Abstract:
Mutant KSR proteins are disclosed. The mutants include single amino acid substitutions, leading to either a loss of kinase activity or a loss of scaffolding activity. Also disclosed are methods of screening compounds for inhibitors of KSR kinase activity or KSR scaffolding activity. In some embodiments, the screening methods include protein complementation assays in which nucleic acids encoding fusion constructs comprising enzyme portions and kinase dimerization domains are expressed in cells. Inhibitors of dimerization can be indicated by loss of enzyme activity.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    The present application is a U.S. Non-Provisional claiming priority to U.S. Provisional Patent Application Ser. No. 61/462,797 filed Feb. 8, 2011, which is herein incorporated by reference in its entirety. 
     
    
     INCORPORATION BY REFERENCE OF SEQUENCE LISTING  
       [0002]    The Sequence Listing, which is a part of the present disclosure, includes a computer readable form and a written sequence listing comprising nucleotide and/or amino acid sequences. The sequence listing information recorded in computer readable form is identical to the written sequence listing. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety. 
       INTRODUCTION  
       [0003]    Mutations in RAS and BRAF represent the majority of oncogenic mutations in most human cancers including malignant melanoma (Brose, M. S. et al., Cancer Res. 62, 6997-7000, 2002). While BRAF-specific inhibitors have shown promise in the clinic, some of them have a paradoxical effect, inhibiting cells with mutated BRAF but accelerating the growth of cells with mutated RAS (Hatzivassiliou, G., et at., Nature 464, 431-435, 2010; Heidorn, S. J., et al., Cell 140, 209-221, 2010; Poulikakos, P. I., et al., Nature 464, 427-430; 2010) About 40% of human melanomas comprise a constitutively active mutation of BRAF, i.e., BRAF V600E. Recent studies suggest that in RAS transformed cells, these BRAF-specific inhibitors can bind to and induce the closed, active conformation of the wild-type forms of BRAF and CRAF RAS (Hatzivassiliou, G., et al., Nature 464, 431-435, 2010; Tsai, J., et al., Proc. Natl. Acad. Sci. USA 105, 3041-3046, 2008). This allows dimers between BRAF and CRAF to form, and through a mechanism that is unknown, dimerization results in the activation of CRAF and downstream signaling pathways. 
         [0004]    Interestingly, one of the drugs tested, PLX4720, induces MEK activation in RAS transformed cells and also induces the closed, active conformation of BRAF but does not induce BRAF/CRAF dimers (Hatzivassiliou, G., et al., Nature 464, 431-435, 2010; Poulikakos, P. I., et al., Nature 464, 427-430, 2010; Tsai, J., et al., Proc. Nat&#39;l. Acad. Sci. USA 105, 3041-3046, 2008). These findings suggest that the mechanism of activation might not be related to BRAF/CRAF dimers but to other proteins that bind to the closed active conformation of BRAF and CRAF. The scaffold protein Kinase Suppressor of RAS (KSR) can form dimers with both RAF isoforms (McKay, M. M., et al., Proc. Natl. Acad. Sci. USA 106, 11022-11027, 2009; Rajakulendran, T., et al., Nature 461, 542-545, 2009). 
         [0005]    KSR was first discovered in  Drosophila  and  C. elegans  as a positive effector of the RAS/MAP kinase signaling pathway (Kornfeld, K., et all., Cell 83, 903-913, 1995; Sundaram, M., et al., Cell 83, 889-901, 1995; Therrien, M., et al., Cell 83, 879-888, 1995). Genetic epistasis experiments place KSR in a position either upstream or parallel with RAF. While KSR is closely related to RAF, the absence of the critical catalytic lysine (in mammalian forms of KSR) and the lack of any convincing evidence for in vitro kinase activity (Michaud, N. R., et al., Proc. Natl. Acad. Sci. USA 94, 12792-12796, 1997) has led to the model that KSR functions mainly as a non-catalytic scaffold for the RAS/MAP kinase signaling pathway. KSR forms stable complexes with RAF and MEK, suggesting that it may function to facilitate MEK phosphorylation by RAF. A recent paper from the Morrison group suggests that BRAF, MEK and KSR form a ternary complex mediated by MEK bound to KSR and an N-terminal domain (CA1) of KSR (McKay, M. M., et al., Proc. Natl. Acad. Sci. USA 106, 11022-11027, 2009). Based on the asymmetric packing of RAF molecules in the crystal structures, Therrien&#39;s group suggested that a side-to-side dimer interface, conserved in KSR and in all isoforms of RAF, mediates the ability of RAF to form dimers with itself or with KSR (Rajakulendran, T., et al., Nature 461, 542-545, 2009). BRAF appears to activate CRAF via dimerization and without requiring kinase activity (Hatzivassiliou, G., et al., Nature 464, 431-435, 2010; Heidorn, S. J., et al., Cell 140, 209-221, 2010; Poulikakos, P.I., et al., Nature 464, 427-430, 2010). 
         [0006]    Understanding how the MAP kinase signaling complex functions has been particularly challenging given there are at least three kinases in the cascade and an even larger number of components identified by genetic epistasis whose function is still unknown (Rubin, G. M., et al. Cold Spring Harb. Symp. Quant. Biol. 62, 347-352, 1997; Therrien, M., et al., Genetics 156, 1231-1242, 2000). While the canonical pathway involving RAS, RAF, MEK and ERK has been known for over a decade, important details about the mechanism of activation are still unknown especially regarding the role of KSR and the function of the different RAF isoforms. 
         [0007]    Recent data suggest that the roles of the three RAF isoforms, ARAF, BRAF and CRAF are more complex than initially thought (Dhomen, N., et al., Curr. Opin. Genet. Dev. 17, 31-39, 2007). BRAF and CRAF are widely expressed, and are expressed together in most cells, while ARAF expression is restricted mainly to germ cells (Niault, T. S., et al., Carcinogenesis 31, 1165-1174, 2010). Originally, each RAF isoform was thought to phosphorylate MEK independently. Recent studies, however suggest that the RAF isoforms have a hierarchy, with BRAF able to activate CRAF but not the other way around (Wan, P. T., et al. Cell 116, 855-867, 2004; Garnett, M. J., et al., Mol. Cell 20, 963-969, 2005). By a mechanism that does not require kinase activity, dimerization of BRAF with CRAF induces the activation of CRAF3 (Wan, P. T., et al. Cell 116, 855-867, 2004; Garnett, M. J., et al., Mol. Cell 20, 963-969, 2005).This is supported by the finding that oncogenic forms of BRAF that lack kinase activity can still drive activation of the pathway (Heidorn, S. J., et al., Cell 140, 209-221, 2010). The function of these catalytically impaired mutants requires RAS presumably to induce the active conformation of BRAF and also the presence of a catalytically active CRAF molecule to convey the signal downstream. The mechanism of CRAF activation is not known but could be either through an allosteric interaction or by the recruitment of accessory proteins that are associated with BRAF (or KSR) to modify and activate CRAF. In contrast, oncogenic forms of BRAF that have enhanced kinase activity like the V600E mutant are both CRAF and RAS independent (Garnett, M. J., et al., Mol. Cell 20, 963-969, 2005) suggesting that they directly phosphorylate and activate MEK. 
         [0008]    Because genetic and biochemical proof for KSR kinase activity has been lacking, KSR has been considered to be a pseudokinase that scaffolds the MAP kinase pathway by binding to RAF, MEK and ERK. Because mutagenesis strategies that impair catalytic activity result in dynamic structures that have impaired scaffold activity, it is difficult to distinguish between the two functions of kinases using traditional mutagenesis approaches. 
       SUMMARY  
       [0009]    Some embodiments of the present teachings include a mutant of KSR that impairs the ability of KSR to bind to ATP and does not reconstitute KSR function. The inventors have found that ATP binding can be required for KSR activity. The inventors have found that activity of the BRAFV600E mutant, found in about 60% of melanoma tumor requires the presence of KSR. The inventors further disclose that the ATP binding site of KSR can be a target for a pharmaceutical compound that can be used to treat diseases such as cancers, including, without limitation, melanoma. 
         [0010]    The present inventors have found that a mechanism for RAF activation can include drug induced dimer formation between CRAF and KSR. The inventors further found that in some embodiments, both CRAF and KSR can be required but that BRAF expression can be dispensable for the effect. 
         [0011]    The present inventors have found that a mechanism for RAF activation can involve induced complex formation between CRAF and KSR. The present inventors disclose that inhibition of CRAF/KSR dimers can inhibit RAF and RAS activation. 
         [0012]    The present inventors have generated mutants, including mutants of kinases and mutants of KSR. In various embodiments, these mutants do not bind ATP. In various embodiments, these mutants can comprise a closed, active conformation of a kinase or a related protein, such as, without limitation, KSR. In some configurations, a mutation can comprise an alanine-to-phenylalanine mutation in the sequence of the KSR polypeptide chain. The present teachings further include alanine-to-phenylalanine mutations at highly conserved homologous sequences not only in KSR, but also in kinases other than KSR, such as BRAF and CRAF. In various aspects, such mutant kinases also do not bind ATP. In various embodiments, these alanine-to-phenylalanine mutations can also comprise a closed, active conformation. A conception of the present inventors includes any kinase and homologous polypeptide comprising an alanine-to-phenylalanine mutation at a homologous sequence which can be a conserved sequence. 
         [0013]    In some embodiment, the present teachings include methods of identifying or designing a compound that can act as an inhibitor of KSR kinase activation. In some configurations, a compound identified by these methods can be used as a cancer therapeutic. In these methods, interacting surfaces of kinase dimers, including KSR/KSR homodimer, KSR/CRAF heterodime, KSR/BRAF heterodimer, BRAF/BRAF homodimer, BRAF/CRAF heterdimer, or CRAF/CRAF homodimer (collectively, KSR/CRAF/BRAF) and models thereof can be used to design inhibitors. 
         [0014]    In some configurations, interaction between an N-terminal sequence of one kinase (residues Y340/W342 in CRAF, D448/W450 in BRAF) which interact with R506/K507 of the alpha-C helix of BRAF or residues R398/K399 of the alpha-C helix in CRAF can be a target for drug design. While D448 of BRAF allows BRAF to activate either BRAF or CRAF constitutively, CRAF requires phosphorylation of Y340 to allow it to phosphorylate CRAF or BRAF. Since KSR lacks an acidic residue in the position corresponding to Y340 in CRAF or D448 in BRAF, KSR can only be activated by BRAF or CRAF but cannot activate BRAF or CRAF. The activation of RAF is in trans and involves acidic residues in the activating partner. In some embodiments, the present teachings include an oligopeptide of sequence MKTLGRRDDDDDWEIPDGGI (SEQ ID NO: 9). This oligopeptide was designed based on structures involved in interaction, in particular the N-terminal activating sequence of BRAF (mktlgrrdssddweipdgq; SEQ ID NO: 10). This oligopeptide can inhibit CRAF kinase activity. In other configurations, an inhibitor identified by these methods can be, without limitation, a small molecule, a peptide, an antibody, or an antigen-binding fragment of an antibody such as a Fab fragment. An antibody of the present teachings can be a polyclonal or a monoclonal antibody. 
