above are consistent with the possibility that KSR and RAF heterodimerize through their kinase domains, it is equally possible that KSR/KSR side-to-side homodimers might instead contribute to RAF activation. To demonstrate that the formation of side-to-side kinase domain heterodimers by KSR and RAF per se leads to RAF activation, we employed the FRB/FKBP fusion protein system to inducibly promote KSR/RAF side-to-side heterodimer formation by the addition of rapamycin in vivo (Muthuswamy, S. K., Gilman, M. & Brugge, J. S. Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo- and heterodimers, Mol Cell Biol, 19, 6845-57 (1999)).

[0287] Towards this end, we fused a region encompassing the minimal kinase domains of KSR and RAF to the FRB and FKBP fragments, respectively (Roy, F., Laberge, G., Douziech, M., Ferland-McCollough, D. & Therrien, M., KSR is a scaffold required for activation of the ERK/MAPK module, Genes Dev, 16, 427-38 (2002)) (See FIG. 5B for schematic). The use of the FRB/FKBP fusion in conjunction with a myristoylation signal on the FKBP fusion construct (to localize it to the membrane) allowed us to tightly modulate heterodimerization of the kinase domains in a rapamycin-dependent manner (Roy, F., Laberge, G., Douziech, M., Ferland-McCollough, D. & Therrien, M., KSR is a scaffold required for activation of the ERK/MAPK module, Genes Dev, 16, 427-38 (2002)). In this setup, we observed that promoting the KSR/RAF heterodimer by addition of rapamycin was indeed sufficient to potently activate RAF as evidenced by the elevated levels of phosphorylated MEK (FIG. 5B). RAF activation was selectively perturbed by ten specific mutations at the side-to-side dimer interface on both KSR (H699E, G700W, R732H, L738R, F739A, F739L, M740W, Y790F, A793E and R794E) and on RAF (H449E, G450W, R481H, L487R, F488A, F488L, M489W, Y538F, A541E and K542E), but not by control mutations outside the side-to-side dimer interface of KSR or RAF (FIG. 5B; FIG. 8). Taken together, these results indicate that formation of the side-to-side heterodimer between KSR and RAF kinase domains is both sufficient and necessary for RAF activation under the conditions tested.

[0288] As both RAF and KSR likely form identical side-to-side dimers, by virtue of having near identical dimerization surfaces (FIG. 3A), it is conceivable that both KSR/RAF heterodimers and RAF/RAF homodimers might equally promote RAF activation, assuming RAF activation and downstream signaling is solely dependent on forming the kinase domain side-to-side dimer. To investigate whether the RAF/RAF homodimers can also lead to RAF activation, we used the FRB/FKBP/rapamycin system to drive side-to-side homodimer formation of RAF kinase domains in vivo (see FIG. 5C for schematic). To ensure our interpretation of side-to-side dimer formation induced activation is not confounded by trans autophosphorylation activity within the RAF/RAF homodimer, we introduced a mutation (K455S) in the FRB-RAF fusion to catalytically impair its kinase activity (i.e. to effectively mimic the kinase dead state of KSR). As shown in FIG. 5C, rapamycin induced formation of RAF/RAF homodimers can indeed drive RAF activation in a manner dependent on the ability to form the side-to-side dimers (FIG. 5C).

[0289] Although RAF/RAF homodimers are competent for activation, the level of activation is not as robust as that resulting from KSR/RAF heterodimers (based on quantification of induced MEK phosphorylation levels in the presence and absence of rapamycin; not shown). If the side-to-side dimer surfaces are in fact functionally equivalent on both KSR and RAF, this observation suggests that the KSR kinase domain may have a second function that is not shared with RAF. Based on the fact that KSR can stably bind MEK while RAF cannot (Roy, F., Laberge, G., Douziech, M., Ferland-McCollough, D. & Therrien, M. KSR is a scaffold required for activation of the ERK/MAPK module. Genes Dev 16, 427-38 (2002)), we reasoned that this may be the root of the difference. Since the side-to-side dimerization surface is comprised mainly by the N-lobe of KSR and RAF kinase domains, and MEK binding function is critically dependent on the C-lobe of KSR (Roy, F., Laberge, G., Douziech, M., Ferland-McCollough, D. & Therrien, M., KSR is a scaffold required for activation of the ERK/MAPK module, Genes Dev, 16, 427-38 (2002)), then a RAF N-lobe--KSR C-lobe chimera might possess both essential functions of the KSR kinase domain. If true, one would predict that substitution of the N-lobe of RAF into KSR, but not the whole kinase domain of RAF into KSR, would lead to the maintenance of KSR's ability to promote RAF mediated phosphorylation of MEK. This indeed proved to be the case. As shown in FIG. 7, overexpression of a form of KSR with a full kinase domain swap with RAF (FIG. 7A, Chimera-A) poorly activated RAF, while overexpression of a form with just an N-lobe swap (FIG. 7A, Chimera-B) was as potent as wild type KSR in promoting MEK phosphorylation by RAF (FIG. 7B). Confirming that MEK binding is indeed constrained to the C-lobe of KSR, Chimera-B but not Chimera-A bound to MEK as assessed by co-immunoprecipitation (FIG. 7B). Taken together, these results highlight two distinct functions for the kinase domain of KSR in RAF signaling. Firstly, the kinase domain of KSR functions as a scaffold whereby it binds to MEK and recruits it to RAF (i.e. KSR mediates RAF substrate targeting). Secondly, the kinase domain of KSR forms a side-to-side heterodimer with the kinase domain of RAF that underlies an allosteric mechanism for RAF catalytic activation.

[0290] Recent studies with mammalian cells, where multiple RAF isoforms exist, have found that RAF activation can also occur upon the physical juxtaposition of two isoforms of RAF mediated by 14-3-3 proteins (Weber, C. K., Slupsky, J. R., Kalmes, H. A. & Rapp, U. R., Active Ras induces heterodimerization of cRaf and Braf., Cancer Res, 61, 3595-8 (2001)), (Rushworth, L. K., Hindley, A. D., O'Neill, E. & Kolch, W., Regulation and role of Raf-1/B-Raf heterodimerization, Mol Cell Biol, 26, 2262-72 (2006)). Intriguingly, this activation route is independent of a phospho-transfer mechanism as reflected by the fact that in such RAF/RAF heterodimers, a kinase-dead isoform of RAF can activate a wild-type isoform of RAF (Chen, C., Lewis, R. E. & White, M. A., IMP modulates KSR1-dependent multivalent complex formation to specify ERK1/2 pathway activation and response thresholds, J Biol Chem, 283, 12789-96 (2008)). This behaviour is highly reminiscent of how KSR activates RAF. We reasoned that 14-3-3 proteins, which are intrinsically dimeric, act to promote the specific side-to-side dimer conformation we see in the RAF crystal structure in a manner analogous to our forced FRB-RAF/FKBP-RAF system (FIG. 5C). Consistent with this possibility, our modeling studies showed that the binding of dimeric 14-3-3 proteins concurrently to the C-terminal extension of two RAF kinase domains is fully compatible with the adoption of a side-to-side dimer configuration (FIG. 10).

[0291] Interestingly, the 14-3-3 consensus binding site in human RAF is conserved in both RAF and KSR molecules in fly and in other organisms (FIG. 9A), suggesting that 14-3-3 could also act to promote RAF homodimers and more potent KSR/RAF heterodimers in flies. Demonstrating that 14-3-3 is indeed relevant for RAF activation in flies, we found that depletion of endogenous 14-3-3 proteins perturbed KSR-dependent RAF activation (FIG. 9B). Consistent with the notion that 14-3-3 mediates dimerization of KSR with RAF, mutation of the consensus 14-3-3 site in both KSR and RAF impaired RAF activation (FIG. 9B). These results suggest that 14-3-3 proteins might act to promote specific KSR/RAF and RAF/RAF side-to-side kinase domain dimers.

[0292] Together, our study indicates that dimerization of the RAF kinase domain with KSR or with other RAF molecules is central to its activation mechanism. We posit that other regulatory events that impinge on RAF activation may also act by modulating dimerization. In this regard, the large group of scaffolding proteins that act together with RAF and KSR, such as 14-3-3 proteins, may serve to spatially and temporally regulate the formation of side-to-side dimers (Douziech, M., Sahmi, M., Laberge, G. & Therrien, M. A, KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila, Genes Dev, 20, 807-19 (2006)), Garnett, M. J., Rana, S., Paterson, H., Barford, D. & Marais, R., Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization, Mol Cell, 20, 963-9 (2005)), Rushworth, L. K., Hindley, A. D., O'Neill, E. & Kolch, W., Regulation and role of Raf-1/B-Raf heterodimerization, Mol Cell Biol, 26, 2262-72 (2006)), Chen, C., Lewis, R. E. & White, M. A., IMP modulates KSR1-dependent multivalent complex formation to specify ERK1/2 pathway activation and response thresholds, J Biol Chem, 283, 12789-96 (2008)). Moreover, the fact that the formation of B-/C-RAF heterodimers appears to depend on RAS activity (Garnett, M. J., Rana, S., Paterson, H., Barford, D. & Marais, R., Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization, Mol Cell, 20, 963-9 (2005)), strongly suggests that RAS may also play a role in forming side-to-side kinase domain dimers. In the absence of RTK/RAS activation, a regulatory element in the N-terminus of RAF engages the C-terminal kinase domain to inhibit catalytic activity by an unknown mechanism (Chong, H. & Guan, K. L., Regulation of Raf through phosphorylation and N terminus-C terminus interaction, J Biol Chem, 278, 36269-76 (2003)). We reason that this autoinhibitory interaction may interfere with the ability of the kinase domain to adopt a productive dimer configuration.

[0293] Although dependent on many more components, the activation mechanism of RAF appears analogous in principle, if not execution, to those employed by the PKR and EGFR protein kinases. In the case of the eIF2 protein kinase PKR, the attainment of a specific dimer configuration by the kinase domain is regulated by the binding of dsRNA viral by-products to regions N-terminal to the kinase domain (Dar, A. C., Dever, T. E. & Sicheri, F., Higher-order substrate recognition of eIF2alpha by the RNA-dependent protein kinase PKR, Cell, 122, 887-900 (2005)), Dey, M. et al., Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition, Cel, 122, 901-13 (2005). In the case of EGFR kinase, adoption of a unique dimer/oligomer configuration by its kinase domain is regulated by the binding of growth factors to the extracellular ligand binding domain of the receptors (Zhang, X., Gureasko, J., Shen, K., Cole, P. A. & Kuriyan, J., An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor, Cell, 125, 1137-49 (2006)) (FIG. 6C). Reflecting the importance of self interaction in the function of all three protein kinase families, residues comprising the self interaction surfaces of the kinase domain in addition to the catalytic infrastructure are evolutionarily conserved within each kinase family. In this regard, KSR is essentially equivalent to a RAF molecule. In effect, we reason that RAF and KSR evolved from a single ancestral progenitor, one that possessed both protein kinase catalytic activity and stable substrate (MEK) binding function. Following a gene duplication event (Claperon, A. & Therrien, M., KSR and CNK: two scaffolds regulating RAS-mediated RAF activation, Oncogene, 26, 3143-58 (2007)), one gene dispensed with phospho-transfer function (i.e. KSR) and the other dispensed with the ability to stably bind MEK substrate (i.e. RAF). However, both maintained the ability to form allosteric dimers and this selective pressure maintained the side-to-side dimer interface and interdependence between KSR and RAF proteins in ERK signaling.

[0294] The mapping of human cancer causing mutations to the activation segment of B-RAF proved unequivocally that the activation segment of RAF is also a key modulator of its catalytic function (Wan, P. T. et al., Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)), (Davies, H. et al., Mutations of the BRAF gene in human cancer, Nature, 417, 949-54 (2002)). Consistent with this, we previously found that a mutation in the activation segment of Drosophila RAF (RAF-AL^ED) strongly hyperactivated its catalytic activity (Douziech, M., Sahmi, M., Laberge, G. & Therrien, M., A KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila, Genes Dev, 20, 807-19 (2006)), suggesting that it likely acts via a similar mechanism as those identified in human cancers (Wan, P. T. et al., Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)). This raises the question of how kinase domain dimerization and the modulation of activation segment conformation are coordinated. Both events may be essential for the transmission of a downstream signal or each event may be sufficient on its own. If both are essential, then oncogenic activation segment mutants of RAF should still be sensitive to dimer interface mutations. Suggesting that this in fact is the case, introduction of a mutation (R481H) within the side-to-side dimer interface in Drosophila RAF effectively nullifies the aberrant signaling properties of RAF-AL^ED (FIG. 11A).

[0295] Intriguingly, while most oncogenic RAF mutations act through modulation of the activation segment, one particular mutation, RAF E558K (E586K in human B-RAF), is located on the opposite surface of the kinase domain from the activation segment (Wan, P. T. et al., Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)) and its mechanism of kinase activation remained enigmatic. Most conspicuously, Glu558 lies on the side-to-side dimer interface (FIG. 11B). If dimerization is indeed critical for RAF activation, we questioned whether RAF_E558K might promote kinase activity by promoting dimerization. We reasoned that mutation of Glu558 to the longer Lys (E558K) could potentially introduce a hydrogen bond with Ser561 (conservative Thr589 in B-RAF) on the second RAF protomer thereby promoting dimer formation (FIG. 11B). Indeed, as tested below we found that the RAF_E558K mutation promoted kinase domain dimerization in solution. Wild type RAF kinase domain is predominantly a dimer in solution (at the micromolar concentrations tested), which prevented a direct test of the RAF_E558K mutant for enhanced dimerization potential (FIG. 3C). To circumvent this problem, we employed the RAF_L487R dimer mutant which displayed a weak monomer-dimer binding equilibrium in solution (FIG. 12).

[0296] Thus, introduction of the E558K mutation (RAF_L487R/E558K double mutant) transitioned RAF_L487R back to a predominantly dimeric state (FIG. 6). To investigate how the RAF_E558K mutation functions to hyperactivate RAF in vivo, we used the FRB/FKBP/rapamycin system to assess RAF activation in S2 cells. When the E558K mutation was introduced in the kinase-dead (K455S) background (FRB-RAF_K455S/E558K double mutant), it displayed no activity when tested alone (not shown), but strongly hyperactivated the FKBP-RAF counterpart in a rapamycin dependent manner (FIG. 11C). Taken together, the ability of the E558K mutant to act in trans (i.e. in the context of a kinase dead mutant) in vivo, and the ability of the E558K mutation to promote kinase domain dimerization in vitro strongly suggests that the mechanism by which the oncogenic RAF_E558K mutation acts is by promoting side-to-side dimers. Givern these results, it is now possible to develop small molecules strategies that are directed at preventing the formation of side-to-side dimers by RAF and which can serve as a therapeutic for RAF-dependent human tumors, one that would complement conventional strategies currently directed at inhibiting RAF enzymatic activity by blocking the catalytic cleft (Wu, S., Guo, W. & Fang, B., Development of small-molecule inhibitors of raf, Recent Patents Anti-Infect Drug Disc, 1, 241-6 (2006)).

[0297] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present discovery and scope of the appended claims.

[0298] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Sequence CWU 1

84114PRTDrosophila melanogaster 1Ile His Arg Ser Ala Ser Glu Pro Asn Leu Thr Gln Ser Gln1 5 10214PRTHomo sapiens 2Leu Asn Arg Arg Leu Ser His Pro Gly His Phe Trp Lys Ser1 5 10314PRTMus musculus 3Leu Asn Arg Arg Leu Ser His Pro Gly His Phe Trp Lys Ser1 5 10414PRTZebrafish 4Leu Asn Arg Arg Leu Ser His Pro Gly His Phe Trp Lys Ser1 5 10514PRTDrosophila melanogaster 5Leu Ala Arg Ser Pro Ser His Pro Val Asn Leu Ser Arg Ser1 5 10614PRTChicken 6Leu Asn Arg Arg Leu Ser His Pro Gly His Phe Trp Lys Ser1 5 107766PRTHomo sapiens 7Met Ala Ala Leu Ser Gly Gly Gly Gly Gly Gly Ala Glu Pro Gly Gln1 5 10 15Ala Leu Phe Asn Gly Asp Met Glu Pro Glu Ala Gly Ala Gly Ala Gly 20 25 30Ala Ala Ala Ser Ser Ala Ala Asp Pro Ala Ile Pro Glu Glu Val Trp 35 40 45Asn Ile Lys Gln Met Ile Lys Leu Thr Gln Glu His Ile Glu Ala Leu 50 55 60Leu Asp Lys Phe Gly Gly Glu His Asn Pro Pro Ser Ile Tyr Leu Glu65 70 75 80Ala Tyr Glu Glu Tyr Thr Ser Lys Leu Asp Ala Leu Gln Gln Arg Glu 85 90 95Gln Gln Leu Leu Glu Ser Leu Gly Asn Gly Thr Asp Phe Ser Val Ser 100 105 110Ser Ser Ala Ser Met Asp Thr Val Thr Ser Ser Ser Ser Ser Ser Leu 115 120 125Ser Val Leu Pro Ser Ser Leu Ser Val Phe Gln Asn Pro Thr Asp Val 130 135 140Ala Arg Ser Asn Pro Lys Ser Pro Gln Lys Pro Ile Val Arg Val Phe145 150 155 160Leu Pro Asn Lys Gln Arg Thr Val Val Pro Ala Arg Cys Gly Val Thr 165 170 175Val Arg Asp Ser Leu Lys Lys Ala Leu Met Met Arg Gly Leu Ile Pro 180 185 190Glu Cys Cys Ala Val Tyr Arg Ile Gln Asp Gly Glu Lys Lys Pro Ile 195 200 205Gly Trp Asp Thr Asp Ile Ser Trp Leu Thr Gly Glu Glu Leu His Val 210 215 220Glu Val Leu Glu Asn Val Pro Leu Thr Thr His Asn Phe Val Arg Lys225 230 235 240Thr Phe Phe Thr Leu Ala Phe Cys Asp Phe Cys Arg Lys Leu Leu Phe 245 250 255Gln Gly Phe Arg Cys Gln Thr Cys Gly Tyr Lys Phe His Gln Arg Cys 260 265 270Ser Thr Glu Val Pro Leu Met Cys Val Asn Tyr Asp Gln Leu Asp Leu 275 280 285Leu Phe Val Ser Lys Phe Phe Glu His His Pro Ile Pro Gln Glu Glu 290 295 300Ala Ser Leu Ala Glu Thr Ala Leu Thr Ser Gly Ser Ser Pro Ser Ala305 310 315 320Pro Ala Ser Asp Ser Ile Gly Pro Gln Ile Leu Thr Ser Pro Ser Pro 325 330 335Ser Lys Ser Ile Pro Ile Pro Gln Pro Phe Arg Pro Ala Asp Glu Asp 340 345 350His Arg Asn Gln Phe Gly Gln Arg Asp Arg Ser Ser Ser Ala Pro Asn 355 360 365Val His Ile Asn Thr Ile Glu Pro Val Asn Ile Asp Asp Leu Ile Arg 370 375 380Asp Gln Gly Phe Arg Gly Asp Gly Gly Ser Thr Thr Gly Leu Ser Ala385 390 395 400Thr Pro Pro Ala Ser Leu Pro Gly Ser Leu Thr Asn Val Lys Ala Leu 405 410 415Gln Lys Ser Pro Gly Pro Gln Arg Glu Arg Lys Ser Ser Ser Ser Ser 420 425 430Glu Asp Arg Asn Arg Met Lys Thr Leu Gly Arg Arg Asp Ser Ser Asp 435 440 445Asp Trp Glu Ile Pro Asp Gly Gln Ile Thr Val Gly Gln Arg Ile Gly 450 455 460Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly Lys Trp His Gly Asp Val465 470 475 480Ala Val Lys Met Leu Asn Val Thr Ala Pro Thr Pro Gln Gln Leu Gln 485 490 495Ala Phe Lys Asn Glu Val Gly Val Leu Arg Lys Thr Arg His Val Asn 500 505 510Ile Leu Leu Phe Met Gly Tyr Ser Thr Lys Pro Gln Leu Ala Ile Val 515 520 525Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr His His Leu His Ile Ile 530 535 540Glu Thr Lys Phe Glu Met Ile Lys Leu Ile Asp Ile Ala Arg Gln Thr545 550 555 560Ala Gln Gly Met Asp Tyr Leu His Ala Lys Ser Ile Ile His Arg Asp 565 570 575Leu Lys Ser Asn Asn Ile Phe Leu His Glu Asp Leu Thr Val Lys Ile 580 585 590Gly Asp Phe Gly Leu Ala Thr Val Lys Ser Arg Trp Ser Gly Ser His 595 600 605Gln Phe Glu Gln Leu Ser Gly Ser Ile Leu Trp Met Ala Pro Glu Val 610 615 620Ile Arg Met Gln Asp Lys Asn Pro Tyr Ser Phe Gln Ser Asp Val Tyr625 630 635 640Ala Phe Gly Ile Val Leu Tyr Glu Leu Met Thr Gly Gln Leu Pro Tyr 645 650 655Ser Asn Ile Asn Asn Arg Asp Gln Ile Ile Phe Met Val Gly Arg Gly 660 665 670Tyr Leu Ser Pro Asp Leu Ser Lys Val Arg Ser Asn Cys Pro Lys Ala 675 680 685Met Lys Arg Leu Met Ala Glu Cys Leu Lys Lys Lys Arg Asp Glu Arg 690 695 700Pro Leu Phe Pro Gln Ile Leu Ala Ser Ile Glu Leu Leu Ala Arg Ser705 710 715 720Leu Pro Lys Ile His Arg Ser Ala Ser Glu Pro Ser Leu Asn Arg Ala 725 730 735Gly Phe Gln Thr Glu Asp Phe Ser Leu Tyr Ala Cys Ala Ser Pro Lys 740 745 750Thr Pro Ile Gln Ala Gly Gly Tyr Gly Ala Phe Pro Val His 755 760 76582949DNAHomo sapiens 8cgcctccctt ccccctcccc gcccgacagc ggccgctcgg gccccggctc tcggttataa 60gatggcggcg ctgagcggtg gcggtggtgg cggcgcggag ccgggccagg ctctgttcaa 120cggggacatg gagcccgagg ccggcgccgg cgccggcgcc gcggcctctt cggctgcgga 180ccctgccatt ccggaggagg tgtggaatat caaacaaatg attaagttga cacaggaaca 240tatagaggcc ctattggaca aatttggtgg ggagcataat ccaccatcaa tatatctgga 300ggcctatgaa gaatacacca gcaagctaga tgcactccaa caaagagaac aacagttatt 360ggaatctctg gggaacggaa ctgatttttc tgtttctagc tctgcatcaa tggataccgt 420tacatcttct tcctcttcta gcctttcagt gctaccttca tctctttcag tttttcaaaa 480tcccacagat gtggcacgga gcaaccccaa gtcaccacaa aaacctatcg ttagagtctt 540cctgcccaac aaacagagga cagtggtacc tgcaaggtgt ggagttacag tccgagacag 600tctaaagaaa gcactgatga tgagaggtct aatcccagag tgctgtgctg tttacagaat 660tcaggatgga gagaagaaac caattggttg ggacactgat atttcctggc ttactggaga 720agaattgcat gtggaagtgt tggagaatgt tccacttaca acacacaact ttgtacgaaa 780aacgtttttc accttagcat tttgtgactt ttgtcgaaag ctgcttttcc agggtttccg 840ctgtcaaaca tgtggttata aatttcacca gcgttgtagt acagaagttc cactgatgtg 900tgttaattat gaccaacttg atttgctgtt tgtctccaag ttctttgaac accacccaat 960accacaggaa gaggcgtcct tagcagagac tgccctaaca tctggatcat ccccttccgc 1020acccgcctcg gactctattg ggccccaaat tctcaccagt ccgtctcctt caaaatccat 1080tccaattcca cagcccttcc gaccagcaga tgaagatcat cgaaatcaat ttgggcaacg 1140agaccgatcc tcatcagctc ccaatgtgca tataaacaca atagaacctg tcaatattga 1200tgacttgatt agagaccaag gatttcgtgg tgatggagga tcaaccacag gtttgtctgc 1260taccccccct gcctcattac ctggctcact aactaacgtg aaagccttac agaaatctcc 1320aggacctcag cgagaaagga agtcatcttc atcctcagaa gacaggaatc gaatgaaaac 1380acttggtaga cgggactcga gtgatgattg ggagattcct gatgggcaga ttacagtggg 1440acaaagaatt ggatctggat catttggaac agtctacaag ggaaagtggc atggtgatgt 1500ggcagtgaaa atgttgaatg tgacagcacc tacacctcag cagttacaag ccttcaaaaa 1560tgaagtagga gtactcagga aaacacgaca tgtgaatatc ctactcttca tgggctattc 1620cacaaagcca caactggcta ttgttaccca gtggtgtgag ggctccagct tgtatcacca 1680tctccatatc attgagacca aatttgagat gatcaaactt atagatattg cacgacagac 1740tgcacagggc atggattact tacacgccaa gtcaatcatc cacagagacc tcaagagtaa 1800taatatattt cttcatgaag acctcacagt aaaaataggt gattttggtc tagctacagt 1860gaaatctcga tggagtgggt cccatcagtt tgaacagttg tctggatcca ttttgtggat 1920ggcaccagaa gtcatcagaa tgcaagataa aaatccatac agctttcagt cagatgtata 1980tgcatttgga attgttctgt atgaattgat gactggacag ttaccttatt caaacatcaa 2040caacagggac cagataattt ttatggtggg acgaggatac ctgtctccag atctcagtaa 2100ggtacggagt aactgtccaa aagccatgaa gagattaatg gcagagtgcc tcaaaaagaa 2160aagagatgag agaccactct ttccccaaat tctcgcctct attgagctgc tggcccgctc 2220attgccaaaa attcaccgca gtgcatcaga accctccttg aatcgggctg gtttccaaac 2280agaggatttt agtctatatg cttgtgcttc tccaaaaaca cccatccagg cagggggata 2340tggtgcgttt cctgtccact gaaacaaatg agtgagagag ttcaggagag tagcaacaaa 2400aggaaaataa atgaacatat gtttgcttat atgttaaatt gaataaaata ctctcttttt 2460ttttaaggtg aaccaaagaa cacttgtgtg gttaaagact agatataatt tttccccaaa 2520ctaaaattta tacttaacat tggattttta acatccaagg gttaaaatac atagacattg 2580ctaaaaattg gcagagcctc ttctagaggc tttactttct gttccgggtt tgtatcattc 2640acttggttat tttaagtagt aaacttcagt ttctcatgca acttttgttg ccagctatca 2700catgtccact agggactcca gaagaagacc ctacctatgc ctgtgtttgc aggtgagaag 2760ttggcagtcg gttagcctgg gttagataag gcaaactgaa cagatctaat ttaggaagtc 2820agtagaattt aataattcta ttattattct taataatttt tctataacta tttcttttta 2880taacaatttg gaaaatgtgg atgtctttta tttccttgaa gcaataaact aagtttcttt 2940ttataaaaa 29499804PRTMus musculus 9Met Ala Ala Leu Ser Gly Gly Gly Gly Ser Ser Ser Gly Gly Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Gly Gly Gly Asp Gly Gly Gly Gly Ala Glu 20 25 30Gln Gly Gln Ala Leu Phe Asn Gly Asp Met Glu Pro Glu Ala Gly Ala 35 40 45Gly Ala Ala Ala Ser Ser Ala Ala Asp Pro Ala Ile Pro Glu Glu Val 50 55 60Trp Asn Ile Lys Gln Met Ile Lys Leu Thr Gln Glu His Ile Glu Ala65 70 75 80Leu Leu Asp Lys Phe Gly Gly Glu His Asn Pro Pro Ser Ile Tyr Leu 85 90 95Glu Ala Tyr Glu Glu Tyr Thr Ser Lys Leu Asp Ala Leu Gln Gln Arg 100 105 110Glu Gln Gln Leu Leu Glu Ser Leu Val Phe Gln Thr Pro Thr Asp Ala 115 120 125Ser Arg Asn Asn Pro Lys Ser Pro Gln Lys Pro Ile Val Arg Val Phe 130 135 140Leu Pro Asn Lys Gln Arg Thr Val Val Pro Ala Arg Cys Gly Val Thr145 150 155 160Val Arg Asp Ser Leu Lys Lys Ala Leu Met Met Arg Gly Leu Ile Pro 165 170 175Glu Cys Cys Ala Val Tyr Arg Ile Gln Asp Gly Glu Lys Lys Pro Ile 180 185 190Gly Trp Asp Thr Asp Ile Ser Trp Leu Thr Gly Glu Glu Leu His Val 195 200 205Glu Val Leu Glu Asn Val Pro Leu Thr Thr His Asn Phe Val Arg Lys 210 215 220Thr Phe Phe Thr Leu Ala Phe Cys Asp Phe Cys Arg Lys Leu Leu Phe225 230 235 240Gln Gly Phe Arg Cys Gln Thr Cys Gly Tyr Lys Phe His Gln Arg Cys 245 250 255Ser Thr Glu Val Pro Leu Met Cys Val Asn Tyr Asp Gln Leu Asp Leu 260 265 270Leu Phe Val Ser Lys Phe Phe Glu His His Pro Val Pro Gln Glu Glu 275 280 285Ala Ser Phe Pro Glu Thr Ala Leu Pro Ser Gly Ser Ser Ser Ala Pro 290 295 300Pro Ser Asp Ser Thr Gly Pro Gln Ile Leu Thr Ser Pro Ser Pro Ser305 310 315 320Lys Ser Ile Pro Ile Pro Gln Pro Phe Arg Pro Ala Asp Glu Asp His 325 330 335Arg Asn Gln Phe Gly Gln Arg Asp Arg Ser Ser Ser Ala Pro Asn Val 340 345 350His Ile Asn Thr Ile Glu Pro Val Asn Ile Asp Glu Lys Phe Pro Glu 355 360 365Val Glu Leu Gln Asp Gln Arg Asp Leu Ile Arg Asp Gln Gly Phe Arg 370 375 380Gly Asp Gly Ala Pro Leu Asn Gln Leu Met Arg Cys Leu Arg Lys Tyr385 390 395 400Gln Ser Arg Thr Pro Ser Pro Leu Leu His Ser Val Pro Ser Glu Ile 405 410 415Val Phe Asp Phe Glu Pro Gly Pro Val Phe Arg Gly Ser Thr Thr Gly 420 425 430Leu Ser Ala Thr Pro Pro Ala Ser Leu Pro Gly Ser Leu Thr Asn Val 435 440 445Lys Ala Leu Gln Lys Ser Pro Gly Pro Gln Arg Glu Arg Lys Ser Ser 450 455 460Ser Ser Ser Ser Ser Glu Asp Arg Ser Arg Met Lys Thr Leu Gly Arg465 470 475 480Arg Asp Ser Ser Asp Asp Trp Glu Ile Pro Asp Gly Gln Ile Thr Val 485 490 495Gly Gln Arg Ile Gly Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly Lys 500 505 510Trp His Gly Asp Val Ala Val Lys Met Leu Asn Val Thr Ala Pro Thr 515 520 525Pro Gln Gln Leu Gln Ala Phe Lys Asn Glu Val Gly Val Leu Arg Lys 530 535 540Thr Arg His Val Asn Ile Leu Leu Phe Met Gly Tyr Ser Thr Lys Pro545 550 555 560Gln Leu Ala Ile Val Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr His 565 570 575His Leu His Ile Ile Glu Thr Lys Phe Glu Met Ile Lys Leu Ile Asp 580 585 590Ile Ala Arg Gln Thr Ala Gln Gly Met Asp Tyr Leu His Ala Lys Ser 595 600 605Ile Ile His Arg Asp Leu Lys Ser Asn Asn Ile Phe Leu His Glu Asp 610 615 620Leu Thr Val Lys Ile Gly Asp Phe Gly Leu Ala Thr Val Lys Ser Arg625 630 635 640Trp Ser Gly Ser His Gln Phe Glu Gln Leu Ser Gly Ser Ile Leu Trp 645 650 655Met Ala Pro Glu Val Ile Arg Met Gln Asp Lys Asn Pro Tyr Ser Phe 660 665 670Gln Ser Asp Val Tyr Ala Phe Gly Ile Val Leu Tyr Glu Leu Met Thr 675 680 685Gly Gln Leu Pro Tyr Ser Asn Ile Asn Asn Arg Asp Gln Ile Ile Phe 690 695 700Met Val Gly Arg Gly Tyr Leu Ser Pro Asp Leu Ser Lys Val Arg Ser705 710 715 720Asn Cys Pro Lys Ala Met Lys Arg Leu Met Ala Glu Cys Leu Lys Lys 725 730 735Lys Arg Asp Glu Arg Pro Leu Phe Pro Gln Ile Leu Ala Ser Ile Glu 740 745 750Leu Leu Ala Arg Ser Leu Pro Lys Ile His Arg Ser Ala Ser Glu Pro 755 760 765Ser Leu Asn Arg Ala Gly Phe Gln Thr Glu Asp Phe Ser Leu Tyr Ala 770 775 780Cys Ala Ser Pro Lys Thr Pro Ile Gln Ala Gly Gly Tyr Gly Glu Phe785 790 795 800Ala Ala Phe Lys109727DNAMus musculus 10ccctcaggct cggctgcgcc ggggccgccg gcgggttcca gaggtggcct ccgccccggc 60cgctccgccc acgccccccg cgcctccgcg cccgcctccg cccgccctgc gcctcccttc 120cccctccccg ccccgcggcg gccgctcggc ccggctcgcg cttcgaagat ggcggcgctg 180agtggcggcg gtggcagcag cagcggtggc ggcggcggcg gtggcggcgg cggtggcggt 240ggcgacggcg gcggcggcgc cgagcagggc caggctctgt tcaatggcga catggagccg 300gaggccggcg ctggcgccgc ggcctcttcg gctgcggacc cggccattcc tgaagaggta 360tggaatatca agcaaatgat taagttgaca caggaacata tagaggccct attggacaaa 420tttggtggag agcataaccc accatcaata tacctggagg cctatgaaga gtacaccagc 480aagctagatg cccttcagca aagagaacag cagcttttgg aatccctggt ttttcaaact 540cccacagatg catcacggaa caaccccaag tcaccacaga aacctatcgt tagagtcttc 600ctgcccaaca aacagaggac agtggtaccc gcaagatgtg gtgttacagt tcgagacagt 660ctaaagaaag cactgatgat gagaggtctc atcccagaat gctgtgctgt ttacagaatt 720caggatggag agaagaaacc aattggctgg gacacggaca tttcctggct tactggagag 780gagttacatg ttgaagtact ggagaatgtc ccacttacaa cacacaactt tgtacggaaa 840acttttttca ccttagcatt ttgtgacttt tgccgaaagc tgcttttcca gggtttccgt 900tgtcaaacat gtggttataa atttcaccag cgttgtagta cagaggttcc actgatgtgt 960gtaaattatg accaacttga tttgctgttt gtctccaagt tctttgagca tcacccagta 1020ccacaggagg aggcctcctt cccagagact gcccttccat ctggatcctc ttccgcaccc 1080ccctcagact ctactgggcc ccaaatcctc accagtccat ctccttcaaa atccattcca 1140attccacagc ccttccgacc agcagatgaa gatcatcgca atcagtttgg gcaacgagac 1200cggtcctcct cagctcccaa tgttcatata aacacaattg agcctgtgaa tatcgatgaa 1260aaattcccag aagtggaatt acaggatcaa agggatttga ttagagacca ggggtttcgt 1320ggtgatggag cccccttgaa ccaactgatg cgctgtcttc ggaaatacca atcccggact 1380cccagccccc tcctccattc tgtccccagt gaaatagtgt ttgattttga gcctggccca 1440gtgttcagag ggtcaaccac aggcttgtcc gccaccccgc ctgcctcatt acctggctca 1500ctcactaacg tgaaagcctt acagaaatct ccaggtcctc agcgggaaag gaagtcatct 1560tcttcctcat cctcggagga cagaagtcgg atgaaaacac ttggtagaag agattcaagt 1620gatgactggg agattcctga tggacagatt acagtgggac agagaattgg atctgggtca 1680tttggaactg tctacaaggg aaagtggcat ggtgatgtgg cagtgaaaat gttgaatgtg 1740acagcaccca cacctcaaca gctacaggcc ttcaaaaatg aagtaggagt gctcaggaaa 1800actcgacatg tgaatatcct ccttttcatg