         [0015]    The present inventors also used molecular modeling which indicated that in various configurations, an alanine-to-phenylalanine mutant of KSR can comprise two stabilized hydrophobic spines. Without being limited by theory, the inventors further hypothesize that the stabilized hydrophobic spines can be critical for a closed active conformation. 
         [0016]    A conception of the present inventors includes any kinase comprising the alanine-to-phenylalanine mutation at the homologous sequence, as well as multimers, such as heterodimers and homodimers comprising a polypeptide comprising the alanine-to-phenylalanine mutation. In various embodiments, a conception of the present inventors includes polypeptides homologous to kinases that comprise the alanine-to-phenylalanine mutation at the homologous sequence, as well as multimers, such as heterodimers and homodimers comprising a polypeptide comprising the alanine-to-phenylalanine mutation. Other aspects of the present teachings include nucleic acids encoding mutations comprising the conserved alanine-to-phenylalanine mutation of KSR, as well as nucleic acids encoding other kinases, or homologues thereof, wherein the kinases or homologues thereof comprise an alanine-to-phenylalanine mutant homologous to A587F of KSR (with amino acid numbering in reference to the sequence of  Mus musculus  KSR). In various configuration, the nucleic acid can be a DNA or an RNA, and can encode, for example, BRAF A481F or CRAF A373F. 
         [0017]    In some embodiments, the present teachings include methods to discriminate between scaffold versus kinase functions of KSR. The inventors found that the alanine-to-phenylalanine mutant of KSR can bind constitutively to RAF and MEK but cannot reconstitute activity. Without being limited by theory, this can imply that the catalytic activity of KSR can be required for its function. 
         [0018]    The present inventors further disclose that two different inhibitors (PLX4720 and GDC0879, Selleck Chemicals, Houston, Tex.) can induce CRAF/KSR dimers. The inventors further disclose that the ability of BRAF-specific inhibitors to activate MEK and ERK in RAS transformed cells require KSR. 
         [0019]    The present inventors generated a mutated form of KSR. In some configurations, this mutated form can dimerize constitutively with CRAF but cannot hind ATP. Without being limited by theory, the failure of this mutant to reconstitute KSR function suggested to the present inventors that the scaffolding function of KSR with CRAF might not be sufficient for its function. The present inventors further disclose that while KSR exhibits no kinase activity when expressed alone, co-expression and dimerization of KSR with CRAF can result in detectable KSR kinase activity for MEK. The inventors further determined that KSR can be a bona fide kinase whose activity can be required for activation of MEK. 
         [0020]    The present inventors have determined that KSR can be a target for a drug for treating a cancer, such as a tumor in which the cells are resistant to BRAF inhibitors such as PLX4032. Furthermore, the inventors have determined that because KSR, CRAF and BRAF can form homodimers and heterodimers, that the interacting surfaces of these polypeptides can be used to identify or design an inhibitor of kinase activation and/or protein scaffolding. The present teachings include the application of structure-based identification of inhibitors that can disrupt a KSR/KSR homodimer interface, a KSR/BRAF heterodimer interface, a KSR/CRAF heterodimer interface, a BRAF/BRAF homodimer interface, a BRAF/CRAF heterodimer interface, or a CRAF/CRAF homodimer interface. 
         [0021]    In various embodiments, the methods developed by the present inventors can involve multilevel investigations, such as analysis of three-dimensional structures and models of kinase homodimer and heterodimer complexes, and various analytical tools, including virtual docking of chemical databases to kinase dimerization domains and in silica screening of chemical structures as potential inhibitors; tests of candidate compounds for inhibitory effects on kinase activity, tests for specificity of candidate compounds, and/or tests to investigate the effects of a candidate inhibitor on dimerization or kinase activity. In various configurations, compounds identified can be, without limitation, a small molecule, an oligopeptide, an aptamer. 
         [0022]    In some aspects, methods of the present teachings can include identifying the binding site involved in hetero- or homo-dimerization, in a computer-based model of kinase dimers. To identify candidate inhibitors, these sites can be targeted by docking and scoring of compounds comprised by one or more libraries of virtual compounds. High scoring candidate compounds can be purchased and/or synthesized. A candidate compound can be tested for its ability to inhibit tumor growth in vitro or in vivo, its ability to inhibit kinase activity of a polypeptide comprising a target sequence, and/or is ability to inhibit dimerization, for example through a chemical cross-linking assay of dimer formation. 
         [0023]    In some aspects, the present inventors have developed methods for designing a drug which inhibits activity of KSR. In various configurations, these methods comprise providing on a digital computer a three-dimensional structure of a KSR/CRAF/BRAF homodimer or heterodimer complex; using software comprised by the digital computer to design a chemical compound which is predicted to bind to a homodimer or heterodimer, and in particular to the interface between binding domains in a dimer. In some aspects, the methods can involve virtual screening not only of an actual 3-dimensional structure of a dimer developed using x-ray crystallography, but also virtual screening of a homology model, whereby candidate inhibitory compounds can be identified using conceptual structures of homodimerized and/or heterodimerized domains of a KSR/C RAF/BRAF homodimer or heterodimer. 
         [0024]    Also disclosed herein are methods for testing a compound as a KSR/CRAF/BRAF inhibitor in a cell or tissue. These methods comprise: selecting a candidate inhibitor of KSR/CRAF/BRAF dimerization and/or kinase activity by performing a structure-based drug design using a three-dimensional structure determined for a crystal comprising an KSR/CRAF/BRAF dimer; contacting the cell or tissue with the candidate inhibitor; and determining a change of an activity of the KSR/CRAF/BRAF dimer comprised by the cell or tissue. 
         [0025]    In yet other aspects, the present teachings include methods for decreasing KSR/CRAF/BRAF dimer activity such as KSR activity in a subject for the treatment of a disease such as a cancer. These methods can comprise selecting a compound identified as an inhibitor of KSR/CRAF/BRAF dimerization using a three-dimensional structure determined for a crystal comprising a KSR/CRAF/BRAF dimer, and administering a therapeutically effective amount of the inhibitor to a subject in need thereof. A disease of these aspects can be, without limitation, a cancer such as a cancer of the breast, a cancer of the ovary or the uterus, or a melanoma. 
         [0026]    In yet other aspects, the present teachings include compounds identified by the screening methods set forth herein, as well as salts thereof such as pharmaceutically acceptable salts. In some configurations, the present teachings include stereoisomers of the compounds, and salts thereof. The compounds can function as inhibitors of KSR/CRAF/BRAF dimerization, such as KSR/BRAF dimerization, and can be used in therapeutic applications such as oncology (such as, for example, breast, ovarian, uterine cancers or melanomas) and/or in a research context. 
         [0027]    In some aspects, a screening method of the present teachings can include a “top-down” approach to identifying lead compounds which inhibit KSR/CRAF/BRAF dimerization. 
         [0028]    First, on level 1, candidate compounds can be selected. Selection of these compounds can comprise virtual docking of a chemical database to a KSR/CRAF/BRAF dimerization “hot-spot.” Level 2 can comprise testing the candidate compounds for activity as inhibitors of KSR/CRAF/BRAF activation. These methods can comprise assays for KSR/CRAF/BRAF activity that are well known to skilled artisans, such as, for example, Western blot assays on kinase autophosphorylation or phosphorylation of a downstream target such as MEK kinase. In level 3, compounds can be tested for selectivity using methods well known to skilled artisans, such as, for example, Western blot assays for effects of a compound on related kinases. In level 4, further analysis of a candidate compound can comprise investigations into mechanism, such as, in non-limiting example, split-luciferase assays, cross-linking assays, and kinase binding assays. In level 5, lead candidate compounds can be optimized. This optimization can comprise performing a structural similarity search for related compounds in at least one additional database, which can be, for example a larger database. The optimization level analysis can also comprise synthesis of a focused combichem library. Because the last level can suggest new compounds to test, in some configurations, these new compounds can be taken through the levels in a new cycle of analysis. 
         [0029]    A KSR of the present teachings can be a mammalian KSR. A KSR of the present teachings can be a human KSR. A KSR (wild type) of the present teachings can have an amino acid sequence as set forth in SEQ ID NO: 1. 
         [0000]                                                (SEQ ID NO: 1)                1   mneakvketl rrcgasgdec grlqyaltcl rkvtglggeh kedsswssld arresgsgps                   61   tdtlsaaslp wppgssqlgr agnsaqgprs isvsalpasd sptpsfsegl sdtciplhas               121   grltpralhs fitppttpql rrhtklkppr tppppsrkvf qllpsfptlt rskshesqlg               181   nriddvssmr fdlshgspqm vrrdiglsvt hrfstkswls qvchvcqksm ifgvkckhcr               241   lkchnkctke apacrisflp ltrlrrtesv psdinnpvdr aaephigtlp kaltkkehpp               301   amnhldsssn pssttsstps spapfptssn pssattppnp spgqrdsrfn fpaayfihhr               361   qqfifpvpsa ghcwkcllia eslkenafni safahaaplp eaadgtrldd qpkadvleah               421   eaeaeepeag kseaeddede vddlpssrrp wrgpisrkas qtsvylqewd ipfeqvelge               481   pigqgrwgrv hrgrwhgeva irllemdghn qdhlklfkke vmnyrqtrhe nvvlfmgacm               541   npphlaiits fckgrtlhsf vrdpktsldi nktrqiaqei ikgmgylhak givhkdlksk               601   nvfydngkvv itdfglfgis gvvregrren qlklshdwlc ylapeivrem tpgkdedqlp               661   fskaadvyaf gtvwyelqar dwplknqaae asiwqigsge gmkrvltsys lgkevseils               721   acwafdlqer psfsllmdml eklpklnrrl shpghfwksa el.                    (NCBI Accession NP_055053.1).                
A human KSR mutant including A587F of the present teachings can have amino acid sequence
 