ggctattcta caaagccaca actggcaatt 1860gttacacagt ggtgtgaggg ctccagctta tatcaccatc tccacatcat tgagaccaaa 1920tttgagatga tcaaacttat agatattgct cggcagactg cacagggcat ggattactta 1980cacgccaagt caatcatcca cagagacctc aagagtaata atatatttct tcatgaagac 2040ctcacggtaa aaataggtga ctttggtcta gccacagtga aatctcggtg gagtgggtcc 2100catcagtttg aacagttgtc tggatctatt ttgtggatgg caccagaagt aatcagaatg 2160caagataaaa acccgtatag ctttcagtca gacgtgtatg cgtttgggat tgttctgtac 2220gaactgatga ccggccagct accttattca aacatcaaca acagggatca gataattttt 2280atggtgggac gaggatacct atctccagat ctcagtaagg tacggagtaa ctgtccaaaa 2340gccatgaaga gattaatggc agagtgcctc aaaaagaaaa gagacgagag accactcttc 2400cccaaattct cgcctccatt gagctgctgg cccgctcatt gccaaaaatt caccgcagtg 2460catcagaacc ttccttgaat cgggctggtt tccaaacaga agattttagt ctgtatgctt 2520gtgcttctcc gaaaacaccc atccaagcag ggggatatgg agaatttgca gccttcaagt 2580agccagtcca tcatggcagc atctactctt tatttcttaa gtcttgtgtt catacagttt 2640gttaacatca aaacacagtt ctgttcctca aaaaattttt taaagataca aaattttcaa 2700tgcataagtt catgtggaac agaatggaat ttcctattca acaaaagagg gaagaatgtt 2760ttaggaacca gaattctctg ctgcccgtgt ttcttcttca acataactat cacgtgcata 2820caagtctgcc cattcccaag aagaaagagg agagaccctg aattctgccc ttttggtggt 2880caggcatgat ggaaagaatt tgctgctgca gcttgggaaa attgctatgg aaagtctgcc 2940agtcgacttt gcccttctaa ccaccagatc agcctgtggc tggtcatctg atggggcgat 3000ttccatcacc aagcatcgtt cttgcctatt ctgggattat gttgtggagc actttccctg 3060tccagcaccg ttcatttctg agggatggag taaatgcagc attcccttgt gtagcgcctg 3120ttcagtcctc agcagctgct gtcacagcga agctttttac agttaagtgg tgggggagag 3180ttgaggagag cctgcctcgg ggcagagaaa agggggtgct gcatcttctt cctcacctcc 3240agctctctca cctcgggttg ccttgctcac tgggctccgc ctaaccactc aggctgctca 3300gtgctggcac acattgcctt cttttctcat tgggtccagc aattgaggag agggttgggg 3360gattgtttcc tcctcaatgt agcaaattct caggaaaata cagtccatat cttcctctca 3420gctcttccag tcaccaaata cttacgtggc tcctttgtcc aggacataaa acaccgtgga 3480caacacctaa ttaaaagcct acaaaactgc ttactgacag ttttgaatgt gagacacttg 3540tgtaatttaa atgtaaggta caggttttaa tttctgagtt tcttctattt ttatttaaaa 3600gaagaaaata attttcagtt ttaattggaa taaatgagta cttcccacaa gactatatac 3660cctgaaaatt atatttttgt taattgtaaa caacttttaa agaataatta ttatcctttt 3720ctctacctaa aaattatggg gaatcttagc ataatgacaa ttatttatac tttttaaata 3780aatggtactt gctggatcca cactaacatc tttgctaaca atcccattgt ttcttccaac 3840ttaactccta cactacatcc tacatcctct ttctagtctt ttatctataa tatgcaacct 3900aaaataaacg tggtggcgtc tccattcatt ctccctcttc ctgttttccc caagcctggt 3960cttcaaaagg ttgggtaatc ggtccctgag ctccctagct ggcaatgcaa ctattaggga 4020cattggagtt gcaggagagc aggaagcctg tccccagctg ttcttctaga accctaaatc 4080ttatctttgc acagatcaaa agtatcacct cgtcacagtt ctccttagcc tttacttaca 4140ggtaatataa ataaaaatca ccatagtagt aaagaaaaca actggatgga ttgatgacca 4200gtacctctca gagccaggaa tcttgaatct ccaggattta tacgtgcaaa tttaaggaga 4260tgtacttagc aacttcaagc caagaacttc caaaatacta gcgaatctaa aataaaatgg 4320aattttgagt tatttttaaa gttcaaatta taattgatac cactatgtat ttaagcctac 4380tcacagcaag ttagatggat tttgctaaac tcattgccag actgtggtgg tggtggtggt 4440agtgtgcacc tttaatccaa gcaactcagc aatcagaatg aggtaaatct ctgtgaatac 4500aaggcctgcc tagtctgcag cgctagttcc aggatagcca gggctacaca cacaaaaacc 4560ctctctcaaa aaaaacaaaa ttaattagtt gataataaaa aataactaaa gtatcatcaa 4620aggaaggcct actggaagtt ttatatattc ccagtaaatt gaaaaatatt ctgaagttat 4680taaccagtta gcaacaatgt gtttttaagt cttacataaa cagagcaaag tcttcaaatg 4740tttcagagct gagaagataa ttgtgcttga tatgaaaaat agcctctcca tatgatgtgc 4800cacattgaaa ggcgtcatta cccttttaaa tacttcttaa tgtggctttg ttccctttac 4860ccaggattag ctagaaagag ctaggtaggc ttcggccaca gttgcacatt tcgggcctgc 4920tgaagaatgg gagctttgaa ggctggcctt ggtggaggag cccctcagtg ctggagggtg 4980gggcgtgtac gcagcatgga agtggtctag acagagtgca aagggacaga cttctttctc 5040attttagtat agggtgatgt ctcacttgaa atgagaaagt agagttgata ttaaacgaag 5100ctgtgcccag aaaccaggct cagggtattg tgagattttc tttttaaata gagaatataa 5160aagatagaaa taaatattta aaccttcctt cttattttct atcaaataga ttttttttat 5220catttgcaaa caacataaaa aaaggtttct tttgtggggt tttctttcct tctttttttt 5280tttttttttt ttttaagact gcagataatc ttgttgagct cctcggaaaa tacaaggaag 5340tccgtgtttg tgcagagcgc tttatgagta actgtataga cagtgtggct gcttcactca 5400tcccagaggg ctgcagctgt cggcccatga agtggctgca gtgcctcgtg agatctgctt 5460tgttttgttt ggagtgaagt ctttgaaagg tttgagtgca actatatagg actgttttta 5520aataagtagt attcctcatg aactttctca ttgttaagct acaggaccca aactctacca 5580ctaagatatt attaacctca aaatgtagtt tatagaagga atttgcaaat agaatatcca 5640gttcgtactt atatgcatct tcaacaaaga ttctctgtga cttgttggat ttggttcctg 5700aacagcccat ttctgtattt gaggttagga gggcataatg aggcatccta aaagacaatc 5760tgatataaac tgtatgctag atgtatgctg gtaggggaga aagcattctg taaagacatg 5820atttaagact tcagctctgt caaccagaaa ccttgtaaat acttcctgtc ttggtgcagc 5880cccgcccctt tgatcacacg atgttgtctt gtgcttgtca gacactgtca gagctgctgt 5940tcgtccctct gcagatctca cctgtcccca ctgcacaccc acctcctgcc tcttgcagac 6000ctcagcatct agctttagtt ggaaacagtt cagggttcag gtgacttctt aaaaaaaaaa 6060aaaaacccta cctcctcaga atgaggtaat gaatagttat ttatttaaag tatgaagagt 6120caggagcgct cgaacatgaa ggtgatttaa gatggttcct ttcgtgtgta ttgtagctga 6180gcacttgttt ttgtcctaaa gggcattata catttaagca gtgattctgt ttaaagatgt 6240ttttctttaa aggtgtagct cagagtatct gttgttggaa ttggtgccag agtctgctta 6300atagatttca gaatcctaag cttaagtcag tcgcatgaag ttaagtagtt atggtaacac 6360tttgctagcc atgatataat tctacttttt aggagtaggt ttggcaaaac tgtatgcctt 6420caaagtgagt tggccacagc tttgtcacat gcacagatac tcatctgaag agactgccca 6480gctaagaggg cggaaggata cccttttttc ctacgattcg cttctttgtc cacgttggca 6540ttgttagtac tagtttatca gcaccttgac cagcagatgt caaccaataa gctattttta 6600aaaccatagc cagagatgga gaggtcactg tgagtagaaa cagcaggacg cttacaggag 6660tgaaatggtg tagggaggct ctagaaaaat atcttgacaa tttgccaaat gatcttactg 6720tgccttcatg atgcaataaa aaagctaaca ttttagcaga aatcagtgat ttacgaagag 6780agtggccagt ctggtttaac tcagctggga taatattttt agagtgcaat ttagactgcg 6840aagataaatg cactaaagag tttatagcca attcacattt gaaaaataag aaaatggtaa 6900attttcagtg aaatattttt ttaaagcaca taatccctag tgtagccaga aatatttacc 6960acatagagca gctaggctga gatacagtcc agtgacattt ctagagaaac cttttctact 7020cccacgggct cctcaaagca tggaaatttt atacaaaatg tttgacattt taagatactg 7080ctgtagttta gttttgaaat agtatgtgct gagcagcaat catgtactaa ctcagagaga 7140gaaaacaaca acaaattgtg catctgattt gttttcagag aaatgctgcc aacttagata 7200ctgagttctc agagcttcaa gtgtaaactt gcctcccaag tcctgtttgc aaatgaagtt 7260ggctagtgct actgactgct ccagcacatg atggaaggca gggggctgtc tctgaagtgt 7320cttctataaa gggacaatag aatagtgaga gacctggtca gtgtgtgtca gctggacact 7380ccatgctatg ggacttgcat cttctgtcct caccatcccc aagacattgt gctttcctca 7440gttgtcctct agctgtttca ctcagacacc aagatgaatt actgatgcca gaaggggcca 7500aaatggccag tgtgttttgg gggttgtatc agttgactgg acaataactt taatagtttc 7560agatcattta tttttacttc cattttgaca gacatttaaa tggaaattta gtcctaactt 7620ttgtcatttg aaaggaaaaa ttaacagttc ctataagata cttttgaggt ggaatctgac 7680atcctaattt tttttctttt cagtgggttt gcagcgaggg tcttgtatgc actaggcaag 7740ggttctacca ctaagccaca tttcccagga aataaaatgt taacagttaa aacatacaca 7800caaatacaca aacaccttat taccacttta gtaaagtgag agatgtgcgt cctttgtctc 7860agtctccacg atttcagctg ccccttgtat gaataactca gtctcgctaa actgtttact 7920tttatttacc tggtttgact agttgcagct atataaccag ttgtgcatga ggacaacagc 7980cagtgtgttt gttttgtttt tggttttttg tggtacattt tttgtaaaga attctgtaga 8040ttgaagtgct ctttgaaaac agaactgaga tatatttatt cttgttagca tcaaaaaaca 8100ttttgtgcaa atgatttgct tttcctggca ggctgagtac catatccagc gcccacaatt 8160gcgggttccc atctaccatg tccacagggg agacagacgg gaagcacatg aggggtgtgt 8220ttacagagtt gtaggagtta tgtagttctc ttgttgcctt ggaaatcact gttgttttaa 8280gactgttgaa cccgtgtgtt tggctgggct gtgagttaca tgaagaaact gcaaactagc 8340atatgcagac aaagctcaca gactaggcgt aaatggagga aaatggacca aaataaggca 8400gggtgacaca taaaccttgg gcttcggaga aaactaaggg tggagatgaa ctataatcac 8460ctgaatacaa tgtaagagtg caataagtgt gcttattcta agctgtgaac ttcttttaaa 8520tcattccttt ctaatacatt tatgtatgtt ccattgctga ctaaaaccag ctatgagaac 8580atatgccttt ttattcatgt taactaccag tttaagtggc taaccttaat gtcttattta 8640tcttcatttt gtattagttt acataccagg tatgtgtgtg tgctgtactc ttcttccctt 8700tatttgaaaa cacttttcac tgggtcatct ccttggccat tccacaacac aactttggtt 8760tggctttcaa tgtcacctta tttgatggcc tgtgtcccag tagcagaatt tatggtattc 8820ccattgctgg ctgctcttcc gaccctttgc ttctacagca cttgtctctc ctaagatagt 8880cagaaactaa ctgatcaggg gatggacttc accattcatc gtgtctcttc aattctatta 8940aatagaccac tcttgggctt tagaccagga aaaaggagac agctctagcc atctaccaag 9000cctcacccta aaaggtcacc cgtacttctt ggtctgagga caagtctcca ctccagtaag 9060ggagagggga ggaaatgctt cctgtttgaa atgcagtgaa ttcctatggc tcctgtttca 9120ccacccgcac ctatggcaac ccatatacat tcctcttgtc tgtaactgcc aaaggttggg 9180tttatgtcac ttcagttcca ctcaagcatt gaaaaggttc tcatggagtc tggggtgtgc 9240ccagtgaaaa gatggggact ttttcattat ccacagacct ctctatacct gctttgcaaa 9300aattataatg gagtaactat ttttaaagct tatttttcaa ttcataagaa aaagacattt 9360attttcaatc aaatggatga tgtctcttat cccttatccc tcaatgtttg cttgaatttt 9420gtttgttccc tatacctact ccctaattct ttagttcctt cctgctcagg tcccttcatt 9480tgtactttgg agtttttctc atgtaaattt gtataatgga aaatattgtt cagtttggat 9540agaaagcatg gagaaataaa taaaaaaaga tagctgaaaa tcaaattgaa gaaatttatt 9600tctgtgtaaa gttatttaaa aactctgtat tatatttaaa gaaaaaagcc caacccccca 9660aaaagtgcta tgtaattgat gtgaatatgc gaatactgct ataataaaga ttgactgcat 9720ggagaaa 972711897PRTHomo sapiens 11Met Asp Arg Ala Ala Leu Arg Ala Ala Ala Met Gly Glu Lys Lys Glu1 5 10 15Gly Gly Gly Gly Gly Asp Ala Ala Glu Gly Gly Ala Gly Ala Ala Ala 20 25 30Ser Arg Ala Leu Gln Gln Cys Gly Gln Leu Gln Lys Leu Ile Asp Ile 35 40 45Ser Ile Gly Ser Leu Arg Gly Leu Arg Thr Lys Cys Ala Val Ser Asn 50 55 60Asp Leu Thr Gln Gln Glu Ile Arg Thr Leu Glu Ala Lys Leu Val Arg65 70 75 80Tyr Ile Cys Lys Gln Arg Gln Cys Lys Leu Ser Val Ala Pro Gly Glu 85 90 95Arg Thr Pro Glu Leu Asn Ser Tyr Pro Arg Phe Ser Asp Trp Leu Tyr 100 105 110Thr Phe Asn Val Arg Pro Glu Val Val Gln Glu Ile Pro Arg Asp Leu 115 120 125Thr Leu Asp Ala Leu Leu Glu Met Asn Glu Ala Lys Val Lys Glu Thr 130 135 140Leu Arg Arg Cys Gly Ala Ser Gly Asp Glu Cys Gly Arg Leu Gln Tyr145 150 155 160Ala Leu Thr Cys Leu Arg Lys Val Thr Gly Leu Gly Gly Glu His Lys 165 170 175Glu Asp Ser Ser Trp Ser Ser Leu Asp Ala Arg Arg Glu Ser Gly Ser 180 185 190Gly Pro Ser Thr Asp Thr Leu Ser Ala Ala Ser Leu Pro Trp Pro Pro 195 200 205Gly Ser Ser Gln Leu Gly Arg Ala Gly Asn Ser Ala Gln Gly Pro Arg 210 215 220Ser Ile Ser Val Ser Ala Leu Pro Ala Ser Asp Ser Pro Thr Pro Ser225 230 235 240Phe Ser Glu Gly Leu Ser Asp Thr Cys Ile Pro Leu His Ala Ser Gly 245 250 255Arg Leu Thr Pro Arg Ala Leu His Ser Phe Ile Thr Pro Pro Thr Thr 260 265 270Pro Gln Leu Arg Arg His Thr Lys Leu Lys Pro Pro Arg Thr Pro Pro 275 280 285Pro Pro Ser Arg Lys Val Phe Gln Leu Leu Pro Ser Phe Pro Thr Leu 290 295 300Thr Arg Ser Lys Ser His Glu Ser Gln Leu Gly Asn Arg Ile Asp Asp305 310 315 320Val Ser Ser Met Arg Phe Asp Leu Ser His Gly Ser Pro Gln Met Val 325 330 335Arg Arg Asp Ile Gly Leu Ser Val Thr His Arg Phe Ser Thr Lys Ser 340 345 350Trp Leu Ser Gln Val Cys His Val Cys Gln Lys Ser Met Ile Phe Gly 355 360 365Val Lys Cys Lys His Cys Arg Leu Lys Cys His Asn Lys Cys Thr Lys 370 375 380Glu Ala Pro Ala Cys Arg Ile Ser Phe Leu Pro Leu Thr Arg Leu Arg385 390 395 400Arg Thr Glu Ser Val Pro Ser Asp Ile Asn Asn Pro Val Asp Arg Ala 405 410 415Ala Glu Pro His Phe Gly Thr Leu Pro Lys Ala Leu Thr Lys Lys Glu 420 425 430His Pro Pro Ala Met Asn His Leu Asp Ser Ser Ser Asn Pro Ser Ser 435 440 445Thr Thr Ser Ser Thr Pro Ser Ser Pro Ala Pro Phe Pro Thr Ser Ser 450 455 460Asn Pro Ser Ser Ala Thr Thr Pro Pro Asn Pro Ser Pro Gly Gln Arg465 470 475 480Asp Ser Arg Phe Asn Phe Pro Ala Ala Tyr Phe Ile His His Arg Gln 485 490 495Gln Phe Ile Phe Pro Val Pro Ser Ala Gly His Cys Trp Lys Cys Leu 500 505 510Leu Ile Ala Glu Ser Leu Lys Glu Asn Ala Phe Asn Ile Ser Ala Phe 515 520 525Ala His Ala Ala Pro Leu Pro Glu Ala Ala Asp Gly Thr Arg Leu Asp 530 535 540Asp Gln Pro Lys Ala Asp Val Leu Glu Ala His Glu Ala Glu Ala Glu545 550 555 560Glu Pro Glu Ala Gly Lys Ser Glu Ala Glu Asp Asp Glu Asp Glu Val 565 570 575Asp Asp Leu Pro Ser Ser Arg Arg Pro Trp Arg Gly Pro Ile Ser Arg 580 585 590Lys Ala Ser Gln Thr Ser Val Tyr Leu Gln Glu Trp Asp Ile Pro Phe 595 600 605Glu Gln Val Glu Leu Gly Glu Pro Ile Gly Gln Gly Arg Trp Gly Arg 610 615 620Val His Arg Gly Arg Trp His Gly Glu Val Ala Ile Arg Leu Leu Glu625 630 635 640Met Asp Gly His Asn Gln Asp His Leu Lys Leu Phe Lys Lys Glu Val 645 650 655Met Asn Tyr Arg Gln Thr Arg His Glu Asn Val Val Leu Phe Met Gly 660 665 670Ala Cys Met Asn Pro Pro His Leu Ala Ile Ile Thr Ser Phe Cys Lys 675 680 685Gly Arg Thr Leu His Ser Phe Val Arg Asp Pro Lys Thr Ser Leu Asp 690 695 700Ile Asn Lys Thr Arg Gln Ile Ala Gln Glu Ile Ile Lys Gly Met Gly705 710 715 720Tyr Leu His Ala Lys Gly Ile Val His Lys Asp Leu Lys Ser Lys Asn 725 730 735Val Phe Tyr Asp Asn Gly Lys Val Val Ile Thr Asp Phe Gly Leu Phe 740 745 750Gly Ile Ser Gly Val Val Arg Glu Gly Arg Arg Glu Asn Gln Leu Lys 755 760 765Leu Ser His Asp Trp Leu Cys Tyr Leu Ala Pro Glu Ile Val Arg Glu 770 775 780Met Thr Pro Gly Lys Asp Glu Asp Gln Leu Pro Phe Ser Lys Ala Ala785 790 795 800Asp Val Tyr Ala Phe Gly Thr Val Trp Tyr Glu Leu Gln Ala Arg Asp 805 810 815Trp Pro Leu Lys Asn Gln Ala Ala Glu Ala Ser Ile Trp Gln Ile Gly 820 825 830Ser Gly Glu Gly Met Lys Arg Val Leu Thr Ser Val Ser Leu Gly Lys 835 840 845Glu Val Ser Glu Ile Leu Ser Ala Cys Trp Ala Phe Asp Leu Gln Glu 850 855 860Arg Pro Ser Phe Ser Leu Leu Met Asp Met Leu Glu Lys Leu Pro Lys865 870 875 880Leu Asn Arg Arg Leu Ser His Pro Gly His Phe Trp Lys Ser Ala Glu 885 890 895Leu124552DNAHomo sapiens 12ctggacccct gccagggaag gggtcctcag acttgaggtt gccagctcag atgtggggct 60gctgatacta ggtgactgga ctgatgttct gttctagatg aaactccttg aggggaccat 120ttgaaaaggc ttgatgtgct gcccaaagcc cccttcagag ctgacttctc cacccccagc 180tgccgtgagc cttggctgct gacagctcat agctgagtcc ctcccgtgaa gtcaccttct 240gctgaagggt acatcctctc ccaaggcgaa gctggtccgt tacatttgta agcagaggca 300gtgcaagctg agcgtggctc ccggtgagag gaccccagag ctcaacagct acccccgctt 360cagcgactgg ctgtacactt tcaacgtgag gccggaggtg gtgcaggaga tcccccgaga 420cctcacgctg gatgccctgc tggagatgaa tgaggccaag gtgaaggaga cgctgcggcg 480ctgtggggcc agcggggatg agtgtggccg tctgcagtat gccctcacct gcctgcggaa 540ggtgacaggc ctgggagggg agcacaagga ggactccagt tggagttcat tggatgcgcg 600gcgggaaagt ggctcagggc cttccacgga caccctctca gcagccagcc tgccctggcc 660cccagggagc tcccagctgg gcagagcagg caacagcgcc cagggcccac gctccatctc 720cgtgtcagct ctgcccgcct cagactcccc cacccccagc ttcagtgagg gcctctcaga 780cacctgtatt cccctgcacg ccagcggccg gctgaccccc cgtgccctgc acagcttcat 840caccccgccc accacacccc agctgcgacg gcacaccaag ctgaagccac cacggacgcc 900ccccccaccc agccgcaagg tcttccagct gctgcccagc ttccccacac tcacccggag 960caagtcccat gagtctcagc tggggaaccg cattgatgac gtctcctcga tgaggtttga 1020tctctcgcat ggatccccac agatggtacg gagggatatc gggctgtcgg tgacgcacag 1080gttctccacc aagtcctggc tgtcgcaggt ctgccacgtg tgccagaaga gcatgatatt 1140tggagtgaag tgcaagcatt gcaggttgaa gtgtcacaac aaatgtacca aagaagcccc 1200tgcctgtaga atatccttcc tgccactaac tcggcttcgg aggacagaat ctgtcccctc 1260ggacatcaac aacccggtgg acagagcagc cgaaccccat tttggaaccc tccccaaagc 1320actgacaaag aaggagcacc ctccggccat gaatcacctg gactccagca gcaacccttc 1380ctccaccacc tcctccacac cctcctcacc ggcgcccttc ccgacatcat ccaacccatc 1440cagcgccacc acgcccccca acccctcacc tggccagcgg gacagcaggt tcaacttccc 1500agctgcctac ttcattcatc atagacagca gtttatcttt ccagtgccat ctgctggcca 1560ttgctggaaa tgcctcctta ttgcagaaag tttaaaggaa aacgctttca