         [0000]                                      (SEQ ID NO: 2)                1   mneakvketl rrcgasgdec grlqyaltcl rkvtglggeh kedsswssld arresgsgps                   61   tdtlsaaslp wppgssqlgr agnsaqgprs isvsalpasd sptpsfsegl sdtciplhas               121   grltpralhs fitppttpql rrhtklkppr tppppsrkvf qllpsfptlt rskshesqlg               181   nriddvssmr fdlshgspqm vrrdiglsvt hrfstkswls qvchvcqksm ifgvkckhcr               241   lkchnkctke apacrisflp ltrlrrtesv psdinnpvdr aaephfgtlp kaltkkehpp               301   amnhldsssn pssttsstps spapfptssn pssattppnp spgqrdsrfn fpaayfihhr               361   qqfifpvpsa ghcwkcllia eslkenafni safahaaplp eaadgtrldd qpkadvleah               421   eaeaeepeag kseaeddede vddlpssrrp wrgpisrkas qtsvylqewd ipfeqvelge               481   pigqgrwgrv hrgrwhgev   f    irllemdghn qdhlklfkke vmnyrqtrhe nvvlfmgacm               541   npphlaiits fckgrtlhsf vrdpktsldi nktrqi   a   qei ikgmgylhak givhkdlksk               601   nvfydngkvv itdfglfgis gvvregrren qlklshdwlc ylapeivrem tpgkdedqlp               661   fskaadvyaf gtvwyelqar dwplknqaae asiwqigsge gmkrvltsvs lgkevseils               721   acwafdlqer psfsllmdml eklpklnrrl shpghfwksa el.            
BRAF (wild type) of the present teachings can have amino acid sequence
 
         [0000]                                                (SEQ ID NO: 3)                1   maalsggggg gaepgqalfn gdmepeagag agaaassaad paipeevwni kqmikltqeh                   61   iealldkfgg ehnppsiyle ayeeytskld alqqreqqll eslgngtdfs vsssasmdtv               121   tsssssslsv lpsslsvfqn ptdvarsnpk spqkpivrvf lpnkqrtvvp arcgvtvrds               181   lkkalmmrgl ipeccavyri qdgekkpigw dtdiswltge elhvevlenv pltthnfvrk               241   tfftlafcdf crkllfqgfr cqtcgyklhq rcstevplmc vnydqldllf vskffehhpi               301   pqeeaslaet altsgsspsa pasdsigpqi ltspspsksi pipqpfrpad edhrnqfgqr               361   drsssapnvh intiepvnid dlirdqgfrg dggsttglsa tppaslpgsl tnvkalqksp               421   gpqrerksss ssedrnrmkt lgrrdssddw eipdgqitvg qrigsgsfgt vykgkwhgdv               481   avkmlnvtap tpqqlqafkn evgvlrktrh vnillfmgys tkpqlaivtq wcegsslyhh               541   lhiietkfem iklidiarqt aqgmdylhak siihrdlksn niflhedltv kigdfglatv               601   ksrwsgshqf eqlsgsilwm apevirmqdk npysfqsdvy afgivlyelm tgqlpysnin               661   nrdqiifmvg rgylspdlsk vrsncpkamk rlmaeclkkk rderplfpqi lasiellars               721   lpkihrsase pslnragfqt edfslyacas pktpiqaggy gafpvh                    (Swiss-Prot Accession P15056.4).                
BRAF A481F, comprising an alanine-to-phenylalanine mutation as described herein which is homologous to KSR A587F can have an amino acid sequence
 