acatttcagc 1620ctttgcacac gcagccccgc tccctgaagc tgccgacggt acccggctcg atgaccagcc 1680gaaagcagat gtgttggaag ctcacgaagc ggaggctgag gagccagagg ctggcaagtc 1740agaggcagaa gacgatgagg acgaggtgga cgacttgccg agctctcgcc ggccctggcg 1800gggccccatc tctcgcaagg ccagccagac cagcgtgtac ctgcaggagt gggacatccc 1860cttcgagcag gtagagctgg gcgagcccat cgggcagggc cgctggggcc gggtgcaccg 1920cggccgctgg catggcgagg tggccattcg cctgctggag atggacggcc acaaccagga 1980ccacctgaag ctcttcaaga aagaggtgat gaactaccgg cagacgcggc atgagaacgt 2040ggtgctcttc atgggggcct gcatgaaccc gccccacctg gccattatca ccagcttctg 2100caaggggcgg acgttgcact cgtttgtgag ggaccccaag acgtctctgg acatcaacaa 2160gacgaggcaa atcgctcagg agatcatcaa gggcatggga tatcttcatg ccaagggcat 2220cgtacacaaa gatctcaaat ctaagaacgt cttctatgac aacggcaagg tggtcatcac 2280agacttcggg ctgtttggga tctcaggcgt ggtccgagag ggacggcgtg agaaccagct 2340aaagctgtcc cacgactggc tgtgctatct ggcccctgag attgtacgcg agatgacccc 2400cgggaaggac gaggatcagc tgccattctc caaagctgct gatgtctatg catttgggac 2460tgtttggtat gagctgcaag caagagactg gcccttgaag aaccaggctg cagaggcatc 2520catctggcag attggaagcg gggaaggaat gaagcgtgtc ctgacttctg tcagcttggg 2580gaaggaagtc agtgagatcc tgtcggcctg ctgggctttc gacctgcagg agagacccag 2640cttcagcctg ctgatggaca tgctggagaa acttcccaag ctgaaccggc ggctctccca 2700ccctggacac ttctggaagt cagctgagtt gtaggcctgg ctgccttgca tgcaccaggg 2760gctttcttcc tcctaatcaa caactcagca ccgtgacttc tgctaaaatg caaaatgaga 2820tgcgggcact aacccagggg atgccacctc tgctgctcca gtcgtctctc tcgaggctac 2880ttcttttgct ttgttttaaa aactggccct ctgccctctc cacgtggcct gcatatgccc 2940aagtaactgc tctcagagga tcccactaac tgagctccct ccaaggcagt ctgggcagct 3000tctaactacc ttcctggaca tgactgattg ctcccgtgtt cttctgaggg ctggtcttgt 3060ttttgtttgg gtggctctgt ctcactgcta acaccttagt gagatgcctt ccaccctcct 3120gagcacacca gcctcccact gggtgtgtgc ctagtgcggg gcgggcggag gttgggaggg 3180tgttggcttg gcttttaacc tgtggggatt ttgtccaaca aggagtggaa tgatttcaga 3240gctgccctga ggctggcacc ctggtcacag gaaccctctg cgctggctcc tgtctcagtc 3300ccctctgtag agttagatca gaagacacag aaagttctgt ggccatgaaa gataccagct 3360tggaagggtt gtgtcttcag tggcaccctc agaaaaattg tcttaaagca aagaggtacc 3420tggctccaga caatttttct gatgaaaaca aagtctctgc cccgtcccca ccctgccacc 3480ctggcaaagt tacttccttt acagctgccc agtgtaccat agaccagacc ccaggtcagc 3540atttgtcaag agcatggctg ctgagtcccc tgtggcagtc aatgcactgt ttaccaaatg 3600caggtttctg ttctccctcc ccagcaagac ctgctgaacc cagatctctg gaatggggcc 3660ctaggaattt gcatttcaac ctgcttccca ggtggccctg atgcacccca gtattagagt 3720ttattgctaa aaggaacatg ccctgtcact cctggtatcc tgggagtcat gtttctcttc 3780tctctcagtt ctacttggag caagagcttt cctgggctgc aaatgagaaa acaattccta 3840ggaacccaca gcagtactga gcatgctggg agcttgggac ttggagatga atgagccacc 3900gttgctgctc caagtaggac tacttggagt gtagctgagg ccttggacgc agtatgacca 3960ggggcagctc tgccagggct gttggccaat cagtcatttt catttcttgt tggaggccag 4020gtcctctgct gaactcattt cctagctagt gttaccctaa ttctgatgaa gatcaatggg 4080gctataattc ttgtttttgt tcctctttgc agcattaaca gcagcaaagt tgtaccccgg 4140tttgaaaggt ttggcttggg cgtcctggag tccagtaatc caaagatgta gccagccata 4200tggtttttcg ctgctgatct ctttcttttt aaaatgtgtt tctgaaacat cccaacaacc 4260accacgacaa aaaaacactg cctgcccagc gctgcaaacc aggagcacac gtcctagatt 4320cagactgttg gccataaacc ccactcggga gatggagctg cacctgctat ttcttaaaat 4380gacaccacca acaaccaaac ctgtcatgac agacagcaaa tgtttacacg tatatttctc 4440ctgagtgaac ctgatgtttt acaataggta ataataaaaa cagtctgtgc aaaaaaaaaa 4500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 455213873PRTMus musculus 13Met Asp Arg Ala Ala Leu Arg Ala Ala Ala Met Gly Glu Lys Lys Glu1 5 10 15Gly Gly Gly Gly Gly Ala Ala Ala Asp Gly Gly Ala Gly Ala Ala Val 20 25 30Ser Arg Ala Leu Gln Gln Cys Gly Gln Leu Gln Lys Leu Ile Asp Ile 35 40 45Ser Ile Gly Ser Leu Arg Gly Leu Arg Thr Lys Cys Ser Val Ser Asn 50 55 60Asp Leu Thr Gln Gln Glu Ile Arg Thr Leu Glu Ala Lys Leu Val Lys65 70 75 80Tyr Ile Cys Lys Gln Gln Gln Ser Lys Leu Ser Val Thr Pro Ser Asp 85 90 95Arg Thr Ala Glu Leu Asn Ser Tyr Pro Arg Phe Ser Asp Trp Leu Tyr 100 105 110Ile Phe Asn Val Arg Pro Glu Val Val Gln Glu Ile Pro Gln Glu Leu 115 120 125Thr Leu Asp Ala Leu Leu Glu Met Asp Glu Ala Lys Ala Lys Glu Met 130 135 140Leu Arg Arg Trp Gly Ala Ser Thr Glu Glu Cys Ser Arg Leu Gln Gln145 150 155 160Ala Leu Thr Cys Leu Arg Lys Val Thr Gly Leu Gly Gly Glu His Lys 165 170 175Met Asp Ser Gly Trp Ser Ser Thr Asp Ala Arg Asp Ser Ser Leu Gly 180 185 190Pro Pro Met Asp Met Leu Ser Ser Leu Gly Arg Ala Gly Ala Ser Thr 195 200 205Gln Gly Pro Arg Ser Ile Ser Val Ser Ala Leu Pro Ala Ser Asp Ser 210 215 220Pro Val Pro Gly Leu Ser Glu Gly Leu Ser Asp Ser Cys Ile Pro Leu225 230 235 240His Thr Ser Gly Arg Leu Thr Pro Arg Ala Leu His Ser Phe Ile Thr 245 250 255Pro Pro Thr Thr Pro Gln Leu Arg Arg His Ala Lys Leu Lys Pro Pro 260 265 270Arg Thr Pro Pro Pro Pro Ser Arg Lys Val Phe Gln Leu Leu Pro Ser 275 280 285Phe Pro Thr Leu Thr Arg Ser Lys Ser His Glu Ser Gln Leu Gly Asn 290 295 300Arg Ile Asp Asp Val Thr Pro Met Lys Phe Glu Leu Pro His Gly Ser305 310 315 320Pro Gln Leu Val Arg Arg Asp Ile Gly Leu Ser Val Thr His Arg Phe 325 330 335Ser Thr Lys Ser Trp Leu Ser Gln Val Cys Asn Val Cys Gln Lys Ser 340 345 350Met Ile Phe Gly Val Lys Cys Lys His Cys Arg Leu Lys Cys His Asn 355 360 365Lys Cys Thr Lys Glu Ala Pro Ala Cys Arg Ile Thr Phe Leu Pro Leu 370 375 380Ala Arg Leu Arg Arg Thr Glu Ser Val Pro Ser Asp Ile Asn Asn Pro385 390 395 400Val Asp Arg Ala Ala Glu Pro His Phe Gly Thr Leu Pro Lys Ala Leu 405 410 415Thr Lys Lys Glu His Pro Pro Ala Met Asn Leu Asp Ser Ser Ser Asn 420 425 430Pro Ser Ser Thr Thr Ser Ser Thr Pro Ser Ser Pro Ala Pro Phe Leu 435 440 445Thr Ser Ser Asn Pro Ser Ser Ala Thr Thr Pro Pro Asn Pro Ser Pro 450 455 460Gly Gln Arg Asp Ser Arg Phe Ser Phe Pro Asp Ile Ser Ala Cys Ser465 470 475 480Gln Ala Ala Pro Leu Ser Ser Thr Ala Asp Ser Thr Arg Leu Asp Asp 485 490 495Gln Pro Lys Thr Asp Val Leu Gly Val His Glu Ala Glu Ala Glu Glu 500 505 510Pro Glu Ala Gly Lys Ser Glu Ala Glu Asp Asp Glu Glu Asp Glu Val 515 520 525Asp Asp Leu Pro Ser Ser Arg Arg Pro Trp Arg Gly Pro Ile Ser Arg 530 535 540Lys Ala Ser Gln Thr Ser Val Tyr Leu Gln Glu Trp Asp Ile Pro Phe545 550 555 560Glu Gln Val Glu Leu Gly Glu Pro Ile Gly Gln Gly Arg Trp Gly Arg 565 570 575Val His Arg Gly Arg Trp His Gly Glu Val Ala Ile Arg Leu Leu Glu 580 585 590Met Asp Gly His Asn Gln Asp His Leu Lys Leu Phe Lys Lys Glu Val 595 600 605Met Asn Tyr Arg Gln Thr Arg His Glu Asn Val Val Leu Phe Met Gly 610 615 620Ala Cys Met Asn Pro Pro His Leu Ala Ile Ile Thr Ser Phe Cys Lys625 630 635 640Gly Arg Thr Leu His Ser Phe Val Arg Asp Pro Lys Thr Ser Leu Asp 645 650 655Ile Asn Lys Thr Arg Gln Ile Ala Gln Glu Ile Ile Lys Gly Met Gly 660 665 670Tyr Leu His Ala Lys Gly Ile Val His Lys Asp Leu Lys Ser Lys Asn 675 680 685Val Phe Tyr Asp Asn Gly Lys Val Val Ile Thr Asp Phe Gly Leu Phe 690 695 700Gly Ile Ser Gly Val Val Arg Glu Glu Arg Arg Glu Asn Gln Leu Lys705 710 715 720Leu Ser His Asp Trp Leu Cys Tyr Leu Ala Pro Glu Ile Val Arg Glu 725 730 735Met Ile Pro Gly Arg Asp Glu Asp Gln Leu Pro Phe Ser Lys Ala Ala 740 745 750Asp Val Tyr Ala Phe Gly Thr Val Trp Tyr Glu Leu Gln Ala Arg Asp 755 760 765Trp Pro Phe Lys His Gln Pro Ala Glu Ala Leu Ile Trp Gln Ile Gly 770 775 780Ser Gly Glu Gly Val Arg Arg Val Leu Ala Ser Val Ser Leu Gly Lys785 790 795 800Glu Val Gly Glu Ile Leu Ser Ala Cys Trp Ala Phe Asp Leu Gln Glu 805 810 815Arg Pro Ser Phe Ser Leu Leu Met Asp Met Leu Glu Arg Leu Pro Lys 820 825 830Leu Asn Arg Arg Leu Ser His Pro Gly His Phe Trp Lys Ser Ala Asp 835 840 845Ile Asn Ser Ser Lys Val Met Pro Arg Phe Glu Arg Phe Gly Leu Gly 850 855 860Thr Leu Glu Ser Gly Asn Pro Lys Met865 870144087DNAMus musculus 14ctcggggctt tcctgccgag gcgcccgtgt ccccgggctc ctcgcctcgg cccccagcgg 60ccccgatgcc gaggcatgga tagagcggcg ttgcgcgcgg cagcgatggg cgagaaaaag 120gagggcggcg gcgggggcgc cgcggcggac gggggcgcag gggccgccgt cagccgggcg 180ctgcagcagt gcggccagct gcagaagctc atcgatatct ccatcggcag tctgcgcggg 240ctgcgcacca agtgctcagt gtctaacgac ctcacacagc aggagatccg gaccctagag 300gcaaagctgg tgaaatacat ttgcaagcag cagcagagca agcttagtgt gaccccaagc 360gacaggaccg ccgagctcaa cagctaccca cgcttcagtg actggctgta catcttcaac 420gtgaggcctg aggtggtgca ggagatcccc caagagctca cactggatgc tctgctggag 480atggacgagg ccaaagccaa ggagatgctg cggcgctggg gggccagcac ggaggagtgc 540agccgcctac agcaagccct tacctgcctt cggaaggtga ctggcctggg aggggagcac 600aaaatggact caggttggag ttcaacagat gctcgagaca gtagcttggg gcctcccatg 660gacatgcttt cctcgctggg cagagcgggt gccagcactc agggaccccg ttccatctcc 720gtgtccgccc tgcctgcctc agactctccg gtccccggcc tcagtgaggg cctctcggac 780tcctgtatcc ccttgcacac cagcggccgg ctgacccccc gggccctgca cagcttcatc 840acgcccccta ccacacccca gctacgacgg cacgccaagc tgaagccacc aaggacaccc 900ccaccgccaa gccgcaaggt cttccagctg ctccccagct tccccacact cacacggagc 960aagtcccacg agtcccagct gggaaaccga atcgacgacg tcaccccgat gaagtttgaa 1020ctccctcatg gatccccaca gctggtacga agggatatcg ggctctcggt gacgcacagg 1080ttctccacaa agtcatggtt gtcacaggtg tgcaacgtgt gccagaagag catgattttt 1140ggcgtgaagt gcaaacactg caggttaaaa tgccataaca agtgcacaaa ggaagctccc 1200gcctgcagga tcaccttcct cccactggcc aggcttcgga ggacagagtc tgtcccgtca 1260gatatcaaca acccagtgga cagagcagca gagccccatt ttggaaccct tcccaaggcc 1320ctgacaaaga aggagcaccc tccagccatg aacctggact ccagcagcaa cccatcctcc 1380accacgtcct ccacaccctc atcgccggca cctttcctga cctcatctaa tccctccagt 1440gccaccacgc ctcccaaccc gtcacctggc cagcgggaca gcaggttcag cttcccagac 1500atttcagcct gttctcaggc agccccgctg tccagcacag ccgacagtac acggctcgac 1560gaccagccca aaacagatgt gctaggtgtt cacgaagcag aggctgagga gcctgaggct 1620ggcaagtcag aggcagagga tgacgaggag gatgaggtgg acgacctccc cagctcccgc 1680cggccctgga ggggccccat ctctcgaaag gccagccaga ccagcgttta cctgcaagag 1740tgggacatcc cctttgaaca ggtggaactg ggcgagccca ttggacaggg tcgctggggc 1800cgggtgcacc gaggccgttg gcatggcgag gtggccattc ggctgctgga gatggacggc 1860cacaatcagg accacctgaa gctgttcaag aaagaggtga tgaactaccg gcagacgcgg 1920catgagaacg tggtgctctt catgggggcc tgcatgaacc cacctcacct ggccattatc 1980accagcttct gcaaggggcg gacattgcat tcattcgtga gggaccccaa gacgtctctg 2040gacatcaata agactaggca gatcgcccag gagatcatca agggcatggg ttatcttcat 2100gcaaaaggca tcgtgcacaa ggacctcaag tccaagaatg tcttctatga caacggcaaa 2160gtggtcatca cagacttcgg gctgtttggg atctcgggtg tggtccgaga ggaacggcgc 2220gagaaccaac tgaaactgtc acatgactgg ctgtgctacc tggcccccga gatcgtacga 2280gaaatgatcc cggggcggga cgaggaccag ctgcccttct ccaaagcagc cgatgtctat 2340gcattcggga ctgtgtggta tgaactacag gcaagagact ggccctttaa gcaccagcct 2400gctgaggcct tgatctggca gattggaagt ggggaaggag tacggcgcgt cctggcatcc 2460gtcagcctgg ggaaggaagt cggcgagatc ctgtctgcct gctgggcttt cgatctgcag 2520gagagaccca gcttcagcct gctgatggac atgctggaga ggctgcccaa gctgaaccgg 2580cggctctccc accctgggca cttttggaag tcggctgaca ttaacagcag caaagtcatg 2640ccccgctttg aaaggtttgg cctggggacc ctggagtccg gtaatccaaa gatgtagcca 2700gccctgcacg ttcatgcaga gagtgtcttc ctttcgaaaa catgatcacg aaacatgcag 2760accaccacct caaggaatca gaagcattgc atcccaagct gcggactggg agcgtgtctc 2820ctccctaaag gacgtgcgtg cgtgcgtgcg tgcgtgcgtg cgtgcgtgcg tcaccaaggt 2880gtgtggagct caggatcgca gccatacacg caactccaga tgataccact accgccagtg 2940tttacacaga ggtttctgcc tggcaagctt ggtattttac agtaggtgaa gatcattctg 3000cagaagggtg ctggcacagt ggagcagcac ggatgtcccc agcccccgtt ctggaagacc 3060ctacagctgt gagaggccca gggttgagcc agatgaaaga aaagctgcgt gggtgtgggc 3120tgtacccgga aaagggcagg tggcaggagg tttgccttgg cctgtgcttg ggccgagaac 3180cacactaagg agcagcagcc tgagttagga atctatctgg attacgggga tcagagttcc 3240tggagagtgg actcagtttc tgctctgatc caggcctgtt gtgctttttt tttttccccc 3300ttaaaaaaaa aaaagtacag acagaatctc agcggcttct agactgatct gatggatctt 3360agcccggctt ctactgcggg ggggaggggg ggagggatag ccacatatct gtggagacac 3420ccacttcttt atctgaggcc tccaggtagg cacaaaggct gtggaactca gcctctatca 3480tcagacaccc ccccccaatg cctcattgac ccccttcccc cagagccaag ggctagccca 3540tcgggtgtgt gtacagtaag ttcttggtga aggagaacag ggacgttggc agaagcagtt 3600tgcagtggcc ctagcatctt aaaacccatt gtctgtcaca ccagaaggtt ctagacctac 3660caccacttcc cttccccatc tcatggaaac cttttagccc attctgaccc ctgtgtgtgc 3720tctgagctca gatcgggtta tgagaccgcc caggcacatc agtcagggag gctctgatgt 3780gagccgcaga cctctgtgtt cattcctatg agctggaggg gctggactgg gtggggtcag 3840atgtgcttgg caggaactgt cagctgctga gcagggtggt ccctgagcgg aggataagca 3900gcatcagact ccacaaccag aggaagaaag aaatggggat ggagcggaga cccacgggct 3960gagtcccgct gtggagtggc cttgcagctc cctctcagtt aaaactccca gtaaagccac 4020agttctccga gcacccaagt ctgctccagc cgtctcttaa aacaggccac tctctgagaa 4080ggaattc 408715739PRTDrosophila melanogaster 15Met Ser Ser Glu Ser Ser Thr Glu Gly Asp Ser Asp Leu Tyr Asp Pro1 5 10 15Leu Ala Glu Glu Leu His Asn Val Gln Leu Val Lys His Val Thr Arg 20 25 30Glu Asn Ile Asp Ala Leu Asn Ala Lys Phe Ala Asn Leu Gln Glu Pro 35 40 45Pro Ala Met Tyr Leu Ile Glu Tyr Gln Glu Leu Thr Ser Lys Leu His 50 55 60Glu Leu Glu Ala Lys Glu Gln Glu Leu Met Glu Arg Leu Asn Ser Gln65 70 75 80Asp Gln Gln Glu Asp Ser Ser Leu Val Glu Arg Phe Lys Glu Gln Pro 85 90 95His Tyr Gln Asn Gln Thr Gln Ile Leu Gln Gln Gln Arg Gln Leu Ala 100 105 110Arg Val His His Gly Asn Asp Leu Thr Asp Ser Leu Gly Ser Gln Pro 115 120 125Gly Ser Gln Cys Gly Thr Leu Thr Arg Gln Pro Lys Ile Leu Leu Arg 130 135 140Ala His Leu Pro Asn Gln Gln Arg Thr Ser Val Glu Val Ile Ser Gly145 150 155 160Val Arg Leu Cys Asp Ala Leu Met Lys Ala Leu Lys Leu Arg Gln Leu 165 170 175Thr Pro Asp Met Cys Glu Val Ser Thr Thr His Ser Gly Arg His Ile 180 185 190Ile Pro Trp His Thr Asp Ile Gly Thr Leu His Val Glu Glu Ile Phe 195 200 205Val Arg Leu Leu Asp Lys Phe Pro Ile Arg Thr His Ile Lys His Gln 210 215 220Ile Ile Arg Lys Thr Phe Phe Ser Leu Val Phe Cys Glu Gly Cys Arg225 230 235 240Arg Leu Leu Phe Thr Gly Phe Tyr Cys Ser Gln Cys Asn Phe Arg Phe 245 250 255His Gln Arg Cys Ala Asn Arg Val Pro Met Leu Cys Gln Pro Phe Pro 260 265 270Met Asp Ser Tyr Tyr Gln Leu Leu Leu Ala Glu Asn Pro Asp Asn Gly 275 280 285Val Gly Phe Pro Gly Arg Gly Thr Ala Val Arg Phe Asn Met Ser Ser 290 295 300Arg Ser Arg Ser Arg Arg Cys Ser Ser Ser Gly Ser Ser Ser Ser Ser305 310 315 320Lys Pro Pro Ser Ser Ser Ser Gly Asn His Arg Gln Gly Arg Pro Pro 325 330 335Arg Ile Ser Gln Asp Asp Arg Ser Asn Ser Ala Pro Asn Val Cys Ile 340 345 350Asn Asn Ile Arg Ser Val Thr Ser Glu Val Gln Arg Ser Leu Ile Met 355 360 365Gln Ala Arg Pro Pro Leu Pro His Pro Cys Thr Asp His Ser Asn Ser 370 375 380Thr Gln Ala Ser Pro Thr Ser Thr Leu Lys His Asn Arg Pro Arg Ala385 390 395 400Arg Ser Ala Asp Glu Ser Asn Lys Asn Leu Leu Leu Arg Asp Ala Lys 405 410 415Ser Ser Glu Glu Asn Trp Asn Ile Leu Ala Glu Glu Ile Leu Ile Gly 420