         [0000]                                      (SEQ ID NO: 4)                1   maalsggggg gaepgqalfn gdmepeagag agaaassaad paipeevwni kqmikltqeh                   61   iealldkfgg ehnppsiyle ayeeytskld alqqreqqll eslgngtdfs vsssasmdtv               121   tsssssslsv lpsslsvfqn ptdvarsnpk spqkpivrvf lpnkqrtvvp arcgvtvrds               181   lkkalmmrgl ipeccavyri qdgekkpigw dtdiswltge elhvevlenv pltthnfvrk               241   tfftlafcdf crkllfqgfr cqtcgykfhq rcstevplmc vnydqldllf vskffehhpi               301   pqeeaslaet altsgsspsa pasdsigpqi ltspspsksi pipqpfrpad edhrnqfgqr               361   drsssapnvh intiepvnid dlirdqgfrg dggsttglsa tppaslpgsl tnvkalqksp               421   gpqrerksss ssedrnrmkt lgrrdssddw eipdgqitvg qrigsgsfgt vykgkwhgdv               481       f   vkmlnvtap tpqqlqafkn evgvlrktrh vnillfmgys tkpqlaivtq wcegsslyhh               541   lhiietkfem iklidiarqt aqgmdylhak siihrdlksn niflhedltv kigdfglatv               601   ksrwsgshqf eqlsgsilwm apevirmqdk npysfqsdvy afgivlyelm tgqlpysnin               661   nrdqiifmvg rgylspdlsk vrsncpkamk rlmaeclkkk rderplfpqi lasiellars               721   lpkihrsase pslnragfqt edfslyacas pktpiqaggy gafpvh.            
CRAF (wild type) of the present teachings can have amino acid sequence
 