425 430Pro Arg Ile Gly Ser Gly Ser Phe Gly Thr Val Tyr Arg Ala His Trp 435 440 445His Gly Pro Val Ala Val Lys Thr Leu Asn Val Lys Thr Pro Ser Pro 450 455 460Ala Gln Leu Gln Ala Phe Lys Asn Glu Val Ala Met Leu Lys Lys Thr465 470 475 480Arg His Cys Asn Ile Leu Leu Phe Met Gly Cys Val Ser Lys Pro Ser 485 490 495Leu Ala Ile Val Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr Lys His 500 505 510Val His Val Ser Glu Thr Lys Phe Lys Leu Asn Thr Leu Ile Asp Ile 515 520 525Gly Arg Gln Val Ala Gln Gly Met Asp Tyr Leu His Ala Lys Asn Ile 530 535 540Ile His Arg Asp Leu Lys Ser Asn Asn Ile Phe Leu His Glu Asp Leu545 550 555 560Ser Val Lys Ile Gly Asp Phe Gly Leu Ala Thr Ala Lys Thr Arg Trp 565 570 575Ser Gly Glu Lys Gln Ala Asn Gln Pro Thr Gly Ser Ile Leu Trp Met 580 585 590Ala Pro Glu Val Ile Arg Met Gln Glu Leu Asn Pro Tyr Ser Phe Gln 595 600 605Ser Asp Val Tyr Ala Phe Gly Ile Val Met Tyr Glu Leu Leu Ala Glu 610 615 620Cys Leu Pro Tyr Gly His Ile Ser Asn Lys Asp Gln Ile Leu Phe Met625 630 635 640Val Gly Arg Gly Leu Leu Arg Pro Asp Met Ser Gln Val Arg Ser Asp 645 650 655Ala Pro Gln Ala Leu Lys Arg Leu Ala Glu Asp Cys Ile Lys Tyr Thr 660 665 670Pro Lys Asp Arg Pro Leu Phe Arg Pro Leu Leu Asn Met Leu Glu Asn 675 680 685Met Leu Arg Thr Leu Pro Lys Ile His Arg Ser Ala Ser Glu Pro Asn 690 695 700Leu Thr Gln Ser Gln Leu Gln Asn Asp Glu Phe Leu Tyr Leu Pro Ser705 710 715 720Pro Lys Thr Pro Val Asn Phe Asn Asn Phe Gln Phe Phe Gly Ser Ala 725 730 735Gly Asn Ile 163420DNADrosophila melanogaster 16cacttgtata tggttagttg attaatagca cgtcagaaac taatttacct gttgccgctc 60gtaccagatc cagatttgta ctatcccgag aagttaaaag ctctaggcaa attaacaatt 120agccgcgaca caaaccccgt ttcgcagagc acctgatacc ctttatcgtt atcgattggt 180acagccgaat cacgcctcct gataacgatt aaacaaaaag tcgaaatgta gtaaaattcg 240cggaaagtaa ataaattgtt atagccaagg tgaaataacg agcggccagc tagtggcgat 300actgatactg ttgcgaacgt tgggcagcca ccgacggtgc cggctggtca ggttgttatc 360gggtaattgg cagctccttt ggaaaatcct caagttcagc tgcttctgca cacactgacc 420ttcattatac atacataccg tatatacgag ctgtttgtgt gcgtgtgtgt gtgtgcgctt 480gcaagtgtgt gggtgcactg aaaaaaggtt ggaaaggata caagccagaa atcagtgaaa 540accgggaata ttgcatcccg gagacggcgg aaaagccgaa aaagcccatt aaaagtcaag 600gacgacatgc tgccctccgc ccacagaagt ggatgtgggt ggctcaccca ttagaactcc 660accaaaacgc aagcgcagga gtttttcctt caagaagtca aggcttcttc gttttcgggg 720tcatggtcac agcgcatagt atataggata aagcaacacc atgtccagcg agtcctccac 780cgaaggcgac agcgatctat acgatccttt ggccgaggag ctgcacaacg tccagttggt 840caaacatgtg acccgcgaga atattgatgc cctgaatgcc aagtttgcca acctgcagga 900gccaccagcc atgtacttaa tagaatacca ggagttgacc tccaagctcc acgaactgga 960ggccaaggag caggaactaa tggagcgact gaactcgcag gaccagcagg aggactcctc 1020cttggtcgag cggttcaagg agcagcccca ctatcaaaat caaactcaaa tcctgcagca 1080acaacggcaa ttggcgcgag tgcaccacgg caacgatcta accgatagct tgggctctca 1140gccgggcagc caatgtggaa ctttgacccg tcagcccaag atccttttgc gagcccacct 1200gcccaatcaa cagcgcactt cagtggaggt aatttcggga gtacgactat gtgatgccct 1260catgaaggcc ctgaaactcc ggcaactaac gccggatatg tgcgaagtaa gcacaactca 1320ttccggaaga catatcatac cctggcacac ggatatcggc actctgcatg tggaggagat 1380ctttgtcagg ctgctggata agtttcccat taggacacac atcaagcacc agatcatacg 1440gaagaccttc ttctcgttgg tattctgcga gggctgtcga aggcttctgt tcaccgggtt 1500ctactgtagc cagtgtaatt ttcgattcca tcagaggtgt gccaatagag tgccgatgct 1560gtgccagccc tttcccatgg atagctacta tcagctactg ctggccgaga atccggataa 1620tggcgttggt ttccccggca gaggcactgc tgtccgcttc aatatgagca gccggagtcg 1680cagtcgtcgt tgcagcagca gtggcagcag cagcagctcg aagccaccat cttcatcctc 1740cggcaatcat cgacagggtc gtccgccgag gatcagccaa gacgatcgat ccaattccgc 1800gccaaatgtg tgcatcaaca acattcgatc ggtcacaagc gaagtgcagc gcagtttgat 1860aatgcaggcc agacctcctt tgccgcatcc gtgcacagat cactccaact ccacgcaagc 1920gtcgcccacg agcaccttga aacacaatcg tcccagggcc aggtccgccg atgagagcaa 1980taaaaatctg cttttaagag acgccaaaag ttccgaggaa aactggaata ttctggcgga 2040ggagatttta attgggccgc gcatcggatc gggttccttt ggaaccgttt atcgcgccca 2100ttggcacggt cccgtggccg taaagacact caacgtgaag acaccgagtc ccgcccagtt 2160gcaggcgttt aagaacgagg tggccatgct gaaaaagacg cgccactgca atatcctcct 2220cttcatgggc tgtgtatcca aaccatctct agcgattgtg acccagtggt gcgagggcag 2280cagtctctac aagcacgtcc atgtcagcga aaccaagttt aaattgaaca cgctcatcga 2340tatcggacgt caggtggccc agggcatgga ttacctgcat gccaagaata tcattcatag 2400agacctcaag tcaaacaaca tctttttgca cgaggatctt tccgtgaaga taggcgactt 2460cggattggcc actgcgaaaa ctcgatggtc gggtgaaaag caagccaatc aacccacggg 2520cagtatttta tggatggctc cagaggtgat tcgcatgcag gagctaaacc cctactcctt 2580ccagtcggac gtttatgcct ttggtatcgt gatgtacgaa ctgttggcgg agtgcttgcc 2640ctacggtcat attagcaaca aggatcagat cctgtttatg gtggggcgag gacttctgcg 2700tccggacatg agtcaagtgc gctcggatgc gccgcaggca ttgaagcgct tggccgagga 2760ttgcattaag tataccccca aggatcgacc gctctttagg ccgctgctca atatgctgga 2820gaacatgctg cgcactttgc ccaaaattca tcgcagtgcc agtgaaccaa acttgacgca 2880atcgcagctg cagaacgatg agtttctgta tctgcccagc ccgaaaacgc cggtgaactt 2940caacaacttt cagttcttcg gcagcgctgg gaatatctag acagcgacct gtacctgtac 3000ttacatatat cctgcgtgat caacgtgatc ctacatctat atactttttg ttcttgtccc 3060tctgtacata agcgattcgc gaaggggacg gctttggttg tccaccaaag tgaaagagag 3120agagagagag agaaagagag agagatgggt tgcctgccga cccgggagcg aaacttgctt 3180ctttccttgg aactgacaaa gtgcatttct gttaccacac acaaaacgac tacaaactgt 3240aaactaaact gcaacgccca tgtgtacata actgcatcat aacttatata cgttaggcaa 3300gactactgaa actaaactaa actaaactaa actagctgat cgcaattaca ttatacacat 3360tatacttata ctacaagaga tggtgttgtt tctggagtcg agcacgatga agaacattta 342017966PRTDrosophila melanogaster 17Met Ser Ser Asn Asn Asn Ala Pro Ala Ser Ala Pro Asp Thr Gly Ser1 5 10 15Thr Asn Ala Asn Asp Pro Ile Ser Gly Ser Leu Ser Val Asp Ser Asn 20 25 30Leu Val Ile Ile Gln Asp Met Ile Asp Leu Ser Ala Asn His Leu Glu 35 40 45Gly Leu Arg Thr Gln Cys Ala Ile Ser Ser Thr Leu Thr Gln Gln Glu 50 55 60Ile Arg Cys Leu Glu Ser Lys Leu Val Arg Tyr Phe Ser Glu Leu Leu65 70 75 80Leu Ala Lys Met Arg Leu Asn Glu Arg Ile Pro Ala Asn Gly Leu Val 85 90 95Pro His Thr Thr Gly Asn Glu Leu Arg Gln Trp Leu Arg Val Val Gly 100 105 110Leu Ser Gln Gly Thr Leu Thr Ala Cys Leu Ala Arg Leu Thr Thr Leu 115 120 125Glu Gln Ser Leu Arg Leu Ser Asp Glu Glu Ile Arg Gln Leu Leu Ala 130 135 140Asp Ser Pro Ser Gln Arg Glu Glu Glu Glu Leu Arg Arg Leu Thr Arg145 150 155 160Ala Met Gln Asn Leu Arg Lys Cys Met Glu Ser Leu Glu Ser Gly Thr 165 170 175Ala Ala Ser Asn Asn Asp Pro Glu Gln Trp His Trp Asp Ser Trp Asp 180 185 190Arg Pro Thr His Ile His Arg Gly Ser Val Gly Asn Ile Gly Leu Gly 195 200 205Asn Asn Ser Thr Ala Ser Pro Arg Thr His His Arg Gln His Gly Val 210 215 220Lys Gly Lys Asn Ser Ala Leu Ala Asn Ser Thr Asn Phe Lys Ser Gly225 230 235 240Arg Gln Ser Pro Ser Ala Thr Glu Glu Leu Asn Ser Thr Gln Gly Ser 245 250 255Gln Leu Thr Leu Thr Leu Thr Pro Ser Pro Pro Asn Ser Pro Phe Thr 260 265 270Pro Ser Ser Gly Leu Ser Ser Ser Leu Asn Gly Thr Pro Gln Arg Ser 275 280 285Arg Gly Thr Pro Pro Pro Ala Arg Lys His Gln Thr Leu Leu Ser Gln 290 295 300Ser His Val Gln Val Asp Gly Glu Gln Leu Ala Arg Asn Arg Leu Pro305 310 315 320Thr Asp Pro Ser Pro Asp Ser His Ser Ser Thr Ser Ser Asp Ile Phe 325 330 335Val Asp Pro Asn Thr Asn Ala Ser Ser Gly Gly Ser Ser Ser Asn Val 340 345 350Leu Met Val Pro Cys Ser Pro Gly Val Gly His Val Gly Met Gly His 355 360 365Ala Ile Lys His Arg Phe Thr Lys Ala Leu Gly Phe Met Ala Thr Cys 370 375 380Thr Leu Cys Gln Lys Gln Val Phe His Arg Trp Met Lys Cys Thr Asp385 390 395 400Cys Lys Tyr Ile Cys His Lys Ser Cys Ala Pro His Val Pro Pro Ser 405 410 415Cys Gly Leu Pro Arg Glu Tyr Val Asp Glu Phe Arg His Ile Lys Glu 420 425 430Gln Gly Gly Tyr Ala Ser Leu Pro His Val His Gly Ala Ala Lys Gly 435 440 445Ser Pro Leu Val Lys Lys Ser Thr Leu Gly Lys Pro Leu His Gln Gln 450 455 460His Gly Asp Ser Ser Ser Pro Ser Ser Ser Cys Thr Ser Ser Thr Pro465 470 475 480Ser Ser Pro Ala Leu Phe Gln Gln Arg Glu Arg Glu Leu Asp Gln Ala 485 490 495Gly Ser Ser Ser Ser Ala Asn Leu Leu Pro Thr Pro Ser Leu Gly Lys 500 505 510His Gln Pro Ser Gln Phe Asn Phe Pro Asn Val Thr Val Thr Ser Ser 515 520 525Gly Gly Ser Gly Gly Val Ser Leu Ile Ser Asn Glu Pro Val Pro Glu 530 535 540Gln Phe Pro Thr Ala Pro Ala Thr Ala Asn Gly Gly Leu Asp Ser Leu545 550 555 560Val Ser Ser Ser Asn Gly His Met Ser Ser Leu Ile Gly Ser Gln Thr 565 570 575Ser Asn Ala Ser Thr Ala Ala Thr Leu Thr Gly Ser Leu Val Asn Ser 580 585 590Thr Thr Thr Thr Ser Thr Cys Ser Phe Phe Pro Arg Lys Leu Ser Thr 595 600 605Ala Gly Val Asp Lys Arg Thr Pro Phe Thr Ser Glu Tyr Thr Asp Thr 610 615 620His Lys Ser Asn Asp Ser Asp Lys Thr Val Ser Leu Ser Gly Ser Ala625 630 635 640Ser Thr Asp Ser Asp Arg Thr Pro Val Arg Val Asp Ser Thr Glu Asp 645 650 655Gly Asp Ser Gly Gln Trp Arg Gln Asn Ser Ile Ser Leu Lys Glu Trp 660 665 670Asp Ile Pro Tyr Gly Asp Leu Leu Leu Leu Glu Arg Ile Gly Gln Gly 675 680 685Arg Phe Gly Thr Val His Arg Ala Leu Trp His Gly Asp Val Ala Val 690 695 700Lys Leu Leu Asn Glu Asp Tyr Leu Gln Asp Glu His Met Leu Glu Thr705 710 715 720Phe Arg Ser Glu Val Ala Asn Phe Lys Asn Thr Arg His Glu Asn Leu 725 730 735Val Leu Phe Met Gly Ala Cys Met Asn Pro Pro Tyr Leu Ala Ile Val 740 745 750Thr Ser Leu Cys Lys Gly Asn Thr Leu Tyr Thr Tyr Ile His Gln Arg 755 760 765Arg Glu Lys Phe Ala Met Asn Arg Thr Leu Leu Ile Ala Gln Gln Ile 770 775 780Ala Gln Gly Met Gly Tyr Leu His Ala Arg Glu Ile Ile His Lys Asp785 790 795 800Leu Arg Thr Lys Asn Ile Phe Ile Glu Asn Gly Lys Val Ile Ile Thr 805 810 815Asp Phe Gly Leu Phe Ser Ser Thr Lys Leu Leu Tyr Cys Asp Met Gly 820 825 830Leu Gly Val Pro His Asn Trp Leu Cys Tyr Leu Ala Pro Glu Leu Ile 835 840 845Arg Ala Leu Gln Pro Glu Lys Pro Arg Gly Glu Cys Leu Glu Phe Thr 850 855 860Pro Tyr Ser Asp Val Tyr Ser Phe Gly Thr Val Trp Tyr Glu Leu Ile865 870 875 880Cys Gly Glu Phe Thr Phe Lys Asp Gln Pro Ala Glu Ser Ile Ile Trp 885 890 895Gln Val Gly Arg Gly Met Lys Gln Ser Leu Ala Asn Leu Gln Ser Gly 900 905 910Arg Asp Val Lys Asp Leu Leu Met Leu Cys Trp Thr Tyr Glu Lys Glu 915 920 925His Arg Pro Gln Phe Ala Arg Leu Leu Ser Leu Leu Glu His Leu Pro 930 935 940Lys Lys Arg Leu Ala Arg Ser Pro Ser His Pro Val Asn Leu Ser Arg945 950 955 960Ser Ala Glu Ser Val Phe 965183737DNADrosophila melanogaster 18cttgggtgta gagaaatttc aacaacagcc gaggaacttg tacaaattat tgctttttcg 60cattgcctaa gccgtttaga gttgcgggcg ttagcgtgcg cgatagccgg agcaccgaac 120gtcaaggtcg cttggcgagg gccacaatgc ggggcggagt cccagccatt ggtcccatcg 180aatcgtcgag tccccgaggg ggcgtctgaa aaaatcaatc gggctccact ccgtcgcgaa 240taagcaggat gagcagcaac aacaacgcac ccgcatcggc tccagacacg ggctccacca 300atgccaacga tcccatctcc ggttcgctgt ccgtagacag caacctggtt atcattcagg 360acatgattga tctctcggcc aaccatctgg agggcctgcg aacgcagtgc gcgatcagct 420ccacgctgac gcagcaggag attcgttgcc tggagtcgaa gctggtgcga tacttctccg 480agctgctgct ggcgaagatg cggctaaatg agcgcatccc ggccaacggg cttgtgcccc 540acacaacggg caacgaactg aggcaatggc tgcgcgtagt gggccttagc caggggactc 600ttaccgcctg ccttgctcgc ctgaccactc tagagcaaag cctgcgtctc agcgacgagg 660agatccgtca actcctggct gacagcccca gccagcgaga ggaggaggaa ctgcgacgcc 720tgaccagggc catgcagaac ttaaggaagt gcatggagtc gctggagagc ggtactgcgg 780ctagcaacaa cgatccagag cagtggcact gggactcctg ggacaggccc acccacattc 840atcgcggcag tgtgggaaac attggactgg gtaacaattc aaccgcctcc ccgagaaccc 900atcatcgcca gcatggtgtc aagggaaaga attccgctct ggccaactcc accaacttca 960aaagtggccg ccaatcgccc tcagcgacag aagagctgaa cagcacacag ggttcccagc 1020tgactttaac ccttacgccc tcgccaccca attcgccctt cacgccttcc agtgggctga 1080gcagcagcct taatggaaca ccacagagga gtcgtggtac cccgccgcca gctagaaagc 1140accagacctt gctgagccag agtcatgtgc aagtggacgg ggagcaatta gcccgcaacc 1200gtttgcccac tgatcccagc cccgatagcc acagctccac cagctcggac atctttgtgg 1260acccaaatac taatgccagc tccggaggaa gttcctcgaa cgtgcttatg gtgccatgct 1320ctccgggcgt gggtcacgtg ggcatgggtc atgcaatcaa gcatcgtttc accaaggccc 1380tgggcttcat ggccacctgt accctgtgcc agaagcaggt ctttcaccgc tggatgaagt 1440gcaccgactg caagtacatc tgccacaagt catgcgcacc gcacgtaccg ccctcctgtg 1500gacttccacg agaatatgtg gacgagtttc ggcacataaa ggagcaggga ggatacgcca 1560gtctgccgca tgtgcatggc gcggcgaaag gatccccttt ggtaaaaaag agcaccctgg 1620gtaagccctt gcatcagcag cacggcgata gcagttcgcc gagttccagc tgcactagtt 1680ccacgcccag cagtccggcg ctgttccagc aaagggagcg cgagctggat caggcgggca 1740gcagctctag cgccaatctg ttacctacgc cttcgcttgg caagcaccag ccgagtcaat 1800tcaactttcc caacgtgacg gtgacgagca gtggcggaag cggtggtgta tcgctcatct 1860ccaatgaacc agtgccagag caattcccca cggcgcctgc aacagccaac ggaggacttg 1920atagtctggt gagcagctcc aacgggcaca tgagctcgct catcggtagc caaacttcaa 1980acgcttctac tgcggccacc ttgacgggca gtctggtcaa tagcacaacc accaccagca 2040cctgcagttt ctttccgcga aaattgagca cagccggtgt ggataagagg acgccgttca 2100ccagcgagta cacggatacc cacaagtcaa atgacagcga caagacagtc tccttgtctg 2160gaagtgccag cacggactcg gaccggacac ccgttcgtgt ggattcaacg gaagacggag 2220actcgggaca atggcgccag aactcgatct cactcaagga atgggacatc ccgtatggtg 2280atctgcttct gctcgagcgg atagggcagg gacgcttcgg caccgtgcat cgagcccttt 2340ggcacggaga tgtggcggtt aagctgctca acgaggacta tctgcaagac gaacacatgc 2400tggagacgtt tcgcagcgag gtagccaact tcaagaacac tcgacacgag aacctggtgc 2460tgttcatggg agcctgcatg aacccaccat atttggccat tgtgacttca ttgtgcaagg 2520gcaacacctt gtatacgtat attcaccagc gtcgggagaa gtttgccatg aaccggactc 2580tcctcattgc ccagcagatc gcccagggca tgggctacct gcacgcaagg gagatcatcc 2640acaaagatct gcgcaccaag aacatcttca tcgagaacgg caaggtgatt atcacggact 2700ttgggctgtt cagctccacc aagctgctct actgtgatat gggcctagga gtgccccaca 2760actggttgtg ctacctggcg ccggagctaa tccgagcatt gcagccggag aagccgcgtg 2820gagagtgtct ggagttcacc ccatactccg atgtctactc tttcggaacc gtttggtacg 2880agctaatctg cggcgagttc acattcaagg atcagccggc ggaatcgatc atctggcagg 2940ttggccgtgg gatgaagcag tcgctggcca acctgcagtc tggacgggat gtcaaggact 3000tgctgatgct gtgctggacc tacgagaagg agcaccggcc gcagttcgca cgcctgctct 3060ccctgctgga gcatcttccc aagaagcgtc tggcgcgcag tccctcccac cccgtcaacc 3120tttcccgttc cgccgagtcc gtgttctgag ggaactgcag catggccact gtcactgtct 3180agtacaattt cgatctacca actaagctag ctcgctttgt gccctcgtcc actctacaca 3240aactctctcc caaggcgaag ttctatcgag ccgagcgaag attgtaaata cataaacgta 3300actaccaaat tatagcaatc cattttaaaa actacataca tatgtgtagg catgtatcgg 3360gagcactcca gttgcagttg ttagcaaacg aaacaaaggc aaatcaaatg ttaactcgaa 3420aaagacaaaa cgcttaaatg tttaagagca gaggcaaaca gagaaggcat agacatacat 3480atacaaacaa acaaacaagc actgtggcaa acataaatgt aaacgttaat caggtgagca 3540atttctaaat tgttaattat gtgtaagaga actatatata tatatatata tatatatata 3600tatatatata tatatataca tgtatataca gcagcaatgt attgtatatg acggactagt 3660gttaaattaa atatatattg tgaattatgt atggtcaagt gtatatagta aatggacttt 3720aaatgcgaaa tcgaaac

37371963PRTArtificial SequenceSynthetic 19Ala Ala Gly Ala Thr Gly Ala Gly Cys Ala Ala Ala Gly Ala Thr Gly1 5 10 15Gly Thr Ala Ala Ala Ala Ala Gly Ala Ala Gly Ala Ala Ala Ala Ala 20 25 30Gly Ala Ala Gly Thr Cys Ala Ala Ala Gly Ala Cys Ala Ala Ala Gly 35 40 45Thr Gly Thr Gly Thr Ala Ala Thr Thr Ala Thr Gly Thr Ala Ala 50 55 602020PRTArtificial SequenceSynthetic 20Lys Met Ser Lys Asp Gly Lys Lys Lys Lys Lys Lys Ser Lys Thr Lys1 5 10 15Cys Val Ile Met 2021729PRTArtificial SequenceSynthetic 21Ala Thr Gly Gly Thr Gly Ala Gly Cys Ala Ala Gly Gly Gly Cys Gly1 5 10 15Ala Gly Gly Ala Gly Cys Thr Gly Thr Thr Cys Ala Cys Cys Gly Gly 20 25 30Gly Gly Thr Gly Gly Thr Gly Cys Cys Cys Ala Thr Cys Cys Thr Gly 35 40 45Gly Thr Cys Gly Ala Gly Cys Thr Gly Gly Ala Cys Gly Gly Cys Gly 50 55 60Ala Cys Gly Thr Ala Ala Ala Cys Gly Gly Cys Cys Ala Cys Ala Ala65 70 75 80Gly Thr Thr Cys Ala Gly Cys Gly Thr Gly Thr Cys Cys Gly Gly Cys 85 90 95Gly Ala Gly Gly Gly Cys Gly Ala Gly Gly Gly Cys Gly Ala Thr Gly 100 105 110Cys Cys Ala Cys Cys Thr Ala Cys Gly Gly Cys Ala Ala Gly Cys Thr 115 120 125Gly Ala Cys Cys Cys Thr Gly Ala Ala Gly Thr Thr Cys Ala Thr Cys 130 135 140Thr Gly Cys Ala Cys Cys Ala Cys Cys Gly Gly Cys Ala Ala Gly Cys145 150 155 160Thr Gly Cys Cys Cys Gly Thr Gly Cys Cys Cys Thr Gly Gly Cys Cys 165 170 175Cys Ala Cys Cys Cys Thr Cys Gly Thr Gly Ala Cys Cys Ala Cys Cys 180 185 190Cys Thr Gly Ala Gly Cys Thr Ala Cys Gly Gly Cys Gly Thr Gly Cys 195 200 205Ala Gly Thr Gly Cys Thr Thr Cys Ala Gly Cys Cys Gly Cys Thr Ala 210 215 220Cys Cys Cys Cys Gly Ala Cys Cys Ala Cys Ala Thr Gly Ala Ala Gly225 230 235 240Cys Ala Gly Cys Ala Cys Gly Ala Cys Thr Thr Cys Thr Thr Cys Ala 245 250 255Ala Gly Thr Cys Cys Gly Cys Cys Ala Thr Gly Cys Cys Cys Gly Ala 260 265 270Ala Gly Gly Cys Thr Ala Cys Gly Thr Cys Cys Ala Gly Gly Ala Gly 275 280 285Cys Gly Cys Ala Cys Cys Ala Thr Cys Thr Thr Cys Thr Thr Cys Ala 290 295 300Ala Gly Gly Ala Cys Gly Ala Cys Gly Gly Cys Ala Ala Cys Thr Ala305 310 315 320Cys Ala Ala Gly Ala Cys Cys Cys Gly Cys Gly Cys Cys Gly Ala Gly 325 330 335Gly Thr Gly Ala Ala Gly Thr Thr Cys Gly Ala Gly Gly Gly Cys Gly 340 345 350Ala Cys Ala Cys Cys Cys Thr Gly Gly Thr Gly Ala Ala Cys Cys Gly 355 360 365Cys Ala Thr Cys Gly Ala Gly Cys Thr Gly Ala Ala Gly Gly Gly Cys 370 375 380Ala Thr Cys Gly Ala Cys Thr Thr Cys Ala Ala Gly Gly Ala Gly Gly385 390 395 400Ala Cys Gly Gly Cys Ala Ala Cys Ala Thr Cys Cys Thr Gly Gly Gly 405 410 415Gly Cys Ala Cys Ala Ala Gly Cys Thr Gly Gly Ala Gly Thr Ala Cys 420 425 430Ala Ala Cys Thr Ala Cys Ala Ala Cys Cys Cys Cys Cys Ala Cys Ala 435 440 445Ala Cys Gly Thr Cys Thr Ala Thr Ala Thr Cys Ala Thr Gly Gly Cys 450 455 460Cys Gly Ala Cys Ala Ala Gly Cys Ala Gly Ala Ala Gly Ala Ala Cys465 470 475 480Gly Gly Cys Ala Thr Cys Ala Ala Gly Gly Thr Gly Ala Ala Cys Thr 485 490 495Thr Cys Ala Ala Gly Ala Thr Cys Cys Gly Cys Cys Ala Cys Ala Ala 500 505 510Cys Ala Thr Cys Gly Ala Gly Gly Ala Cys Gly Gly Cys Ala Gly Cys 515 520 525Gly Thr Gly Cys Ala Gly Cys Thr Cys Gly Cys Cys Gly Ala Cys Cys 530 535 540Ala Cys Thr Ala Cys Cys Ala Gly Cys Ala Gly Ala Ala Cys Ala Cys545 550 555 560Cys Cys Cys Cys Ala Thr Cys Gly Gly Cys Gly Ala Cys Gly Gly Cys 565 570 575Cys Cys Cys Gly Thr Gly Cys Thr Gly Cys Thr Gly Cys Cys Cys Gly 580 585 590Ala Cys Ala Ala Cys Cys Ala Cys Thr Ala Cys Cys Thr Gly Thr Thr 595 600 605Cys Ala Cys Cys Cys Ala Gly Thr Cys Cys Gly Cys Cys Cys Thr Gly 610 615 620Ala Gly Cys Ala Ala Ala Gly Ala Cys Cys Cys Cys Ala Ala Cys Gly625 630 635 640Ala Gly Ala Ala Gly Cys Gly Cys Gly Ala Thr Cys Ala Cys Ala Thr 645 650 655Gly Gly Thr Cys Cys Thr Gly Cys Thr Gly Gly Ala Gly Thr Thr Cys 660 665 670Gly Thr Gly Ala Cys Cys Gly Cys Cys Gly Cys Cys Gly Gly Gly Ala 675 680 685Thr Cys Ala Cys Thr Cys Thr Cys Gly Gly Cys Ala Thr Gly Gly Ala 690 695 700Cys Gly Ala Gly Cys Thr Gly Thr Ala Cys Ala Ala Gly Gly Gly Ala705 710 715 720Thr Cys Cys Gly Cys Cys Thr Ala Gly 72522242PRTArtificial SequenceSynthetic 22Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu1 5 10 15Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60Leu Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys65 70 75 80Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140Asn Tyr Asn Pro His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150 155 160Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Phe Thr Gln Ser Ala Leu 195 200 205Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly225 230 235 240Ser Ala23945DNAArtificial SequenceSynthetic 23atgaccagca aggtgtacga ccccgagcag aggaagagga tgatcaccgg cccccagtgg 60tgggccaggt gcaagcagat gaacgtgctg gacagcttca tcaactacta cgacagcgag 120aagcacgccg agaacgccgt gatcttcctg cacggcaacg ccactagcag ctacctgtgg 180aggcacgtgg tgccccacat cgagcccgtg gccaggtgca tcatccccga tctgatcggc 240atgggcaaga gcggcaagag cggcaacggc agctacaggc tgctggacca ctacaagtac 300ctgaccgcct ggttcgagct cctgaacctg cccaagaaga tcatcttcgt gggccacgac 360tggggcgccg cactggcctt ccactacagc tacgagcacc aggacaagat caaggccatc 420gtgcacgccg agagcgtggt tgacgtgatc gagagctggg acgagtggcc agacatcgag 480gaggacatcg ccctgatcaa gagcgaggag ggcgagaaga tggtgctgga gaacaacttc 540ttcgtggaga ccgttctgcc cagcaagatc atgagaaagc tggagcccga ggagttcgcc 600gcctacctgg agcccttcaa ggagaagggc gaggtgagaa gacccaccct gagctggccc 660agagagatcc ccctggtgaa gggcggcaag cccgacgtgg tgcagatcgt gagaaactac 720aacgcctacc tgagagccag cgacgacctg cccaagatgt tcatcgagag cgaccccggc 780ttcttcagca acgccatcgt ggagggcgcc aagaagttcc ccaacaccga gttcgtgaag 840gtgaagggcc tgcacttcag ccaggaggac gcccccgacg agatgggcaa gtacatcaag 900agcttcgtgg agagagtgct gaagaacgag cagggatccg cctag 94524314PRTArtificial SequenceSynthetic 24Met Thr Ser Lys Val Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr1 5 10 15Gly Pro Gln Trp Trp Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser 20 25 30Phe Ile Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile 35 40 45Phe Leu His Gly Asn Ala Thr Ser Ser Tyr Leu Trp Arg His Val Val 50 55 60Pro His Ile Glu Pro Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly65 70 75 80Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp 85 90 95His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys 100 105 110Lys Ile Ile Phe Val Gly His Asp Trp Gly Ala Ala Leu Ala Phe His 115 120 125Tyr Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val His Ala Glu 130 135 140Ser Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu145 150 155 160Glu Asp Ile Ala Leu Ile Lys Ser Glu Glu Gly Glu Lys Met Val Leu 165 170 175Glu Asn Asn Phe Phe Val Glu Thr Val Leu Pro Ser Lys Ile Met Arg 180 185 190Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu 195 200 205Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro 210 215 220Leu Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr225 230 235 240Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe Ile Glu 245 250 255Ser Asp Pro Gly Phe Phe Ser Asn Ala Ile Val Glu Gly Ala Lys Lys 260 265 270Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gln 275 280 285Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr Ile Lys Ser Phe Val Glu 290 295 300Arg Val Leu Lys Asn Glu Gln Gly Ser Ala305 31025960DNAHomo sapiens 25atggatgatt gggagattcc tgatgggcag attacagtgg gacaaagaat tggatctgga 60tcatttggaa cagtctacaa gggaaagtgg catggtgatg tggcagtgaa aatgttgaat 120gtgacagcac ctacacctca gcagttacaa gccttcaaaa atgaagtagg agtactcagg 180aaaacacgac atgtgaatat cctactcttc atgggctatt ccacaaagcc acaactggct 240attgttaccc agtggtgtga gggctccagc ttgtatcacc atctccatat cattgagacc 300aaatttgaga tgatcaaact tatagatatt gcacgacaga ctgcacaggg catggattac 360ttacacgcca agtcaatcat ccacagagac ctcaagagta ataatatatt tcttcatgaa 420gacctcacag taaaaatagg tgattttggt ctagctacag tgaaatctcg atggagtggg 480tcccatcagt ttgaacagtt gtctggatcc attttgtgga tggcaccaga agtcatcaga 540atgcaagata aaaatccata cagctttcag tcagatgtat atgcatttgg aattgttctg 600tatgaattga tgactggaca gttaccttat tcaaacatca acaacaggga ccagataatt 660tttatggtgg gacgaggata cctgtctcca gatctcagta aggtacggag taactgtcca 720aaagccatga agagattaat ggcagagtgc ctcaaaaaga aaagagatga gagaccactc 780tttccccaaa ttctcgcctc tattgagctg ctggcccgct cattgccaaa aattcaccgc 840agtgcatcag aaccctcctt gaatcgggct ggtttccaaa cagaggattt tagtctatat 900gcttgtgctt ctccaaaaac acccatccag gcagggggat atggtgcgtt tcctgtccac 96026320PRTHomo sapiens 26Met Asp Asp Trp Glu Ile Pro Asp Gly Gln Ile Thr Val Gly Gln Arg1 5 10 15Ile Gly Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly Lys Trp His Gly 20 25 30Asp Val Ala Val Lys Met Leu Asn Val Thr Ala Pro Thr Pro Gln Gln 35 40 45Leu Gln Ala Phe Lys Asn Glu Val Gly Val Leu Arg Lys Thr Arg His 50 55 60Val Asn Ile Leu Leu Phe Met Gly Tyr Ser Thr Lys Pro Gln Leu Ala65 70 75 80Ile Val Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr His His Leu His 85 90 95Ile Ile Glu Thr Lys Phe Glu Met Ile Lys Leu Ile Asp Ile Ala Arg 100 105 110Gln Thr Ala Gln Gly Met Asp Tyr Leu His Ala Lys Ser Ile Ile His 115 120 125Arg Asp Leu Lys Ser Asn Asn Ile Phe Leu His Glu Asp Leu Thr Val 130 135 140Lys Ile Gly Asp Phe Gly Leu Ala Thr Val Lys Ser Arg Trp Ser Gly145 150 155 160Ser His Gln Phe Glu Gln Leu Ser Gly Ser Ile Leu Trp Met Ala Pro 165 170 175Glu Val Ile Arg Met Gln Asp Lys Asn Pro Tyr Ser Phe Gln Ser Asp 180 185 190Val Tyr Ala Phe Gly Ile Val Leu Tyr Glu Leu Met Thr Gly Gln Leu 195 200 205Pro Tyr Ser Asn Ile Asn Asn Arg Asp Gln Ile Ile Phe Met Val Gly 210 215 220Arg Gly Tyr Leu Ser Pro Asp Leu Ser Lys Val Arg Ser Asn Cys Pro225 230 235 240Lys Ala Met Lys Arg Leu Met Ala Glu Cys Leu Lys Lys Lys Arg Asp 245 250 255Glu Arg Pro Leu Phe Pro Gln Ile Leu Ala Ser Ile Glu Leu Leu Ala 260 265 270Arg Ser Leu Pro Lys Ile His Arg Ser Ala Ser Glu Pro Ser Leu Asn 275 280 285Arg Ala Gly Phe Gln Thr Glu Asp Phe Ser Leu Tyr Ala Cys Ala Ser 290 295 300Pro Lys Thr Pro Ile Gln Ala Gly Gly Tyr Gly Ala Phe Pro Val His305 310 315 32027960DNAHomo sapiens 27atggatgatt gggagattcc tgatgggcag attacagtgg gacaaagaat tggatctgga 60tcatttggaa cagtctacaa gggaaagtgg catggtgatg tggcagtgaa aatgttgaat 120gtgacagcac ctacacctca gcagttacaa gccttcaaaa atgaagtagg agtactcagg 180aaaacacatc atgtgaatat cctactcttc atgggctatt ccacaaagcc acaactggct 240attgttaccc agtggtgtga gggctccagc ttgtatcacc atctccatat cattgagacc 300aaatttgaga tgatcaaact tatagatatt gcacgacaga ctgcacaggg catggattac 360ttacacgcca agtcaatcat ccacagagac ctcaagagta ataatatatt tcttcatgaa 420gacctcacag taaaaatagg tgattttggt ctagctacag tgaaatctcg atggagtggg 480tcccatcagt ttgaacagtt gtctggatcc attttgtgga tggcaccaga agtcatcaga 540atgcaagata aaaatccata cagctttcag tcagatgtat atgcatttgg aattgttctg 600tatgaattga tgactggaca gttaccttat tcaaacatca acaacaggga ccagataatt 660tttatggtgg gacgaggata cctgtctcca gatctcagta aggtacggag taactgtcca 720aaagccatga agagattaat ggcagagtgc ctcaaaaaga aaagagatga gagaccactc 780tttccccaaa ttctcgcctc tattgagctg ctggcccgct cattgccaaa aattcaccgc 840agtgcatcag aaccctcctt gaatcgggct ggtttccaaa cagaggattt tagtctatat 900gcttgtgctt ctccaaaaac acccatccag gcagggggat atggtgcgtt tcctgtccac 96028320PRTHomo sapiens 28Met Asp Asp Trp Glu Ile Pro Asp Gly Gln Ile Thr Val Gly Gln Arg1 5 10 15Ile Gly Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly Lys Trp His Gly 20 25 30Asp Val Ala Val Lys Met Leu Asn Val Thr Ala Pro Thr Pro Gln Gln 35 40 45Leu Gln Ala Phe Lys Asn Glu Val Gly Val Leu Arg Lys Thr His His 50 55 60Val Asn Ile Leu Leu Phe Met Gly Tyr Ser Thr Lys Pro Gln Leu Ala65 70 75 80Ile Val Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr His His Leu His 85 90 95Ile Ile Glu Thr Lys Phe Glu Met Ile Lys Leu Ile Asp Ile Ala Arg 100 105 110Gln Thr Ala Gln Gly Met Asp Tyr Leu His Ala Lys Ser Ile Ile His 115 120 125Arg Asp Leu Lys Ser Asn Asn Ile Phe Leu His Glu Asp Leu Thr Val 130 135 140Lys Ile Gly Asp Phe Gly Leu Ala Thr Val Lys Ser Arg Trp Ser Gly145 150 155 160Ser His Gln Phe Glu Gln Leu Ser Gly Ser Ile Leu Trp Met Ala Pro 165 170 175Glu Val Ile Arg Met Gln Asp Lys Asn Pro Tyr Ser Phe Gln Ser Asp 180 185 190Val Tyr Ala Phe Gly Ile Val Leu Tyr Glu Leu Met Thr Gly Gln Leu 195 200 205Pro Tyr Ser Asn Ile Asn Asn Arg Asp Gln Ile Ile Phe Met Val Gly 210 215 220Arg Gly Tyr Leu Ser Pro Asp Leu Ser Lys Val Arg Ser Asn Cys Pro225 230 235 240Lys Ala Met Lys Arg Leu Met Ala Glu Cys Leu Lys Lys Lys Arg Asp 245 250 255Glu Arg Pro Leu Phe Pro Gln Ile Leu Ala Ser Ile Glu Leu Leu Ala 260 265