         [0000]                                                (SEQ ID NO: 5)                1   mehiqgawkt isngfgfkda vfdgsscisp tivqqfgyqr rasddgkltd psktsntirv                   61   flpnkqrtvv nvrngmslhd clmkalkvrg lqpeccavfr llhehkgkka rldwntdaas               121   ligeelqvdf ldhvpltthn farktflkla fcdicqkfll ngfrcqtcgy kfhehcstkv               181   ptmcvdwsni rqlllfpnst igdsgvpalp sltmrrmres vsrmpvssqh rystphaftf               241   ntsspssegs lsqrqrstst pnvhmvsttl pvdsrmieda irshsesasp salssspnnl               301   sptgwsqpkt pvpaqrerap vsgtqeknki rprgqrdssy yweieasevm lstrigsgsf               361   gtvykgkwhg dvavkilkvv dptpeqfqaf rnevavlrkt rhvnillfmg ymtkdnlaiv               421   tqwcegssly khlhvqetkf qmfqlidiar qtaqgmdylh akniihrdmk snniflhegl               481   tvkigdfgla tvksrwsgsq qveqptgsvl wmapevirmq dnnpfsfqsd vysygivlye               541   lmtgelpysh innrdqiifm vgrgyaspdl sklykncpka mkrlvadcvk kvkeerplfp               601   qilssiellq hslpkinrsa sepslhraah tedinactlt tsprlpvf                    (NCBI Accession NP_002871.1).                
CRAF A373F, comprising an alanine-to-phenylalanine mutation as described herein which is homologous to KSR A587F can have an amino acid sequence
 
         [0000]                                      (SEQ ID NO: 6)                1   mehiqgawkt isngfgfkda vfdgsscisp tivqqfgyqr rasddgkltd psktsntirv                   61   flpnkqrtvv nvrngmslhd clmkalkvrg lqpeccavfr llhehkgkka rldwntdaas               121   ligeelqvdf ldhvpltthn farktflkla fcdicqkfll ngfrcqtcgy kfhehcstkv               181   ptmcvdwsni rqlllfpnst igdsgvpalp sltmrrmres vsrmpvssqh rystphaftf               241   ntsspssegs lsqrqrstst pnvhmvsttl pvdsrmieda irshsesasp salssspnnl               301   sptgwsqpkt pvpaqrerap vsgtqeknki rprgqrdssy yweieasevm lstrigsgsf               361   gtvykgkwhg dv   f   vkilkvv dptpeqfqaf rnevavlrkt rhvnillfmg ymtkdnlaiv               421   tqwcegssly khlhvqetkf qmfqlidiar qtaqgmdylh akniihrdmk snniflhegl               481   tvkigdfgla tvksrwsgsq qveqptgsvl wmapevirmq dnnpfsfqsd vysygivlye               541   lmtgelpysh innrdqiifm vgrgyaspdl sklykncpka mkrlvadcvk kvkeerplfp               601   qilssiellq hslpkinrsa sepslhraah tedinactlt tsprlpvf.            
A nucleic acid of the present teachings can encode KSR, and have a nucleotide sequence such as
 
         [0000]    
       
         
               
               
             
               
               
               
             
               
               
             
           
               
                 (SEQ ID NO: 7) 
                   
               
             
          
           
               
                 1 
                 ctggacccct gccagggaag gggtcctcag acttgaggtt gccagctcag atgtggggct 
                   
               
               
                   
               
               
                 61 
                 gctgatacta ggtgactgga ctgatgttct gttctagatg aaactccttg aggggaccat 
               
               
                   
               
               
                 121 
                 ttgaaaaggc ttgatgtgct gcccaaagcc cccttcagag ctgacttctc cacccccagc 
               
               
                   
               
               
                 181 
                 tgccgtgagc cttggctgct gacagctcat agctgagtcc ctcccgtgaa gtcaccttct 
               
               
                   
               
               
                 241 
                 gctgaagggt acatcctctc ccaaggcgaa gctggtccgt tacatttgta agcagaggca 
               
               
                   
               
               
                 301 
                 gtgcaagctg agcgtggctc ccggtgagag gaccccagag ctcaacagct acccccgctt 
               
               
                   
               
               
                 361 
                 cagcgactgg ctgtacactt tcaacgtgag gccggaggtg gtgcaggaga tcccccgaga 
               
               
                   
               
               
                 421 
                 cctcacgctg gatgccctgc tggagatgaa tgaggccaag gtgaaggaga cgctgcggcg 
               
               
                   
               
               
                 481 
                 ctgtggggcc agcggggatg agtgtggccg tctgcagtat gccctcacct gcctgcggaa 
               
               
                   
               
               
                 541 
                 ggtgacaggc ctgggagggg agcacaagga ggactccagt tggagttcat tggatgcgcg 
               
               
                   
               
               
                 601 
                 gcgggaaagt ggctcagggc cttccacgga caccctctca gcagccagcc tgccctggcc 
               
               
                   
               
               
                 661 
                 cccagggagc tcccagctgg gcagagcagg caacagcgcc cagggcccac gctccatctc 
               
               
                   
               
               
                 721 
                 cgtgtcagct ctgcccgcct cagactcccc cacccccagc ttcagtgagg gcctctcaga 
               
               
                   
               
               
                 781 
                 cacctgtatt cccctgcacg ccagcggccg gctgaccccc cgtgccctgc acagcttcat 
               
               
                   
               
               
                 841 
                 caccccgccc accacacccc agctgcgacg gcacaccaag ctgaagccac cacggacgcc 
               
               
                   
               
               
                 901 
                 ccccccaccc agccgcaagg tcttccagct gctgcccagc ttccccacac tcacccggag 
               
               
                   
               
               
                 961 
                 caagtcccat gagtctcagc tggggaaccg cattgatgac gtctcctcga tgaggtttga 
               
               
                   
               
               
                 T1021 
                 tctctcgcat ggatccccac agatggtacg gagggatatc gggctgtcgg tgacgcacag 
               
               
                   
               
               
                 1081 
                 gttctccacc aagtcctggc tgtcgcaggt ctgccacgtg tgccagaaga gcatgatatt 
               
               
                   
               
               