270Arg Ser Leu Pro Lys Ile His Arg Ser Ala Ser Glu Pro Ser Leu Asn 275 280 285Arg Ala Gly Phe Gln Thr Glu Asp Phe Ser Leu Tyr Ala Cys Ala Ser 290 295 300Pro Lys Thr Pro Ile Gln Ala Gly Gly Tyr Gly Ala Phe Pro Val His305 310 315 320291755DNAHomo sapiens 29atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180ctcgtgacca ccctgagcta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420aagctggagt acaactacaa cccccacaac gtctatatca tggccgacaa gcagaagaac 480ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600tacctgttca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggga 720tccgccggta ccatggatga ttgggagatt cctgatgggc agattacagt gggacaaaga 780attggatctg gatcatttgg aacagtctac aagggaaagt ggcatggtga tgtggcagtg 840aaaatgttga atgtgacagc acctacacct cagcagttac aagccttcaa aaatgaagta 900ggagtactca ggaaaacacg acatgtgaat atcctactct tcatgggcta ttccacaaag 960ccacaactgg ctattgttac ccagtggtgt gagggctcca gcttgtatca ccatctccat 1020atcattgaga ccaaatttga gatgatcaaa cttatagata ttgcacgaca gactgcacag 1080ggcatggatt acttacacgc caagtcaatc atccacagag acctcaagag taataatata 1140tttcttcatg aagacctcac agtaaaaata ggtgattttg gtctagctac agtgaaatct 1200cgatggagtg ggtcccatca gtttgaacag ttgtctggat ccattttgtg gatggcacca 1260gaagtcatca gaatgcaaga taaaaatcca tacagctttc agtcagatgt atatgcattt 1320ggaattgttc tgtatgaatt gatgactgga cagttacctt attcaaacat caacaacagg 1380gaccagataa tttttatggt gggacgagga tacctgtctc cagatctcag taaggtacgg 1440agtaactgtc caaaagccat gaagagatta atggcagagt gcctcaaaaa gaaaagagat 1500gagagaccac tctttcccca aattctcgcc tctattgagc tgctggcccg ctcattgcca 1560aaaattcacc gcagtgcatc agaaccctcc ttgaatcggg ctggtttcca aacagaggat 1620tttagtctat atgcttgtgc ttctccaaaa acacccatcc aggcaggggg atatggtgcg 1680tttcctgtcc acaagatgag caaagatggt aaaaagaaga aaaagaagtc aaagacaaag 1740tgtgtaatta tgtaa 175530584PRTHomo sapiens 30Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu1 5 10 15Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60Leu Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys65 70 75 80Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140Asn Tyr Asn Pro His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150 155 160Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Phe Thr Gln Ser Ala Leu 195 200 205Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly225 230 235 240Ser Ala Gly Thr Met Asp Asp Trp Glu Ile Pro Asp Gly Gln Ile Thr 245 250 255Val Gly Gln Arg Ile Gly Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly 260 265 270Lys Trp His Gly Asp Val Ala Val Lys Met Leu Asn Val Thr Ala Pro 275 280 285Thr Pro Gln Gln Leu Gln Ala Phe Lys Asn Glu Val Gly Val Leu Arg 290 295 300Lys Thr Arg His Val Asn Ile Leu Leu Phe Met Gly Tyr Ser Thr Lys305 310 315 320Pro Gln Leu Ala Ile Val Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr 325 330 335His His Leu His Ile Ile Glu Thr Lys Phe Glu Met Ile Lys Leu Ile 340 345 350Asp Ile Ala Arg Gln Thr Ala Gln Gly Met Asp Tyr Leu His Ala Lys 355 360 365Ser Ile Ile His Arg Asp Leu Lys Ser Asn Asn Ile Phe Leu His Glu 370 375 380Asp Leu Thr Val Lys Ile Gly Asp Phe Gly Leu Ala Thr Val Lys Ser385 390 395 400Arg Trp Ser Gly Ser His Gln Phe Glu Gln Leu Ser Gly Ser Ile Leu 405 410 415Trp Met Ala Pro Glu Val Ile Arg Met Gln Asp Lys Asn Pro Tyr Ser 420 425 430Phe Gln Ser Asp Val Tyr Ala Phe Gly Ile Val Leu Tyr Glu Leu Met 435 440 445Thr Gly Gln Leu Pro Tyr Ser Asn Ile Asn Asn Arg Asp Gln Ile Ile 450 455 460Phe Met Val Gly Arg Gly Tyr Leu Ser Pro Asp Leu Ser Lys Val Arg465 470 475 480Ser Asn Cys Pro Lys Ala Met Lys Arg Leu Met Ala Glu Cys Leu Lys 485 490 495Lys Lys Arg Asp Glu Arg Pro Leu Phe Pro Gln Ile Leu Ala Ser Ile 500 505 510Glu Leu Leu Ala Arg Ser Leu Pro Lys Ile His Arg Ser Ala Ser Glu 515 520 525Pro Ser Leu Asn Arg Ala Gly Phe Gln Thr Glu Asp Phe Ser Leu Tyr 530 535 540Ala Cys Ala Ser Pro Lys Thr Pro Ile Gln Ala Gly Gly Tyr Gly Ala545 550 555 560Phe Pro Val His Lys Met Ser Lys Asp Gly Lys Lys Lys Lys Lys Lys 565 570 575Ser Lys Thr Lys Cys Val Ile Met 580311755DNAHomo sapiens 31atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180ctcgtgacca ccctgagcta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420aagctggagt acaactacaa cccccacaac gtctatatca tggccgacaa gcagaagaac 480ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600tacctgttca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggga 720tccgccggta ccatggatga ttgggagatt cctgatgggc agattacagt gggacaaaga 780attggatctg gatcatttgg aacagtctac aagggaaagt ggcatggtga tgtggcagtg 840aaaatgttga atgtgacagc acctacacct cagcagttac aagccttcaa aaatgaagta 900ggagtactca ggaaaacaca tcatgtgaat atcctactct tcatgggcta ttccacaaag 960ccacaactgg ctattgttac ccagtggtgt gagggctcca gcttgtatca ccatctccat 1020atcattgaga ccaaatttga gatgatcaaa cttatagata ttgcacgaca gactgcacag 1080ggcatggatt acttacacgc caagtcaatc atccacagag acctcaagag taataatata 1140tttcttcatg aagacctcac agtaaaaata ggtgattttg gtctagctac agtgaaatct 1200cgatggagtg ggtcccatca gtttgaacag ttgtctggat ccattttgtg gatggcacca 1260gaagtcatca gaatgcaaga taaaaatcca tacagctttc agtcagatgt atatgcattt 1320ggaattgttc tgtatgaatt gatgactgga cagttacctt attcaaacat caacaacagg 1380gaccagataa tttttatggt gggacgagga tacctgtctc cagatctcag taaggtacgg 1440agtaactgtc caaaagccat gaagagatta atggcagagt gcctcaaaaa gaaaagagat 1500gagagaccac tctttcccca aattctcgcc tctattgagc tgctggcccg ctcattgcca 1560aaaattcacc gcagtgcatc agaaccctcc ttgaatcggg ctggtttcca aacagaggat 1620tttagtctat atgcttgtgc ttctccaaaa acacccatcc aggcaggggg atatggtgcg 1680tttcctgtcc acaagatgag caaagatggt aaaaagaaga aaaagaagtc aaagacaaag 1740tgtgtaatta tgtaa 175532584PRTHomo sapiens 32Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu1 5 10 15Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60Leu Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys65 70 75 80Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140Asn Tyr Asn Pro His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150 155 160Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Phe Thr Gln Ser Ala Leu 195 200 205Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly225 230 235 240Ser Ala Gly Thr Met Asp Asp Trp Glu Ile Pro Asp Gly Gln Ile Thr 245 250 255Val Gly Gln Arg Ile Gly Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly 260 265 270Lys Trp His Gly Asp Val Ala Val Lys Met Leu Asn Val Thr Ala Pro 275 280 285Thr Pro Gln Gln Leu Gln Ala Phe Lys Asn Glu Val Gly Val Leu Arg 290 295 300Lys Thr His His Val Asn Ile Leu Leu Phe Met Gly Tyr Ser Thr Lys305 310 315 320Pro Gln Leu Ala Ile Val Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr 325 330 335His His Leu His Ile Ile Glu Thr Lys Phe Glu Met Ile Lys Leu Ile 340 345 350Asp Ile Ala Arg Gln Thr Ala Gln Gly Met Asp Tyr Leu His Ala Lys 355 360 365Ser Ile Ile His Arg Asp Leu Lys Ser Asn Asn Ile Phe Leu His Glu 370 375 380Asp Leu Thr Val Lys Ile Gly Asp Phe Gly Leu Ala Thr Val Lys Ser385 390 395 400Arg Trp Ser Gly Ser His Gln Phe Glu Gln Leu Ser Gly Ser Ile Leu 405 410 415Trp Met Ala Pro Glu Val Ile Arg Met Gln Asp Lys Asn Pro Tyr Ser 420 425 430Phe Gln Ser Asp Val Tyr Ala Phe Gly Ile Val Leu Tyr Glu Leu Met 435 440 445Thr Gly Gln Leu Pro Tyr Ser Asn Ile Asn Asn Arg Asp Gln Ile Ile 450 455 460Phe Met Val Gly Arg Gly Tyr Leu Ser Pro Asp Leu Ser Lys Val Arg465 470 475 480Ser Asn Cys Pro Lys Ala Met Lys Arg Leu Met Ala Glu Cys Leu Lys 485 490 495Lys Lys Arg Asp Glu Arg Pro Leu Phe Pro Gln Ile Leu Ala Ser Ile 500 505 510Glu Leu Leu Ala Arg Ser Leu Pro Lys Ile His Arg Ser Ala Ser Glu 515 520 525Pro Ser Leu Asn Arg Ala Gly Phe Gln Thr Glu Asp Phe Ser Leu Tyr 530 535 540Ala Cys Ala Ser Pro Lys Thr Pro Ile Gln Ala Gly Gly Tyr Gly Ala545 550 555 560Phe Pro Val His Lys Met Ser Lys Asp Gly Lys Lys Lys Lys Lys Lys 565 570 575Ser Lys Thr Lys Cys Val Ile Met 580331971DNAHomo sapiens 33atgaccagca aggtgtacga ccccgagcag aggaagagga tgatcaccgg cccccagtgg 60tgggccaggt gcaagcagat gaacgtgctg gacagcttca tcaactacta cgacagcgag 120aagcacgccg agaacgccgt gatcttcctg cacggcaacg ccactagcag ctacctgtgg 180aggcacgtgg tgccccacat cgagcccgtg gccaggtgca tcatccccga tctgatcggc 240atgggcaaga gcggcaagag cggcaacggc agctacaggc tgctggacca ctacaagtac 300ctgaccgcct ggttcgagct cctgaacctg cccaagaaga tcatcttcgt gggccacgac 360tggggcgccg cactggcctt ccactacagc tacgagcacc aggacaagat caaggccatc 420gtgcacgccg agagcgtggt tgacgtgatc gagagctggg acgagtggcc agacatcgag 480gaggacatcg ccctgatcaa gagcgaggag ggcgagaaga tggtgctgga gaacaacttc 540ttcgtggaga ccgttctgcc cagcaagatc atgagaaagc tggagcccga ggagttcgcc 600gcctacctgg agcccttcaa ggagaagggc gaggtgagaa gacccaccct gagctggccc 660agagagatcc ccctggtgaa gggcggcaag cccgacgtgg tgcagatcgt gagaaactac 720aacgcctacc tgagagccag cgacgacctg cccaagatgt tcatcgagag cgaccccggc 780ttcttcagca acgccatcgt ggagggcgcc aagaagttcc ccaacaccga gttcgtgaag 840gtgaagggcc tgcacttcag ccaggaggac gcccccgacg agatgggcaa gtacatcaag 900agcttcgtgg agagagtgct gaagaacgag cagggatccg ccggtaccat ggatgattgg 960gagattcctg atgggcagat tacagtggga caaagaattg gatctggatc atttggaaca 1020gtctacaagg gaaagtggca tggtgatgtg gcagtgaaaa tgttgaatgt gacagcacct 1080acacctcagc agttacaagc cttcaaaaat gaagtaggag tactcaggaa aacacgacat 1140gtgaatatcc tactcttcat gggctattcc acaaagccac aactggctat tgttacccag 1200tggtgtgagg gctccagctt gtatcaccat ctccatatca ttgagaccaa atttgagatg 1260atcaaactta tagatattgc acgacagact gcacagggca tggattactt acacgccaag 1320tcaatcatcc acagagacct caagagtaat aatatatttc ttcatgaaga cctcacagta 1380aaaataggtg attttggtct agctacagtg aaatctcgat ggagtgggtc ccatcagttt 1440gaacagttgt ctggatccat tttgtggatg gcaccagaag tcatcagaat gcaagataaa 1500aatccataca gctttcagtc agatgtatat gcatttggaa ttgttctgta tgaattgatg 1560actggacagt taccttattc aaacatcaac aacagggacc agataatttt tatggtggga 1620cgaggatacc tgtctccaga tctcagtaag gtacggagta actgtccaaa agccatgaag 1680agattaatgg cagagtgcct caaaaagaaa agagatgaga gaccactctt tccccaaatt 1740ctcgcctcta ttgagctgct ggcccgctca ttgccaaaaa ttcaccgcag tgcatcagaa 1800ccctccttga atcgggctgg tttccaaaca gaggatttta gtctatatgc ttgtgcttct 1860ccaaaaacac ccatccaggc agggggatat ggtgcgtttc ctgtccacaa gatgagcaaa 1920gatggtaaaa agaagaaaaa gaagtcaaag acaaagtgtg taattatgta a 197134656PRTHomo sapiens 34Met Thr Ser Lys Val Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr1 5 10 15Gly Pro Gln Trp Trp Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser 20 25 30Phe Ile Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile 35 40 45Phe Leu His Gly Asn Ala Thr Ser Ser Tyr Leu Trp Arg His Val Val 50 55 60Pro His Ile Glu Pro Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly65 70 75 80Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp 85 90 95His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys 100 105 110Lys Ile Ile Phe Val Gly His Asp Trp Gly Ala Ala Leu Ala Phe His 115 120 125Tyr Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val His Ala Glu 130 135 140Ser Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu145 150 155 160Glu Asp Ile Ala Leu Ile Lys Ser Glu Glu Gly Glu Lys Met Val Leu 165 170 175Glu Asn Asn Phe Phe Val Glu Thr Val Leu Pro Ser Lys Ile Met Arg 180 185 190Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu 195 200 205Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro 210 215 220Leu Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr225 230 235 240Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe Ile Glu 245 250 255Ser Asp Pro Gly Phe Phe Ser Asn Ala Ile Val Glu Gly Ala Lys Lys 260 265 270Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gln 275 280 285Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr Ile Lys Ser Phe Val Glu 290 295 300Arg Val Leu Lys Asn Glu Gln Gly Ser Ala Gly Thr Met Asp Asp Trp305 310 315 320Glu Ile Pro Asp Gly Gln Ile

Thr Val Gly Gln Arg Ile Gly Ser Gly 325 330 335Ser Phe Gly Thr Val Tyr Lys Gly Lys Trp His Gly Asp Val Ala Val 340 345 350Lys Met Leu Asn Val Thr Ala Pro Thr Pro Gln Gln Leu Gln Ala Phe 355 360 365Lys Asn Glu Val Gly Val Leu Arg Lys Thr Arg His Val Asn Ile Leu 370 375 380Leu Phe Met Gly Tyr Ser Thr Lys Pro Gln Leu Ala Ile Val Thr Gln385 390 395 400Trp Cys Glu Gly Ser Ser Leu Tyr His His Leu His Ile Ile Glu Thr 405 410 415Lys Phe Glu Met Ile Lys Leu Ile Asp Ile Ala Arg Gln Thr Ala Gln 420 425 430Gly Met Asp Tyr Leu His Ala Lys Ser Ile Ile His Arg Asp Leu Lys 435 440 445Ser Asn Asn Ile Phe Leu His Glu Asp Leu Thr Val Lys Ile Gly Asp 450 455 460Phe Gly Leu Ala Thr Val Lys Ser Arg Trp Ser Gly Ser His Gln Phe465 470 475 480Glu Gln Leu Ser Gly Ser Ile Leu Trp Met Ala Pro Glu Val Ile Arg 485 490 495Met Gln Asp Lys Asn Pro Tyr Ser Phe Gln Ser Asp Val Tyr Ala Phe 500 505 510Gly Ile Val Leu Tyr Glu Leu Met Thr Gly Gln Leu Pro Tyr Ser Asn 515 520 525Ile Asn Asn Arg Asp Gln Ile Ile Phe Met Val Gly Arg Gly Tyr Leu 530 535 540Ser Pro Asp Leu Ser Lys Val Arg Ser Asn Cys Pro Lys Ala Met Lys545 550 555 560Arg Leu Met Ala Glu Cys Leu Lys Lys Lys Arg Asp Glu Arg Pro Leu 565 570 575Phe Pro Gln Ile Leu Ala Ser Ile Glu Leu Leu Ala Arg Ser Leu Pro 580 585 590Lys Ile His Arg Ser Ala Ser Glu Pro Ser Leu Asn Arg Ala Gly Phe 595 600 605Gln Thr Glu Asp Phe Ser Leu Tyr Ala Cys Ala Ser Pro Lys Thr Pro 610 615 620Ile Gln Ala Gly Gly Tyr Gly Ala Phe Pro Val His Lys Met Ser Lys625 630 635 640Asp Gly Lys Lys Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met645 650 655351971DNAHomo sapiens 35atgaccagca aggtgtacga ccccgagcag aggaagagga tgatcaccgg cccccagtgg 60tgggccaggt gcaagcagat gaacgtgctg gacagcttca tcaactacta cgacagcgag 120aagcacgccg agaacgccgt gatcttcctg cacggcaacg ccactagcag ctacctgtgg 180aggcacgtgg tgccccacat cgagcccgtg gccaggtgca tcatccccga tctgatcggc 240atgggcaaga gcggcaagag cggcaacggc agctacaggc tgctggacca ctacaagtac 300ctgaccgcct ggttcgagct cctgaacctg cccaagaaga tcatcttcgt gggccacgac 360tggggcgccg cactggcctt ccactacagc tacgagcacc aggacaagat caaggccatc 420gtgcacgccg agagcgtggt tgacgtgatc gagagctggg acgagtggcc agacatcgag 480gaggacatcg ccctgatcaa gagcgaggag ggcgagaaga tggtgctgga gaacaacttc 540ttcgtggaga ccgttctgcc cagcaagatc atgagaaagc tggagcccga ggagttcgcc 600gcctacctgg agcccttcaa ggagaagggc gaggtgagaa gacccaccct gagctggccc 660agagagatcc ccctggtgaa gggcggcaag cccgacgtgg tgcagatcgt gagaaactac 720aacgcctacc tgagagccag cgacgacctg cccaagatgt tcatcgagag cgaccccggc 780ttcttcagca acgccatcgt ggagggcgcc aagaagttcc ccaacaccga gttcgtgaag 840gtgaagggcc tgcacttcag ccaggaggac gcccccgacg agatgggcaa gtacatcaag 900agcttcgtgg agagagtgct gaagaacgag cagggatccg ccggtaccat ggatgattgg 960gagattcctg atgggcagat tacagtggga caaagaattg gatctggatc atttggaaca 1020gtctacaagg gaaagtggca tggtgatgtg gcagtgaaaa tgttgaatgt gacagcacct 1080acacctcagc agttacaagc cttcaaaaat gaagtaggag tactcaggaa aacacatcat 1140gtgaatatcc tactcttcat gggctattcc acaaagccac aactggctat tgttacccag 1200tggtgtgagg gctccagctt gtatcaccat ctccatatca ttgagaccaa atttgagatg 1260atcaaactta tagatattgc acgacagact gcacagggca tggattactt acacgccaag 1320tcaatcatcc acagagacct caagagtaat aatatatttc ttcatgaaga cctcacagta 1380aaaataggtg attttggtct agctacagtg aaatctcgat ggagtgggtc ccatcagttt 1440gaacagttgt ctggatccat tttgtggatg gcaccagaag tcatcagaat gcaagataaa 1500aatccataca gctttcagtc agatgtatat gcatttggaa ttgttctgta tgaattgatg 1560actggacagt taccttattc aaacatcaac aacagggacc agataatttt tatggtggga 1620cgaggatacc tgtctccaga tctcagtaag gtacggagta actgtccaaa agccatgaag 1680agattaatgg cagagtgcct caaaaagaaa agagatgaga gaccactctt tccccaaatt 1740ctcgcctcta ttgagctgct ggcccgctca ttgccaaaaa ttcaccgcag tgcatcagaa 1800ccctccttga atcgggctgg tttccaaaca gaggatttta gtctatatgc ttgtgcttct 1860ccaaaaacac ccatccaggc agggggatat ggtgcgtttc ctgtccacaa gatgagcaaa 1920gatggtaaaa agaagaaaaa gaagtcaaag acaaagtgtg taattatgta a 197136656PRTHomo sapiens 36Met Thr Ser Lys Val Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr1 5 10 15Gly Pro Gln Trp Trp Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser 20 25 30Phe Ile Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile 35 40 45Phe Leu His Gly Asn Ala Thr Ser Ser Tyr Leu Trp Arg His Val Val 50 55 60Pro His Ile Glu Pro Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly65 70 75 80Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp 85 90 95His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys 100 105 110Lys Ile Ile Phe Val Gly His Asp Trp Gly Ala Ala Leu Ala Phe His 115 120 125Tyr Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val His Ala Glu 130 135 140Ser Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu145 150 155 160Glu Asp Ile Ala Leu Ile Lys Ser Glu Glu Gly Glu Lys Met Val Leu 165 170 175Glu Asn Asn Phe Phe Val Glu Thr Val Leu Pro Ser Lys Ile Met Arg 180 185 190Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu 195 200 205Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro 210 215 220Leu Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr225 230 235 240Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe Ile Glu 245 250 255Ser Asp Pro Gly Phe Phe Ser Asn Ala Ile Val Glu Gly Ala Lys Lys 260 265 270Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gln 275 280 285Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr Ile Lys Ser Phe Val Glu 290 295 300Arg Val Leu Lys Asn Glu Gln Gly Ser Ala Gly Thr Met Asp Asp Trp305 310 315 320Glu Ile Pro Asp Gly Gln Ile Thr Val Gly Gln Arg Ile Gly Ser Gly 325 330 335Ser Phe Gly Thr Val Tyr Lys Gly Lys Trp His Gly Asp Val Ala Val 340 345 350Lys Met Leu Asn Val Thr Ala Pro Thr Pro Gln Gln Leu Gln Ala Phe 355 360 365Lys Asn Glu Val Gly Val Leu Arg Lys Thr His His Val Asn Ile Leu 370 375 380Leu Phe Met Gly Tyr Ser Thr Lys Pro Gln Leu Ala Ile Val Thr Gln385 390 395 400Trp Cys Glu Gly Ser Ser Leu Tyr His His Leu His Ile Ile Glu Thr 405 410 415Lys Phe Glu Met Ile Lys Leu Ile Asp Ile Ala Arg Gln Thr Ala Gln 420 425 430Gly Met Asp Tyr Leu His Ala Lys Ser Ile Ile His Arg Asp Leu Lys 435 440 445Ser Asn Asn Ile Phe Leu His Glu Asp Leu Thr Val Lys Ile Gly Asp 450 455 460Phe Gly Leu Ala Thr Val Lys Ser Arg Trp Ser Gly Ser His Gln Phe465 470 475 480Glu Gln Leu Ser Gly Ser Ile Leu Trp Met Ala Pro Glu Val Ile Arg 485 490 495Met Gln Asp Lys Asn Pro Tyr Ser Phe Gln Ser Asp Val Tyr Ala Phe 500 505 510Gly Ile Val Leu Tyr Glu Leu Met Thr Gly Gln Leu Pro Tyr Ser Asn 515 520 525Ile Asn Asn Arg Asp Gln Ile Ile Phe Met Val Gly Arg Gly Tyr Leu 530 535 540Ser Pro Asp Leu Ser Lys Val Arg Ser Asn Cys Pro Lys Ala Met Lys545 550 555 560Arg Leu Met Ala Glu Cys Leu Lys Lys Lys Arg Asp Glu Arg Pro Leu 565 570 575Phe Pro Gln Ile Leu Ala Ser Ile Glu Leu Leu Ala Arg Ser Leu Pro 580 585 590Lys Ile His Arg Ser Ala Ser Glu Pro Ser Leu Asn Arg Ala Gly Phe 595 600 605Gln Thr Glu Asp Phe Ser Leu Tyr Ala Cys Ala Ser Pro Lys Thr Pro 610 615 620Ile Gln Ala Gly Gly Tyr Gly Ala Phe Pro Val His Lys Met Ser Lys625 630 635 640Asp Gly Lys Lys Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met 645 650 65537933DNAHomo sapiens 37atgtattatt gggaaataga agccagtgaa gtgatgctgt ccactcggat tgggtcaggc 60tcttttggaa ctgtttataa gggtaaatgg cacggagatg ttgcagtaaa gatcctaaag 120gttgtcgacc caaccccaga gcaattccag gccttcagga atgaggtggc tgttctgcgc 180aaaacacggc atgtgaacat tctgcttttc atggggtaca tgacaaagga caacctggca 240attgtgaccc agtggtgcga gggcagcagc ctctacaaac acctgcatgt ccaggagacc 300aagtttcaga tgttccagct aattgacatt gcccggcaga cggctcaggg aatggactat 360ttgcatgcaa agaacatcat ccatagagac atgaaatcca acaatatatt tctccatgaa 420ggcttaacag tgaaaattgg agattttggt ttggcaacag taaagtcacg ctggagtggt 480tctcagcagg ttgaacaacc tactggctct gtcctctgga tggccccaga ggtgatccga 540atgcaggata acaacccatt cagtttccag tcggatgtct actcctatgg catcgtattg 600tatgaactga tgacggggga gcttccttat tctcacatca acaaccgaga tcagatcatc 660ttcatggtgg gccgaggata tgcctcccca gatcttagta agctatataa gaactgcccc 720aaagcaatga agaggctggt agctgactgt gtgaagaaag taaaggaaga gaggcctctt 780tttccccaga tcctgtcttc cattgagctg ctccaacact ctctaccgaa gatcaaccgg 840agcgcttccg agccatcctt gcatcgggca gcccacactg aggatatcaa tgcttgcacg 900ctgaccacgt ccccgaggct gcctgtcttc tag 93338310PRTHomo sapiens 38Met Tyr Tyr Trp Glu Ile Glu Ala Ser Glu Val Met Leu Ser Thr Arg1 5 10 15Ile Gly Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly Lys Trp His Gly 20 25 30Asp Val Ala Val Lys Ile Leu Lys Val Val Asp Pro Thr Pro Glu Gln 35 40 45Phe Gln Ala Phe Arg Asn Glu Val Ala Val Leu Arg Lys Thr Arg His 50 55 60Val Asn Ile Leu Leu Phe Met Gly Tyr Met Thr Lys Asp Asn Leu Ala65 70 75 80Ile Val Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr Lys His Leu His 85 90 95Val Gln Glu Thr Lys Phe Gln Met Phe Gln Leu Ile Asp Ile Ala Arg 100 105 110Gln Thr Ala Gln Gly Met Asp Tyr Leu His Ala Lys Asn Ile Ile His 115 120 125Arg Asp Met Lys Ser Asn Asn Ile Phe Leu His Glu Gly Leu Thr Val 130 135 140Lys Ile Gly Asp Phe Gly Leu Ala Thr Val Lys Ser Arg Trp Ser Gly145 150 155 160Ser Gln Gln Val Glu Gln Pro Thr Gly Ser Val Leu Trp Met Ala Pro 165 170 175Glu Val Ile Arg Met Gln Asp Asn Asn Pro Phe Ser Phe Gln Ser Asp 180 185 190Val Tyr Ser Tyr Gly Ile Val Leu Tyr Glu Leu Met Thr Gly Glu Leu 195 200 205Pro Tyr Ser His Ile Asn Asn Arg Asp Gln Ile Ile Phe Met Val Gly 210 215 220Arg Gly Tyr Ala Ser Pro Asp Leu Ser Lys Leu Tyr Lys Asn Cys Pro225 230 235 240Lys Ala Met Lys Arg Leu Val Ala Asp Cys Val Lys Lys Val Lys Glu 245 250 255Glu Arg Pro Leu Phe Pro Gln Ile Leu Ser Ser Ile Glu Leu Leu Gln 260 265 270His Ser Leu Pro Lys Ile Asn Arg Ser Ala Ser Glu Pro Ser Leu His 275 280 285Arg Ala Ala His Thr Glu Asp Ile Asn Ala Cys Thr Leu Thr Thr Ser 290 295 300Pro Arg Leu Pro Val Phe305 310391725DNAHomo sapiens 39atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180ctcgtgacca ccctgagcta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420aagctggagt acaactacaa cccccacaac gtctatatca tggccgacaa gcagaagaac 480ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600tacctgttca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggga 720tccgccggta ccatgtatta ttgggaaata gaagccagtg aagtgatgct gtccactcgg 780attgggtcag gctcttttgg aactgtttat aagggtaaat ggcacggaga tgttgcagta 840aagatcctaa aggttgtcga cccaacccca gagcaattcc aggccttcag gaatgaggtg 900gctgttctgc gcaaaacacg gcatgtgaac attctgcttt tcatggggta catgacaaag 960gacaacctgg caattgtgac ccagtggtgc gagggcagca gcctctacaa acacctgcat 1020gtccaggaga ccaagtttca gatgttccag ctaattgaca ttgcccggca gacggctcag 1080ggaatggact atttgcatgc aaagaacatc atccatagag acatgaaatc caacaatata 1140tttctccatg aaggcttaac agtgaaaatt ggagattttg gtttggcaac agtaaagtca 1200cgctggagtg gttctcagca ggttgaacaa cctactggct ctgtcctctg gatggcccca 1260gaggtgatcc gaatgcagga taacaaccca ttcagtttcc agtcggatgt ctactcctat 1320ggcatcgtat tgtatgaact gatgacgggg gagcttcctt attctcacat caacaaccga 1380gatcagatca tcttcatggt gggccgagga tatgcctccc cagatcttag taagctatat 1440aagaactgcc ccaaagcaat gaagaggctg gtagctgact gtgtgaagaa agtaaaggaa 1500gagaggcctc tttttcccca gatcctgtct tccattgagc tgctccaaca ctctctaccg 1560aagatcaacc ggagcgcttc cgagccatcc ttgcatcggg cagcccacac tgaggatatc 1620aatgcttgca cgctgaccac gtccccgagg ctgcctgtct tcaagatgag caaagatggt 1680aaaaagaaga aaaagaagtc aaagacaaag tgtgtaatta tgtaa 172540574PRTHomo sapiens 40Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu1 5 10 15Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60Leu Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys65 70 75 80Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140Asn Tyr Asn Pro His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150 155 160Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Phe Thr Gln Ser Ala Leu 195 200 205Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly225 230 235 240Ser Ala Gly Thr Met Tyr Tyr Trp Glu Ile Glu Ala Ser Glu Val Met 245 250 255Leu Ser Thr Arg Ile Gly Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly 260 265 270Lys Trp His Gly Asp Val Ala Val Lys Ile Leu Lys Val Val Asp Pro 275 280 285Thr Pro Glu Gln Phe Gln Ala Phe Arg Asn Glu Val Ala Val Leu Arg 290 295 300Lys Thr Arg His Val Asn Ile Leu Leu Phe Met Gly Tyr Met Thr Lys305 310 315 320Asp Asn Leu Ala Ile Val Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr 325 330 335Lys His Leu His Val Gln Glu Thr Lys Phe Gln Met Phe Gln Leu Ile 340 345 350Asp Ile Ala Arg Gln Thr Ala Gln Gly Met Asp Tyr Leu His Ala Lys 355 360 365Asn Ile Ile His Arg Asp Met Lys Ser Asn Asn Ile Phe Leu His Glu 370 375 380Gly Leu Thr Val Lys Ile Gly Asp Phe Gly

Leu Ala Thr Val Lys Ser385 390 395 400Arg Trp Ser Gly Ser Gln Gln Val Glu Gln Pro Thr Gly Ser Val Leu 405 410 415Trp Met Ala Pro Glu Val Ile Arg Met Gln Asp Asn Asn Pro Phe Ser 420 425 430Phe Gln Ser Asp Val Tyr Ser Tyr Gly Ile Val Leu Tyr Glu Leu Met 435 440 445Thr Gly Glu Leu Pro Tyr Ser His Ile Asn Asn Arg Asp Gln Ile Ile 450 455 460Phe Met Val Gly Arg Gly Tyr Ala Ser Pro Asp Leu Ser Lys Leu Tyr465 470 475 480Lys Asn Cys Pro Lys Ala Met Lys Arg Leu Val Ala Asp Cys Val Lys 485 490 495Lys Val Lys Glu Glu Arg Pro Leu Phe Pro Gln Ile Leu Ser Ser Ile 500 505 510Glu Leu Leu Gln His Ser Leu Pro Lys Ile Asn Arg Ser Ala Ser Glu 515 520 525Pro Ser Leu His Arg Ala Ala His Thr Glu Asp Ile Asn Ala Cys Thr 530 535 540Leu Thr Thr Ser Pro Arg Leu Pro Val Phe Lys Met Ser Lys Asp Gly545 550 555 560Lys Lys Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met565 570411941DNAHomo sapiens 41atgaccagca aggtgtacga ccccgagcag aggaagagga tgatcaccgg cccccagtgg 60tgggccaggt gcaagcagat gaacgtgctg gacagcttca tcaactacta cgacagcgag 120aagcacgccg agaacgccgt gatcttcctg cacggcaacg ccactagcag ctacctgtgg 180aggcacgtgg tgccccacat cgagcccgtg gccaggtgca tcatccccga tctgatcggc 240atgggcaaga gcggcaagag cggcaacggc agctacaggc tgctggacca ctacaagtac 300ctgaccgcct ggttcgagct cctgaacctg cccaagaaga tcatcttcgt gggccacgac 360tggggcgccg cactggcctt ccactacagc tacgagcacc aggacaagat caaggccatc 420gtgcacgccg agagcgtggt tgacgtgatc gagagctggg acgagtggcc agacatcgag 480gaggacatcg ccctgatcaa gagcgaggag ggcgagaaga tggtgctgga gaacaacttc 540ttcgtggaga ccgttctgcc cagcaagatc atgagaaagc tggagcccga ggagttcgcc 600gcctacctgg agcccttcaa ggagaagggc gaggtgagaa gacccaccct gagctggccc 660agagagatcc ccctggtgaa gggcggcaag cccgacgtgg tgcagatcgt gagaaactac 720aacgcctacc tgagagccag cgacgacctg cccaagatgt tcatcgagag cgaccccggc 780ttcttcagca acgccatcgt ggagggcgcc aagaagttcc ccaacaccga gttcgtgaag 840gtgaagggcc tgcacttcag ccaggaggac gcccccgacg agatgggcaa gtacatcaag 900agcttcgtgg agagagtgct gaagaacgag cagggatccg ccggtaccat gtattattgg 960gaaatagaag ccagtgaagt gatgctgtcc actcggattg ggtcaggctc ttttggaact 1020gtttataagg gtaaatggca cggagatgtt gcagtaaaga tcctaaaggt tgtcgaccca 1080accccagagc aattccaggc cttcaggaat gaggtggctg ttctgcgcaa aacacggcat 1140gtgaacattc tgcttttcat ggggtacatg acaaaggaca acctggcaat tgtgacccag 1200tggtgcgagg gcagcagcct ctacaaacac ctgcatgtcc aggagaccaa gtttcagatg 1260ttccagctaa ttgacattgc ccggcagacg gctcagggaa tggactattt gcatgcaaag 1320aacatcatcc atagagacat gaaatccaac aatatatttc tccatgaagg cttaacagtg 1380aaaattggag attttggttt ggcaacagta aagtcacgct ggagtggttc tcagcaggtt 1440gaacaaccta ctggctctgt cctctggatg gccccagagg tgatccgaat gcaggataac 1500aacccattca gtttccagtc ggatgtctac tcctatggca tcgtattgta tgaactgatg 1560acgggggagc ttccttattc tcacatcaac aaccgagatc agatcatctt catggtgggc 1620cgaggatatg cctccccaga tcttagtaag ctatataaga actgccccaa agcaatgaag 1680aggctggtag ctgactgtgt gaagaaagta aaggaagaga ggcctctttt tccccagatc 1740ctgtcttcca ttgagctgct ccaacactct ctaccgaaga tcaaccggag cgcttccgag 1800ccatccttgc atcgggcagc ccacactgag gatatcaatg cttgcacgct gaccacgtcc 1860ccgaggctgc ctgtcttcaa gatgagcaaa gatggtaaaa agaagaaaaa gaagtcaaag 1920acaaagtgtg taattatgta a 194142646PRTHomo sapiens 42Met Thr Ser Lys Val Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr1 5 10 15Gly Pro Gln Trp Trp Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser 20 25 30Phe Ile Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile 35 40 45Phe Leu His Gly Asn Ala Thr Ser Ser Tyr Leu Trp Arg His Val Val 50 55 60Pro His Ile Glu Pro Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly65 70 75 80Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp 85 90 95His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys 100 105 110Lys Ile Ile Phe Val Gly His Asp Trp Gly Ala Ala Leu Ala Phe His 115 120 125Tyr Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val His Ala Glu 130 135 140Ser Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu145 150 155 160Glu Asp Ile Ala Leu Ile Lys Ser Glu Glu Gly Glu Lys Met Val Leu 165 170 175Glu Asn Asn Phe Phe Val Glu Thr Val Leu Pro Ser Lys Ile Met Arg 180 185 190Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu 195 200 205Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro 210 215 220Leu Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr225 230 235 240Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe Ile Glu 245 250 255Ser Asp Pro Gly Phe Phe Ser Asn Ala Ile Val Glu Gly Ala Lys Lys 260 265 270Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gln 275 280 285Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr Ile Lys Ser Phe Val Glu 290 295 300Arg Val Leu Lys Asn Glu Gln Gly Ser Ala Gly Thr Met Tyr Tyr Trp305 310 315 320Glu Ile Glu Ala Ser Glu Val Met Leu Ser Thr Arg Ile Gly Ser Gly 325 330 335Ser Phe Gly Thr Val Tyr Lys Gly Lys Trp His Gly Asp Val Ala Val 340 345 350Lys Ile Leu Lys Val Val Asp Pro Thr Pro Glu Gln Phe Gln Ala Phe 355 360 365Arg Asn Glu Val Ala Val Leu Arg Lys Thr Arg His Val Asn Ile Leu 370 375 380Leu Phe Met Gly Tyr Met Thr Lys Asp Asn Leu Ala Ile Val Thr Gln385 390 395 400Trp Cys Glu Gly Ser Ser Leu Tyr Lys His Leu His Val Gln Glu Thr 405 410 415Lys Phe Gln Met Phe Gln Leu Ile Asp Ile Ala Arg Gln Thr Ala Gln 420 425 430Gly Met Asp Tyr Leu His Ala Lys Asn Ile Ile His Arg Asp Met Lys 435 440 445Ser Asn Asn Ile Phe Leu His Glu Gly Leu Thr Val Lys Ile Gly Asp 450 455 460Phe Gly Leu Ala Thr Val Lys Ser Arg Trp Ser Gly Ser Gln Gln Val465 470 475 480Glu Gln Pro Thr Gly Ser Val Leu Trp Met Ala Pro Glu Val Ile Arg 485 490 495Met Gln Asp Asn Asn Pro Phe Ser Phe Gln Ser Asp Val Tyr Ser Tyr 500 505 510Gly Ile Val Leu Tyr Glu Leu Met Thr Gly Glu Leu Pro Tyr Ser His 515 520 525Ile Asn Asn Arg Asp Gln Ile Ile Phe Met Val Gly Arg Gly Tyr Ala 530 535 540Ser Pro Asp Leu Ser Lys Leu Tyr Lys Asn Cys Pro Lys Ala Met Lys545 550 555 560Arg Leu Val Ala Asp Cys Val Lys Lys Val Lys Glu Glu Arg Pro Leu 565 570 575Phe Pro Gln Ile Leu Ser Ser Ile Glu Leu Leu Gln His Ser Leu Pro 580 585 590Lys Ile Asn Arg Ser Ala Ser Glu Pro Ser Leu His Arg Ala Ala His 595 600 605Thr Glu Asp Ile Asn Ala Cys Thr Leu Thr Thr Ser Pro Arg Leu Pro 610 615 620Val Phe Lys Met Ser Lys Asp Gly Lys Lys Lys Lys Lys Lys Ser Lys625 630 635 640Thr Lys Cys Val Ile Met64543930DNAHomo sapiens 43cccatctctc gcaaggccag ccagaccagc gtgtacctgc aggagtggga catccccttc 60gagcaggtag agctgggcga gcccatcggg cagggccgct ggggccgggt gcaccgcggc 120cgctggcatg gcgaggtggc cattcgcctg ctggagatgg acggccacaa ccaggaccac 180ctgaagctct tcaagaaaga ggtgatgaac taccggcaga cgcggcatga gaacgtggtg 240ctcttcatgg gggcctgcat gaacccgccc cacctggcca ttatcaccag cttctgcaag 300gggcggacgt tgcactcgtt tgtgagggac cccaagacgt ctctggacat caacaagacg 360aggcaaatcg ctcaggagat catcaagggc atgggatatc ttcatgccaa gggcatcgta 420cacaaagatc tcaaatctaa gaacgtcttc tatgacaacg gcaaggtggt catcacagac 480ttcgggctgt ttgggatctc aggcgtggtc cgagagggac ggcgtgagaa ccagctaaag 540ctgtcccacg actggctgtg ctatctggcc cctgagattg tacgcgagat gacccccggg 600aaggacgagg atcagctgcc attctccaaa gctgctgatg tctatgcatt tgggactgtt 660tggtatgagc tgcaagcaag agactggccc ttgaagaacc aggctgcaga ggcatccatc 720tggcagattg gaagcgggga aggaatgaag cgtgtcctga cttctgtcag cttggggaag 780gaagtcagtg agatcctgtc ggcctgctgg gctttcgacc tgcaggagag acccagcttc 840agcctgctga tggacatgct ggagaaactt cccaagctga accggcggct ctcccaccct 900ggacacttct ggaagtcagc tgagttgtag 93044309PRTHomo sapiens 44Pro Ile Ser Arg Lys Ala Ser Gln Thr Ser Val Tyr Leu Gln Glu Trp1 5 10 15Asp Ile Pro Phe Glu Gln Val Glu Leu Gly Glu Pro Ile Gly Gln Gly 20 25 30Arg Trp Gly Arg Val His Arg Gly Arg Trp His Gly Glu Val Ala Ile 35 40 45Arg Leu Leu Glu Met Asp Gly His Asn Gln Asp His Leu Lys Leu Phe 50 55 60Lys Lys Glu Val Met Asn Tyr Arg Gln Thr Arg His Glu Asn Val Val65 70 75 80Leu Phe Met Gly Ala Cys Met Asn Pro Pro His Leu Ala Ile Ile Thr 85 90 95Ser Phe Cys Lys Gly Arg Thr Leu His Ser Phe Val Arg Asp Pro Lys 100 105 110Thr Ser Leu Asp Ile Asn Lys Thr Arg Gln Ile Ala Gln Glu Ile Ile 115 120 125Lys Gly Met Gly Tyr Leu His Ala Lys Gly Ile Val His Lys Asp Leu 130 135 140Lys Ser Lys Asn Val Phe Tyr Asp Asn Gly Lys Val Val Ile Thr Asp145 150 155 160Phe Gly Leu Phe Gly Ile Ser Gly Val Val Arg Glu Gly Arg Arg Glu 165 170 175Asn Gln Leu Lys Leu Ser His Asp Trp Leu Cys Tyr Leu Ala Pro Glu 180 185 190Ile Val Arg Glu Met Thr Pro Gly Lys Asp Glu Asp Gln Leu Pro Phe 195 200 205Ser Lys Ala Ala Asp Val Tyr Ala Phe Gly Thr Val Trp Tyr Glu Leu 210 215 220Gln Ala Arg Asp Trp Pro Leu Lys Asn Gln Ala Ala Glu Ala Ser Ile225 230 235 240Trp Gln Ile Gly Ser Gly Glu Gly Met Lys Arg Val Leu Thr Ser Val 245 250 255Ser Leu Gly Lys Glu Val Ser Glu Ile Leu Ser Ala Cys Trp Ala Phe 260 265 270Asp Leu Gln Glu Arg Pro Ser Phe Ser Leu Leu Met Asp Met Leu Glu 275 280 285Lys Leu Pro Lys Leu Asn Arg Arg Leu Ser His Pro Gly His Phe Trp 290 295 300Lys Ser Ala Glu Leu30545933DNAHomo sapiens 45atgcccatct ctcgcaaggc cagccagacc agcgtgtacc tgcaggagtg ggacatcccc 60ttcgagcagg tagagctggg cgagcccatc gggcagggcc gctggggccg ggtgcaccgc 120ggccgctggc atggcgaggt ggccattcgc ctgctggaga tggacggcca caaccaggac 180cacctgaagc tcttcaagaa agaggtgatg aactaccggc agacgcggca tgagaacgtg 240gtgctcttca tgggggcctg catgaacccg ccccacctgg ccattatcac cagcttctgc 300aaggggcgga cgttgcactc gtttgtgagg gaccccaaga cgtctctgga catcaacaag 360acgaggcaaa tcgctcagga gatcatcaag ggcatgggat atcttcatgc caagggcatc 420gtacacaaag atctcaaatc taagaacgtc ttctatgaca acggcaaggt ggtcatcaca 480gacttcgggc tgtttgggat ctcaggcgtg gtccgagagg gacggcgtga gaaccagcta 540aagctgtccc acgactggct gtgctatctg gcccctgaga ttgtacgcga gatgaccccc 600gggaaggacg aggatcagct gccattctcc aaagctgctg atgtctatgc atttgggact 660gtttggtatg agctgcaagc aagagactgg cccttgaaga accaggctgc agaggcatcc 720atctggcaga ttggaagcgg ggaaggaatg aagcgtgtcc tgacttctgt cagcttgggg 780aaggaagtca gtgagatcct gtcggcctat tgggctttcg acctgcagga gagacccagc 840ttcagcctgc tgatggacat gctggagaaa cttcccaagc tgaaccggcg gctctcccac 900cctggacact tctggaagtc agctgagttg tag 93346310PRTHomo sapiens 46Met Pro Ile Ser Arg Lys Ala Ser Gln Thr Ser Val Tyr Leu Gln Glu1 5 10 15Trp Asp Ile Pro Phe Glu Gln Val Glu Leu Gly Glu Pro Ile Gly Gln 20 25 30Gly Arg Trp Gly Arg Val His Arg Gly Arg Trp His Gly Glu Val Ala 35 40 45Ile Arg Leu Leu Glu Met Asp Gly His Asn Gln Asp His Leu Lys Leu 50 55 60Phe Lys Lys Glu Val Met Asn Tyr Arg Gln Thr Arg His Glu Asn Val65 70 75 80Val Leu Phe Met Gly Ala Cys Met Asn Pro Pro His Leu Ala Ile Ile 85 90 95Thr Ser Phe Cys Lys Gly Arg Thr Leu His Ser Phe Val Arg Asp Pro 100 105 110Lys Thr Ser Leu Asp Ile Asn Lys Thr Arg Gln Ile Ala Gln Glu Ile 115 120 125Ile Lys Gly Met Gly Tyr Leu His Ala Lys Gly Ile Val His Lys Asp 130 135 140Leu Lys Ser Lys Asn Val Phe Tyr Asp Asn Gly Lys Val Val Ile Thr145 150 155 160Asp Phe Gly Leu Phe Gly Ile Ser Gly Val Val Arg Glu Gly Arg Arg 165 170 175Glu Asn Gln Leu Lys Leu Ser His Asp Trp Leu Cys Tyr Leu Ala Pro 180 185 190Glu Ile Val Arg Glu Met Thr Pro Gly Lys Asp Glu Asp Gln Leu Pro 195 200 205Phe Ser Lys Ala Ala Asp Val Tyr Ala Phe Gly Thr Val Trp Tyr Glu 210 215 220Leu Gln Ala Arg Asp Trp Pro Leu Lys Asn Gln Ala Ala Glu Ala Ser225 230 235 240Ile Trp Gln Ile Gly Ser Gly Glu Gly Met Lys Arg Val Leu Thr Ser 245 250 255Val Ser Leu Gly Lys Glu Val Ser Glu Ile Leu Ser Ala Tyr Trp Ala 260 265 270Phe Asp Leu Gln Glu Arg Pro Ser Phe Ser Leu Leu Met Asp Met Leu 275 280 285Glu Lys Leu Pro Lys Leu Asn Arg Arg Leu Ser His Pro Gly His Phe 290 295 300Trp Lys Ser Ala Glu Leu305 310471881DNAHomo sapiens 47atgcccatct ctcgcaaggc cagccagacc agcgtgtacc tgcaggagtg ggacatcccc 60ttcgagcagg tagagctggg cgagcccatc gggcagggcc gctggggccg ggtgcaccgc 120ggccgctggc atggcgaggt ggccattcgc ctgctggaga tggacggcca caaccaggac 180cacctgaagc tcttcaagaa agaggtgatg aactaccggc agacgcggca tgagaacgtg 240gtgctcttca tgggggcctg catgaacccg ccccacctgg ccattatcac cagcttctgc 300aaggggcgga cgttgcactc gtttgtgagg gaccccaaga cgtctctgga catcaacaag 360acgaggcaaa tcgctcagga gatcatcaag ggcatgggat atcttcatgc caagggcatc 420gtacacaaag atctcaaatc taagaacgtc ttctatgaca acggcaaggt ggtcatcaca 480gacttcgggc tgtttgggat ctcaggcgtg gtccgagagg gacggcgtga gaaccagcta 540aagctgtccc acgactggct gtgctatctg gcccctgaga ttgtacgcga gatgaccccc 600gggaaggacg aggatcagct gccattctcc aaagctgctg atgtctatgc atttgggact 660gtttggtatg agctgcaagc aagagactgg cccttgaaga accaggctgc agaggcatcc 720atctggcaga ttggaagcgg ggaaggaatg aagcgtgtcc tgacttctgt cagcttgggg 780aaggaagtca gtgagatcct gtcggcctgc tgggctttcg acctgcagga gagacccagc 840ttcagcctgc tgatggacat gctggagaaa cttcccaagc tgaaccggcg gctctcccac 900cctggacact tctggaagtc agctgagttg tctagaggag gggggatgac cagcaaggtg 960tacgaccccg agcagaggaa gaggatgatc accggccccc agtggtgggc caggtgcaag 1020cagatgaacg tgctggacag cttcatcaac tactacgaca gcgagaagca cgccgagaac 1080gccgtgatct tcctgcacgg caacgccact agcagctacc tgtggaggca cgtggtgccc 1140cacatcgagc ccgtggccag gtgcatcatc cccgatctga tcggcatggg caagagcggc 1200aagagcggca acggcagcta caggctgctg gaccactaca agtacctgac cgcctggttc 1260gagctcctga acctgcccaa gaagatcatc ttcgtgggcc acgactgggg cgccgcactg 1320gccttccact acagctacga gcaccaggac aagatcaagg ccatcgtgca cgccgagagc 1380gtggttgacg tgatcgagag ctgggacgag tggccagaca tcgaggagga catcgccctg 1440atcaagagcg aggagggcga gaagatggtg ctggagaaca acttcttcgt ggagaccgtt 1500ctgcccagca agatcatgag aaagctggag cccgaggagt tcgccgccta cctggagccc 1560ttcaaggaga agggcgaggt gagaagaccc accctgagct ggcccagaga gatccccctg 1620gtgaagggcg gcaagcccga cgtggtgcag atcgtgagaa actacaacgc ctacctgaga 1680gccagcgacg acctgcccaa gatgttcatc gagagcgacc ccggcttctt cagcaacgcc 1740atcgtggagg gcgccaagaa gttccccaac accgagttcg tgaaggtgaa gggcctgcac 1800ttcagccagg aggacgcccc cgacgagatg ggcaagtaca tcaagagctt cgtggagaga 1860gtgctgaaga acgagcagta g 188148626PRTHomo sapiens 48Met Pro Ile Ser Arg Lys Ala Ser Gln Thr Ser Val Tyr Leu Gln Glu1 5 10 15Trp Asp Ile Pro Phe Glu Gln Val Glu Leu Gly Glu Pro Ile Gly Gln 20 25 30Gly Arg Trp Gly Arg Val His Arg Gly Arg Trp His Gly Glu Val Ala 35 40 45Ile Arg Leu Leu Glu Met Asp Gly His