                 1141 
                 tggagtgaag tgcaagcatt gcaggttgaa gtgtcacaac aaatgtacca aagaagcccc 
               
               
                   
               
               
                 1201 
                 tgcctgtaga atatccttcc tgccactaac tcggcttcgg aggacagaat ctgtcccctc 
               
               
                   
               
               
                 1261 
                 ggacatcaac aacccggtgg acagagcagc cgaaccccat tttggaaccc tccccaaagc 
               
               
                   
               
               
                 1321 
                 actgacaaag aaggagcacc ctccggccat gaatcacctg gactccagca gcaacccttc 
               
               
                   
               
               
                 1381 
                 ctccaccacc tcctccacac cctcctcacc ggcgcccttc ccgacatcat ccaacccatc 
               
               
                   
               
               
                 1441 
                 cagcgccacc acgcccccca acccctcacc tggccagcgg gacagcaggt tcaacttccc 
               
               
                   
               
               
                 1501 
                 agctgcctac ttcattcatc atagacagca gtttatcttt ccagtgccat ctgctggcca 
               
               
                   
               
               
                 1561 
                 ttgctggaaa tgcctcctta ttgcagaaag tttaaaggaa aacgctttca acatttcagc 
               
               
                   
               
               
                 1621 
                 ctttgcacac gcagccccgc tccctgaagc tgccgacggt acccggctcg atgaccagcc 
               
               
                   
               
               
                 1681 
                 gaaagcagat gtgttggaag ctcacgaagc ggaggctgag gagccagagg ctggcaagtc 
               
               
                   
               
               
                 1741 
                 agaggcagaa gacgatgagg acgaggtgga cgacttgccg agctctcgcc ggccctggcg 
               
               
                   
               
               
                 1801 
                 gggccccatc tctcgcaagg ccagccagac cagcgtgtac ctgcaggagt gggacatccc 
               
               
                   
               
               
                 1861 
                 cttcgagcag gtagagctgg gcgagcccat cgggcagggc cgctggggcc gggtgcaccg 
               
               
                   
               
               
                 1921 
                 cggccgctgg catggcgagg tggccattcg cctgctggag atggacggcc acaaccagga 
               
               
                   
               
               
                 1981 
                 ccacctgaag ctcttcaaga aagaggtgat gaactaccgg cagacgcggc atgagaacgt 
               
               
                   
               
               
                 2041 
                 ggtgctcttc atgggggcct gcatgaaccc gccccacctg gccattatca ccagcttctg 
               
               
                   
               
               
                 2101 
                 caaggggcgg acgttgcact cgtttgtgag ggaccccaag acgtctctgg acatcaacaa 
               
               
                   
               
               
                 2161 
                 gacgaggcaa atcgctcagg agatcatcaa gggcatggga tatcttcatg ccaagggcat 
               
               
                   
               
               
                 2221 
                 cgtacacaaa gatctcaaat ctaagaacgt cttctatgac aacggcaagg tggtcatcac 
               
               
                   
               
               
                 2281 
                 agacttcggg ctgtttggga tctcaggcgt ggtccgagag ggacggcgtg agaaccagct 
               
               
                   
               
               
                 2341 
                 aaagctgtcc cacgactggc tgtgctatct ggcccctgag attgtacgcg agatgacccc 
               
               
                   
               
               
                 2401 
                 cgggaaggac gaggatcagc tgccattctc caaagctgct gatgtctatg catttgggac 
               
               
                   
               
               
                 2461 
                 tgtttggtat gagctgcaag caagagactg gcccttgaag aaccaggctg cagaggcatc 
               
               
                   
               
               
                 2521 
                 catctggcag attggaagcg gggaaggaat gaagcgtgtc ctgacttctg tcagcttggg 
               
               
                   
               
               
                 2581 
                 gaaggaagtc agtgagatcc tgtcggcctg ctgggctttc gacctgcagg agagacccag 
               
               
                   
               
               
                 2641 
                 cttcagcctg ctgatggaca tgctggagaa acttcccaag ctgaaccggc ggctctccca 
               
               
                   
               
               
                 2701 
                 ccctggacac ttctggaagt cagctgagtt gtaggcctgg ctgccttgca tgcaccaggg 
               
               
                   
               
               
                 2761 
                 gctttcttcc tcctaatcaa caactcagca ccgtgacttc tgctaaaatg caaaatgaga 
               
               
                   
               
               
                 2821 
                 tgcgggcact aacccagggg atgccacctc tgctgctcca gtcgtctctc tcgaggctac 
               
               
                   
               
               
                 2881 
                 ttcttttgct ttgttttaaa aactggccct ctgccctctc cacgtggcct gcatatgccc 
               
               
                   
               
               
                 2941 
                 aagtaactgc tctcagagga tcccactaac tgagctccct ccaaggcagt ctgggcagct 
               
               
                   
               
               
                 3001 
                 tctaactacc ttcctggaca tgactgattg ctcccgtgtt cttctgaggg ctggtcttgt 
               
               
                   
               
               
                 3061 
                 ttttgtttgg gtggctctgt ctcactgcta acaccttagt gagatgcctt ccaccctcct 
               
               
                   
               
               
                 3121 
                 gagcacacca gcctcccact gggtgtgtgc ctagtgcggg gcgggcggag gttgggaggg 
               
               
                   
               
               
                 3181 
                 tgttggcttg gcttttaacc tgtggggatt ttgtccaaca aggagtggaa tgatttcaga 
               
               
                   
               
               