Asn Gln Asp His Leu Lys Leu 50 55 60Phe Lys Lys Glu Val Met Asn Tyr Arg Gln Thr Arg His Glu Asn Val65 70 75 80Val Leu Phe Met Gly Ala Cys Met Asn Pro Pro His Leu Ala Ile Ile 85 90 95Thr Ser Phe Cys Lys Gly Arg Thr Leu His Ser Phe Val Arg Asp Pro 100 105 110Lys Thr Ser Leu Asp Ile Asn Lys Thr Arg Gln Ile Ala Gln Glu Ile 115 120 125Ile Lys Gly Met Gly Tyr Leu His Ala Lys Gly Ile Val His Lys Asp 130 135 140Leu Lys Ser Lys Asn Val Phe Tyr Asp Asn Gly Lys Val Val Ile Thr145 150 155 160Asp Phe Gly Leu Phe Gly Ile Ser Gly Val Val Arg Glu Gly Arg Arg 165 170 175Glu Asn Gln Leu Lys Leu Ser His Asp Trp Leu Cys Tyr Leu Ala Pro 180 185 190Glu Ile Val Arg Glu Met Thr Pro Gly Lys Asp Glu Asp Gln Leu Pro 195 200 205Phe Ser Lys Ala Ala Asp Val Tyr Ala Phe Gly Thr Val Trp Tyr Glu 210 215 220Leu Gln Ala Arg Asp Trp Pro Leu Lys Asn Gln Ala Ala Glu Ala Ser225 230 235 240Ile Trp Gln Ile Gly Ser Gly Glu Gly Met Lys Arg Val Leu Thr Ser 245 250 255Val Ser Leu Gly Lys Glu Val Ser Glu Ile Leu Ser Ala Cys Trp Ala 260 265 270Phe Asp Leu Gln Glu Arg Pro Ser Phe Ser Leu Leu Met Asp Met Leu 275 280 285Glu Lys Leu Pro Lys Leu Asn Arg Arg Leu Ser His Pro Gly His Phe 290 295 300Trp Lys Ser Ala Glu Leu Ser Arg Gly Gly Gly Met Thr Ser Lys Val305 310 315 320Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr Gly Pro Gln Trp Trp 325 330 335Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser Phe Ile Asn Tyr Tyr 340 345 350Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile Phe Leu His Gly Asn 355 360 365Ala Thr Ser Ser Tyr Leu Trp Arg His Val Val Pro His Ile Glu Pro 370 375 380Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly Met Gly Lys Ser Gly385 390 395 400Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu 405 410 415Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys Ile Ile Phe Val 420 425 430Gly His Asp Trp Gly Ala Ala Leu Ala Phe His Tyr Ser Tyr Glu His 435 440 445Gln Asp Lys Ile Lys Ala Ile Val His Ala Glu Ser Val Val Asp Val 450 455 460Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu Glu Asp Ile Ala Leu465 470 475 480Ile Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe 485 490 495Val Glu Thr Val Leu Pro Ser Lys Ile Met Arg Lys Leu Glu Pro Glu 500 505 510Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg 515 520 525Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro Leu Val Lys Gly Gly 530 535 540Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr Asn Ala Tyr Leu Arg545 550 555 560Ala Ser Asp Asp Leu Pro Lys Met Phe Ile Glu Ser Asp Pro Gly Phe 565 570 575Phe Ser Asn Ala Ile Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu 580 585 590Phe Val Lys Val Lys Gly Leu His Phe Ser Gln Glu Asp Ala Pro Asp 595 600 605Glu Met Gly Lys Tyr Ile Lys Ser Phe Val Glu Arg Val Leu Lys Asn 610 615 620Glu Gln625491881DNAHomo sapiens 49atgcccatct ctcgcaaggc cagccagacc agcgtgtacc tgcaggagtg ggacatcccc 60ttcgagcagg tagagctggg cgagcccatc gggcagggcc gctggggccg ggtgcaccgc 120ggccgctggc atggcgaggt ggccattcgc ctgctggaga tggacggcca caaccaggac 180cacctgaagc tcttcaagaa agaggtgatg aactaccggc agacgcggca tgagaacgtg 240gtgctcttca tgggggcctg catgaacccg ccccacctgg ccattatcac cagcttctgc 300aaggggcgga cgttgcactc gtttgtgagg gaccccaaga cgtctctgga catcaacaag 360acgaggcaaa tcgctcagga gatcatcaag ggcatgggat atcttcatgc caagggcatc 420gtacacaaag atctcaaatc taagaacgtc ttctatgaca acggcaaggt ggtcatcaca 480gacttcgggc tgtttgggat ctcaggcgtg gtccgagagg gacggcgtga gaaccagcta 540aagctgtccc acgactggct gtgctatctg gcccctgaga ttgtacgcga gatgaccccc 600gggaaggacg aggatcagct gccattctcc aaagctgctg atgtctatgc atttgggact 660gtttggtatg agctgcaagc aagagactgg cccttgaaga accaggctgc agaggcatcc 720atctggcaga ttggaagcgg ggaaggaatg aagcgtgtcc tgacttctgt cagcttgggg 780aaggaagtca gtgagatcct gtcggcctat tgggctttcg acctgcagga gagacccagc 840ttcagcctgc tgatggacat gctggagaaa cttcccaagc tgaaccggcg gctctcccac 900cctggacact tctggaagtc agctgagttg tctagaggag gggggatgac cagcaaggtg 960tacgaccccg agcagaggaa gaggatgatc accggccccc agtggtgggc caggtgcaag 1020cagatgaacg tgctggacag cttcatcaac tactacgaca gcgagaagca cgccgagaac 1080gccgtgatct tcctgcacgg caacgccact agcagctacc tgtggaggca cgtggtgccc 1140cacatcgagc ccgtggccag gtgcatcatc cccgatctga tcggcatggg caagagcggc 1200aagagcggca acggcagcta caggctgctg gaccactaca agtacctgac cgcctggttc 1260gagctcctga acctgcccaa gaagatcatc ttcgtgggcc acgactgggg cgccgcactg 1320gccttccact acagctacga gcaccaggac aagatcaagg ccatcgtgca cgccgagagc 1380gtggttgacg tgatcgagag ctgggacgag tggccagaca tcgaggagga catcgccctg 1440atcaagagcg aggagggcga gaagatggtg ctggagaaca acttcttcgt ggagaccgtt 1500ctgcccagca agatcatgag aaagctggag cccgaggagt tcgccgccta cctggagccc 1560ttcaaggaga agggcgaggt gagaagaccc accctgagct ggcccagaga gatccccctg 1620gtgaagggcg gcaagcccga cgtggtgcag atcgtgagaa actacaacgc ctacctgaga 1680gccagcgacg acctgcccaa gatgttcatc gagagcgacc ccggcttctt cagcaacgcc 1740atcgtggagg gcgccaagaa gttccccaac accgagttcg tgaaggtgaa gggcctgcac 1800ttcagccagg aggacgcccc cgacgagatg ggcaagtaca tcaagagctt cgtggagaga 1860gtgctgaaga acgagcagta g 188150626PRTHomo sapiens 50Met Pro Ile Ser Arg Lys Ala Ser Gln Thr Ser Val Tyr Leu Gln Glu1 5 10 15Trp Asp Ile Pro Phe Glu Gln Val Glu Leu Gly Glu Pro Ile Gly Gln 20 25 30Gly Arg Trp Gly Arg Val His Arg Gly Arg Trp His Gly Glu Val Ala 35 40 45Ile Arg Leu Leu Glu Met Asp Gly His Asn Gln Asp His Leu Lys Leu 50 55 60Phe Lys Lys Glu Val Met Asn Tyr Arg Gln Thr Arg His Glu Asn Val65 70 75 80Val Leu Phe Met Gly Ala Cys Met Asn Pro Pro His Leu Ala Ile Ile 85 90 95Thr Ser Phe Cys Lys Gly Arg Thr Leu His Ser Phe Val Arg Asp Pro 100 105 110Lys Thr Ser Leu Asp Ile Asn Lys Thr Arg Gln Ile Ala Gln Glu Ile 115 120 125Ile Lys Gly Met Gly Tyr Leu His Ala Lys Gly Ile Val His Lys Asp 130 135 140Leu Lys Ser Lys Asn Val Phe Tyr Asp Asn Gly Lys Val Val Ile Thr145 150 155 160Asp Phe Gly Leu Phe Gly Ile Ser Gly Val Val Arg Glu Gly Arg Arg 165 170 175Glu Asn Gln Leu Lys Leu Ser His Asp Trp Leu Cys Tyr Leu Ala Pro 180 185 190Glu Ile Val Arg Glu Met Thr Pro Gly Lys Asp Glu Asp Gln Leu Pro 195 200 205Phe Ser Lys Ala Ala Asp Val Tyr Ala Phe Gly Thr Val Trp Tyr Glu 210 215 220Leu Gln Ala Arg Asp Trp Pro Leu Lys Asn Gln Ala Ala Glu Ala Ser225 230 235 240Ile Trp Gln Ile Gly Ser Gly Glu Gly Met Lys Arg Val Leu Thr Ser 245 250 255Val Ser Leu Gly Lys Glu Val Ser Glu Ile Leu Ser Ala Tyr Trp Ala 260 265 270Phe Asp Leu Gln Glu Arg Pro Ser Phe Ser Leu Leu Met Asp Met Leu 275 280 285Glu Lys Leu Pro Lys Leu Asn Arg Arg Leu Ser His Pro Gly His Phe 290 295 300Trp Lys Ser Ala Glu Leu Ser Arg Gly Gly Gly Met Thr Ser Lys Val305 310 315 320Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr Gly Pro Gln Trp Trp 325 330 335Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser Phe Ile Asn Tyr Tyr 340 345 350Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile Phe Leu His Gly Asn 355 360 365Ala Thr Ser Ser Tyr Leu Trp Arg His Val Val Pro His Ile Glu Pro 370 375 380Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly Met Gly Lys Ser Gly385 390 395 400Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu 405 410 415Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys Ile Ile Phe Val 420 425 430Gly His Asp Trp Gly Ala Ala Leu Ala Phe His Tyr Ser Tyr Glu His 435 440 445Gln Asp Lys Ile Lys Ala Ile Val His Ala Glu Ser Val Val Asp Val 450 455 460Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu Glu Asp Ile Ala Leu465 470 475 480Ile Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe 485 490 495Val Glu Thr Val Leu Pro Ser Lys Ile Met Arg Lys Leu Glu Pro Glu 500 505 510Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg 515 520 525Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro Leu Val Lys Gly Gly 530 535 540Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr Asn Ala Tyr Leu Arg545 550 555 560Ala Ser Asp Asp Leu Pro Lys Met Phe Ile Glu Ser Asp Pro Gly Phe 565 570 575Phe Ser Asn Ala Ile Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu 580 585 590Phe Val Lys Val Lys Gly Leu His Phe Ser Gln Glu Asp Ala Pro Asp 595 600 605Glu Met Gly Lys Tyr Ile Lys Ser Phe Val Glu Arg Val Leu Lys Asn 610 615 620Glu Gln625511182DNAHomo sapiens 51atgcccaaga agaagccgac gcccatccag ctgaacccgg cccccgacgg ctctgcagtt 60aacgggacca gctctgcgga gaccaacttg gaggccttgc agaagaagct ggaggagcta 120gagcttgatg agcagcagcg aaagcgcctt gaggcctttc ttacccagaa gcagaaggtg 180ggagaactga aggatgacga ctttgagaag atcagtgagc tgggggctgg caatggcggt 240gtggtgttca aggtctccca caagccttct ggcctggtca tggccagaaa gctaattcat 300ctggagatca aacccgcaat ccggaaccag atcataaggg agctgcaggt tctgcatgag 360tgcaactctc cgtacatcgt gggcttctat ggtgcgttct acagcgatgg cgagatcagt 420atctgcatgg agcacatgga tggaggttct ctggatcaag tcctgaagaa agctggaaga 480attcctgaac aaattttagg aaaagttagc attgctgtaa taaaaggcct gacatatctg 540agggagaagc acaagatcat gcacagagat gtcaagccct ccaacatcct agtcaactcc 600cgtggggaga tcaagctctg tgactttggg gtcagcgggc agctcatcga ctccatggcc 660aactccttcg tgggcacaag gtcctacatg tcgccagaaa gactccaggg gactcattac 720tctgtgcagt cagacatctg gagcatggga ctgtctctgg tagagatggc ggttgggagg 780tatcccatcc ctcctccaga tgccaaggag ctggagctga tgtttgggtg ccaggtggaa 840ggagatgcgg ctgagacccc acccaggcca aggacccccg ggaggcccct tagctcatac 900ggaatggaca gccgacctcc catggcaatt tttgagttgt tggattacat agtcaacgag 960cctcctccaa aactgcccag tggagtgttc agtctggaat ttcaagattt tgtgaataaa 1020tgcttaataa aaaaccccgc agagagagca gatttgaagc aactcatggt tcatgctttt 1080atcaagagat ctgatgctga ggaagtggat tttgcaggtt ggctctgctc caccatcggc 1140cttaaccagc ccagcacacc aacccatgct gctggcgtct aa 118252393PRTHomo sapiens 52Met Pro Lys Lys Lys Pro Thr Pro Ile Gln Leu Asn Pro Ala Pro Asp1 5 10 15Gly Ser Ala Val Asn Gly Thr Ser Ser Ala Glu Thr Asn Leu Glu Ala 20 25 30Leu Gln Lys Lys Leu Glu Glu Leu Glu Leu Asp Glu Gln Gln Arg Lys 35 40 45Arg Leu Glu Ala Phe Leu Thr Gln Lys Gln Lys Val Gly Glu Leu Lys 50 55 60Asp Asp Asp Phe Glu Lys Ile Ser Glu Leu Gly Ala Gly Asn Gly Gly65 70 75 80Val Val Phe Lys Val Ser His Lys Pro Ser Gly Leu Val Met Ala Arg 85 90 95Lys Leu Ile His Leu Glu Ile Lys Pro Ala Ile Arg Asn Gln Ile Ile 100 105 110Arg Glu Leu Gln Val Leu His Glu Cys Asn Ser Pro Tyr Ile Val Gly 115 120 125Phe Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Ile Ser Ile Cys Met Glu 130 135 140His Met Asp Gly Gly Ser Leu Asp Gln Val Leu Lys Lys Ala Gly Arg145 150 155 160Ile Pro Glu Gln Ile Leu Gly Lys Val Ser Ile Ala Val Ile Lys Gly 165 170 175Leu Thr Tyr Leu Arg Glu Lys His Lys Ile Met His Arg Asp Val Lys 180 185 190Pro Ser Asn Ile Leu Val Asn Ser Arg Gly Glu Ile Lys Leu Cys Asp 195 200 205Phe Gly Val Ser Gly Gln Leu Ile Asp Ser Met Ala Asn Ser Phe Val 210 215 220Gly Thr Arg Ser Tyr Met Ser Pro Glu Arg Leu Gln Gly Thr His Tyr225 230 235 240Ser Val Gln Ser Asp Ile Trp Ser Met Gly Leu Ser Leu Val Glu Met 245 250 255Ala Val Gly Arg Tyr Pro Ile Pro Pro Pro Asp Ala Lys Glu Leu Glu 260 265 270Leu Met Phe Gly Cys Gln Val Glu Gly Asp Ala Ala Glu Thr Pro Pro 275 280 285Arg Pro Arg Thr Pro Gly Arg Pro Leu Ser Ser Tyr Gly Met Asp Ser 290 295 300Arg Pro Pro Met Ala Ile Phe Glu Leu Leu Asp Tyr Ile Val Asn Glu305 310 315 320Pro Pro Pro Lys Leu Pro Ser Gly Val Phe Ser Leu Glu Phe Gln Asp 325 330 335Phe Val Asn Lys Cys Leu Ile Lys Asn Pro Ala Glu Arg Ala Asp Leu 340 345 350Lys Gln Leu Met Val His Ala Phe Ile Lys Arg Ser Asp Ala Glu Glu 355 360 365Val Asp Phe Ala Gly Trp Leu Cys Ser Thr Ile Gly Leu Asn Gln Pro 370 375 380Ser Thr Pro Thr His Ala Ala Gly Val385 390531915DNAHomo sapiens 53caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 60ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 120ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 180caccctcgtg accaccctga gctacggcgt gcagtgcttc agccgctacc ccgaccacat 240gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 300cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 360cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 420gcacaagctg gagtacaact acaaccccca caacgtctat atcatggccg acaagcagaa 480gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 540cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 600ccactacctg ttcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 660ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa 720gggatccgcc ggtaccccca agaagaagcc gacgcccatc cagctgaacc cggcccccga 780cggctctgca gttaacggga ccagctctgc ggagaccaac ttggaggcct tgcagaagaa 840gctggaggag ctagagcttg atgagcagca gcgaaagcgc cttgaggcct ttcttaccca 900gaagcagaag gtgggagaac tgaaggatga cgactttgag aagatcagtg agctgggggc 960tggcaatggc ggtgtggtgt tcaaggtctc ccacaagcct tctggcctgg tcatggccag 1020aaagctaatt catctggaga tcaaacccgc aatccggaac cagatcataa gggagctgca 1080ggttctgcat gagtgcaact ctccgtacat cgtgggcttc tatggtgcgt tctacagcga 1140tggcgagatc agtatctgca tggagcacat ggatggaggt tctctggatc aagtcctgaa 1200gaaagctgga agaattcctg aacaaatttt aggaaaagtt agcattgctg taataaaagg 1260cctgacatat ctgagggaga agcacaagat catgcacaga gatgtcaagc cctccaacat 1320cctagtcaac tcccgtgggg agatcaagct ctgtgacttt ggggtcagcg ggcagctcat 1380cgactccatg gccaactcct tcgtgggcac aaggtcctac atgtcgccag aaagactcca 1440ggggactcat tactctgtgc agtcagacat ctggagcatg ggactgtctc tggtagagat 1500ggcggttggg aggtatccca tccctcctcc agatgccaag gagctggagc tgatgtttgg 1560gtgccaggtg gaaggagatg cggctgagac cccacccagg ccaaggaccc ccgggaggcc 1620ccttagctca tacggaatgg acagccgacc tcccatggca atttttgagt tgttggatta 1680catagtcaac gagcctcctc caaaactgcc cagtggagtg ttcagtctgg aatttcaaga 1740ttttgtgaat aaatgcttaa taaaaaaccc cgcagagaga gcagatttga agcaactcat 1800ggttcatgct tttatcaaga gatctgatgc tgaggaagtg gattttgcag gttggctctg 1860ctccaccatc ggccttaacc agcccagcac accaacccat gctgctggcg tctaa 191554636PRTHomo sapiens 54Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu1 5 10 15Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60Leu Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys65 70 75 80Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140Asn Tyr Asn Pro His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150 155 160Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Phe Thr Gln Ser Ala Leu 195 200 205Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly225 230 235 240Ser Ala Gly Thr Pro Lys Lys Lys Pro Thr Pro Ile Gln Leu Asn Pro 245 250 255Ala Pro Asp Gly Ser Ala Val Asn Gly Thr Ser Ser Ala Glu Thr Asn 260 265 270Leu Glu Ala Leu Gln Lys Lys Leu Glu Glu Leu Glu Leu Asp Glu Gln 275 280 285Gln Arg Lys Arg Leu Glu Ala Phe Leu Thr Gln Lys Gln Lys Val Gly 290 295 300Glu Leu Lys Asp Asp Asp Phe Glu Lys Ile Ser Glu Leu Gly Ala Gly305 310 315 320Asn Gly Gly Val Val Phe Lys Val Ser His Lys Pro Ser Gly Leu Val 325 330 335Met Ala Arg Lys Leu Ile His Leu Glu Ile Lys Pro Ala Ile Arg Asn 340 345 350Gln Ile Ile Arg Glu Leu Gln Val Leu His Glu Cys Asn Ser Pro Tyr 355 360 365Ile Val Gly Phe Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Ile Ser Ile 370 375 380Cys Met Glu His Met Asp Gly Gly Ser Leu Asp Gln Val Leu Lys Lys385 390 395 400Ala Gly Arg Ile Pro Glu Gln Ile Leu Gly Lys Val Ser Ile Ala Val 405 410 415Ile Lys Gly Leu Thr Tyr Leu Arg Glu Lys His Lys Ile Met His Arg 420 425 430Asp Val Lys Pro Ser Asn Ile Leu Val Asn Ser Arg Gly Glu Ile Lys 435 440 445Leu Cys Asp Phe Gly Val Ser Gly Gln Leu Ile Asp Ser Met Ala Asn 450 455 460Ser Phe Val Gly Thr Arg Ser Tyr Met Ser Pro Glu Arg Leu Gln Gly465 470 475 480Thr His Tyr Ser Val Gln Ser Asp Ile Trp Ser Met Gly Leu Ser Leu 485 490 495Val Glu Met Ala Val Gly Arg Tyr Pro Ile Pro Pro Pro Asp Ala Lys 500 505 510Glu Leu Glu Leu Met Phe Gly Cys Gln Val Glu Gly Asp Ala Ala Glu 515 520 525Thr Pro Pro Arg Pro Arg Thr Pro Gly Arg Pro Leu Ser Ser Tyr Gly 530 535 540Met Asp Ser Arg Pro Pro Met Ala Ile Phe Glu Leu Leu Asp Tyr Ile545 550 555 560Val Asn Glu Pro Pro Pro Lys Leu Pro Ser Gly Val Phe Ser Leu Glu 565 570 575Phe Gln Asp Phe Val Asn Lys Cys Leu Ile Lys Asn Pro Ala Glu Arg 580 585 590Ala Asp Leu Lys Gln Leu Met Val His Ala Phe Ile Lys Arg Ser Asp 595 600 605Ala Glu Glu Val Asp Phe Ala Gly Trp Leu Cys Ser Thr Ile Gly Leu 610 615 620Asn Gln Pro Ser Thr Pro Thr His Ala Ala Gly Val625 630 6355536DNAArtificial SequenceSynthetic 55ggggtaccat ggatgattgg gagattcctg atgggc 365692DNAArtificial SequenceSynthetic 56gctctagatt acataattac acactttgtc tttgacttct ttttcttctt tttaccatct 60ttgctcatct tgtggacagg aaacgcacca ta 925732DNAArtificial SequenceSynthetic 57ggggtaccat gtattattgg gaaatagaag cc 325892DNAArtificial SequenceSynthetic 58gctctagatt acataattac acactttgtc tttgacttct ttttcttctt tttaccatct 60ttgctcatct tgaagacagg cagcctcggg ga 925932DNAArtificial SequenceSynthetic 59ggggtacccc catctctcgc aaggccagcc ag 326033DNAArtificial SequenceSynthetic 60gctctagact acaactcagc tgacttccag aag 336132DNAArtificial SequenceSynthetic 61ggggtacccc caagaagaag ccgacgccca tc 326232DNAArtificial SequenceSynthetic 62gctctagatt agacgccagc agcatgggtt gg 326324DNAArtificial SequenceSynthetic 63gggtttccgc tgtcaaacat gtgg 246424DNAArtificial SequenceSynthetic 64tattgttacc cagtggtgtg aggg 246524DNAArtificial SequenceSynthetic 65acacctaatg tccacatggt cagc 246624DNAArtificial SequenceSynthetic 66tttccagtcg gatgtctact ccta 246757DNAArtificial SequenceSynthetic 67aatgaagtag gagtactcag gaaaacacat catgtgaata tcctactctt catgggc 576857DNAArtificial SequenceSynthetic 68gcccatgaag agtaggatat tcacatgatg tgttttcctg agtactccta cttcatt 576951DNAArtificial SequenceSynthetic 69gaagtcagtg agatcctgtc ggcctattgg gctttcgacc tgcaggagag a 517051DNAArtificial SequenceSynthetic 70tctctcctgc aggtcgaaag cccaataggc cgacaggatc tcactgactt c 517121DNAArtificial SequenceSynthetic 71cgtaaacggc cacaagttca g 217221DNAArtificial SequenceSynthetic 72acgaactcca gcaggaccat g 217321DNAArtificial SequenceSynthetic 73aatacaaact ggtcgtcgtt g 217421DNAArtificial SequenceSynthetic 74aatctacgat tcggcttgtt c 217521DNAArtificial SequenceSynthetic 75tttggacaga tctatatgca g 217619DNAArtificial SequenceSynthetic 76tcggttcaaa ggtctccag 197720DNAArtificial SequenceSynthetic 77ccgattgtgt cacccctaat 207820DNAArtificial SequenceSynthetic 78ccacttgagc acatcgctaa 207925DNAArtificial SequenceSynthetic 79gacacttcct agtgcggctc gcgtg 258025DNAArtificial SequenceSynthetic 80gtaatcatac atctggtaat ctacc 258121DNAArtificial SequenceSynthetic 81tgactgagcg cgagaacaat g 218221DNAArtificial SequenceSynthetic 82tcttctgcct gcatatcgga c 218323DNAArtificial SequenceSynthetic 83gacagtcgat aaggaagagc tgg 238420DNAArtificial SequenceSynthetic 84tcgttcagtg tgtccagctc 20

Claims

1. A composition comprising: an aqueous solution of RAF/RAF homodimer.

2-4. (canceled)

5. A composition comprising: an aqueous solution of RAF/KSR heterodimer.

6-40. (canceled)

41. A method of detecting the presence of a mutation in a RAF kinase domain, the method comprising: a) providing a WT RAF kinase domain and a suspected mutant RAF kinase domain, each domain having a cysteine residue located at its N-terminus; b) incubating the WT RAF kinase domain and the suspected mutant RAF kinase domain with different cross-linking detectable labels; c) incubating together equimolar amounts of the labeled WT RAF kinase domain and detecting a signal from the detectable label so as to provide a dimerization reference signal; and d) incubating equimolar amounts of the labeled suspected mutant B-RAF kinase domain and detecting a signal from the detectable labels, an absent signal or a reduce signal compared to that of the dimerization reference signal being an indication that a mutant B-RAF kinase domain is present.

42. A method of monitoring the formation of RAF/RAF or RAF/KSR kinase domain dimers to detect mutations inhibiting dimerization or drug-like molecules interfering with dimerization, the method comprising: a) using either (i) a RAF kinase domain or (ii) a KSR kinase domain at either of their N- or C-termini to a BRET donor or a BRET acceptor to produce donor labeled and acceptor labeled fusion proteins; b) expressing the fusion proteins to identify combinations that provide specific BRET signals; c) introducing dimer interface mutations into either of the labeled fusion proteins; d) expressing the labeled mutated fusion proteins with WT RAF or KSR kinase domains; e) measuring the BRET signals, a loss or significant reduction of the BRET signal using dimer interface mutations as opposed to mutations remote from the interface, being an indication that a specific BRET signal which depends on the RAF/RAF or RAF/KSR dimerization interface has been obtained.

43. The method, according to claim 42, in which the BRET donor is renilla luciferase variant II or rlucII.

44. The method, according to claim 42, in which the BRET acceptor is GFP10.

45. The method, according to claim 42, in which the acceptor label is Yellow Fluorescent Protein (YFP).

46. The method, according to claim 42, in which the donor labeled fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO. 24, SEQ ID NO. 34, SEQ ID NO. 42 and SEQ ID NO. 48.

47. The method, according to claim 42, in which the acceptor labeled fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO. 22, SEQ ID NO. 30, SEQ ID NO. 40 and SEQ ID NO. 54.

48. The method, according to claim 42, in which the donor labeled mutated fusion proteins comprise sequences SEQ ID NO. 36 and SEQ ID NO. 50.

49. The method, according to claim 42, in which the acceptor labeled mutated fusion proteins comprises a sequence of SEQ ID NO. 32.

50. A method of identifying a potential inhibitor of RAF/RAF homodimerization, the method comprising. a) fusing a RAF kinase domain at either of its N- or C-termini to a BRET donor or a BRET acceptor to produce donor labeled and acceptor labeled fusion proteins; b) expressing the fusion proteins to identify combinations that provide specific BRET signals; c) introducing dimer interface mutations into either of the labeled fusion proteins; d) expressing the labeled mutated fusion proteins with WT RAF kinase domains; e) contacting the interface with the potential inhibitor; and f) measuring the BRET signals, a loss or significant reduction of the BRET signal for the wild-type RAF/RAF BRET pair being an indication that the inhibitor is specifically bound to the interface.

51. A method of identifying a potential inhibitor of RAF/RAF homodimerization, the method comprising: a) detectably labeling at least one of the dimerization interface residues to generate a detectably labeled RAF monomer; b) incubating the detectably labeled RAF monomer with the potential inhibitor and a non-labeled RAF monomer; c) measuring a signal from the detectable label; d) contacting the RAF dimerization interface with the inhibitor to determine the ability of the potential inhibitor to inhibit RAF/RAF homodimerization.

52. The method, according to claim 51, in which the interface residues include H449, G450, R481, L487, F488, M489, Y538, A541 or K542.

53. A method of identifying a potential inhibitor of RAF/RAF homodimerization, the method comprising: a) fusing a RAF kinase domain at either of its N- or C-termini to a BRET donor or a BRET acceptor to produce donor labeled and acceptor labeled fusion proteins; b) expressing the fusion proteins to identify combinations that provide specific BRET signals; c) introducing dimer interface mutations into either of the labeled fusion proteins; d) expressing the labeled mutated fusion proteins with WT RAF kinase domains; e) contacting the interface with the potential inhibitor; and f) measuring the BRET signals, a loss or significant reduction of the BRET signal for the wild-type RAF/RAF BRET pair being an indication that the inhibitor is specifically bound to the interface.

54. A method of identifying compounds that bind to a RAF or a KSR dimerization interface, the method comprising: a) contacting the interface with a probe to form a probe: interface complex, the probe being displaceable by a test compound; b) measuring a signal from the probe so as to establish a reference level; c) incubating the probe:interface complex with the test compound; d) measuring the signal from the probe; e) comparing the signal from step d) with the reference level, a modulation of the signal being an indication that the test compound binds to the BIR domain, wherein the probe is a compound labeled with a detectable label or an affinity label.

55. A method of identifying a potential inhibitor of RAF/RAF homodimerization, the method comprising: a) using the atomic coordinates of at least one of the interface residues to generate a three dimensional structure of a RAF dimerization interface; b) using the three-dimensional structure to design or select the potential inhibitor; c) synthesizing the inhibitor; and d) contacting the RAF dimierization interface with the inhibitor to determine the ability of the potential inhibitor to inhibit RAF/RAF homodimerization.

56. The method, according to claim 55, in which the interface residues are H449, G450, R481, L487, F488, M489, Y538, A541 or K542.

57. A method of identifying a potential inhibitor of RAF/KSR heterodimerization, the method comprising: a) using the atomic coordinates of at least one of interface residues to generate a three dimensional structure of a KSR dimerization interface; b) using the three-dimensional structure to design or select the potential inhibitor; c) synthesizing the inhibitor; and d) contacting the KSR dimerization interface with the inhibitor to determine the ability of the potential inhibitor to inhibit RAF/KSR heterodimerization.

58. The method, according to claim 57, in which the interface residues are H699, G700, R732, L738, F739, M740, Y790, A793 or R794.

59-61. (canceled)

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