                 3241 
                 gctgccctga ggctggcacc ctggtcacag gaaccctctg cgctggctcc tgtctcagtc 
               
               
                   
               
               
                 3301 
                 ccctctgtag agttagatca gaagacacag aaagttctgt ggccatgaaa gataccagct 
               
               
                   
               
               
                 3361 
                 tggaagggtt gtgtcttcag tggcaccctc agaaaaattg tcttaaagca aagaggtacc 
               
               
                   
               
               
                 3421 
                 tggctccaga caatttttct gatgaaaaca aagtctctgc cccgtcccca ccctgccacc 
               
               
                   
               
               
                 3481 
                 ctggcaaagt tacttccttt acagctgccc agtgtaccat agaccagacc ccaggtcagc 
               
               
                   
               
               
                 3541 
                 atttgtcaag agcatggctg ctgagtcccc tgtggcagtc aatgcactgt ttaccaaatg 
               
               
                   
               
               
                 3601 
                 caggtttctg ttctccctcc ccagcaagac ctgctgaacc cagatctctg gaatggggcc 
               
               
                   
               
               
                 3661 
                 ctaggaattt gcatttcaac ctgcttccca ggtggccctg atgcacccca gtattagagt 
               
               
                   
               
               
                 3721 
                 ttattgctaa aaggaacatg ccctgtcact cctggtatcc tgggagtcat gtttctcttc 
               
               
                   
               
               
                 3781 
                 tctctcagtt ctacttggag caagagcttt cctgggctgc aaatgagaaa acaattccta 
               
               
                   
               
               
                 3841 
                 ggaacccaca gcagtactga gcatgctggg agcttgggac ttggagatga atgagccacc 
               
               
                   
               
               
                 3901 
                 gttgctgctc caagtaggac tacttggagt gtagctgagg ccttggacgc agtatgacca 
               
               
                   
               
               
                 3961 
                 ggggcagctc tgccagggct gttggccaat cagtcatttt catttcttgt tggaggccag 
               
               
                   
               
               
                 4021 
                 gtcctctgct gaactcattt cctagctagt gttaccctaa ttctgatgaa gatcaatggg 
               
               
                   
               
               
                 4081 
                 gctataattc ttgtttttgt tcctctttgc agcattaaca gcagcaaagt tgtaccccgg 
               
               
                   
               
               
                 4141 
                 tttgaaaggt ttggcttggg cgtcctggag tccagtaatc caaagatgta gccagccata 
               
               
                   
               
               
                 4201 
                 tggtttttcg ctgctgatct ctttcttttt aaaatgtgtt tctgaaacat cccaacaacc 
               
               
                   
               
               
                 4261 
                 accacgacaa aaaaacactg cctgcccagc gctgcaaacc aggagcacac gtcctagatt 
               
               
                   
               
               
                 4321 
                 cagactgttg gccataaacc ccactcggga gatggagctg cacctgctat ttcttaaaat 
               
               
                   
               
               
                 4381 
                 gacaccacca acaaccaaac ctgtcatgac agacagcaaa tgtttacacg tatatttctc 
               
               
                   
               
               
                 4441 
                 ctgagtgaac ctgatgtttt acaataggta ataataaaaa cagtctgtgc aaaaaaaaaa 
               
               
                   
               
               
                 4501 
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 
               
               
                   
               
             
          
           
               
                 (NCBI Accession NM _014238). 
                   
               
             
          
         
       
     
         [0030]    The present teachings include a nucleic acid sequence encoding KSR comprising an A587F mutation. The present teachings also include a nucleic acid sequence encoding KSR comprising an L591F mutation. Such mutations can be introduced into a nucleic acid encoding KSR using routine methods well known to skilled artisans, such as, for example, introduction of mutant sequences using polymerase chain reactions. 
         [0031]    The present teachings also include nucleic acids encoding kinases or pseudokinases, wherein the encoded polypeptide comprises a mutation homologous to A587F of KSR. Such nucleic acids can comprise a DNA or an RNA. 
         [0032]    In various configurations, a nucleic acid of the present teachings can be an oligonucleotide which comprises at least 10 nucleotides, at least 15 nucleotides, or at least 20 nucleotides, up to 50 nucleotides, about 50 nucleotides, up to 60 nucleotides, about 60 nucleotides, up to 70 nucleotides, or about 70 nucleotides, and can comprise a sequence encoding an amino acid sequence comprising A587F of KSR, or a homologous alanine-to-phenylalanine mutation of another kinase or pseudokinase. In some configurations, an oligonucleotide can be complementary to a sequence encoding an A587F of KSR, or a homologous alanine-to-phenylalanine mutation of another kinase or pseudokinase. 
         [0033]    In some embodiments, the present teachings include a mutated form of KSR comprising a leucine 591 to phenylalanine mutation (L591 F, with amino acid numbering in reference to the sequence of  Mus musculus  KSR). In various configurations, the ability of this KSR mutant to act as a scaffold can be impaired. In various configurations, this mutant of KSR can have constitutively active kinase activity which is independent of BRAF and CRAF. In some configurations, this mutated form does not dimerize with CRAF. Without being limited by theory, the failure of this mutant to dimerize with CRAF suggested to the present inventors that the scaffolding function of KSR with CRAF might not be sufficient for KSR&#39;s function. The present inventors further disclose that while wild type KSR exhibits no kinase activity when expressed alone, homodimerization of KSR can result in KSR kinase activity, such as phosphorylation of a substrate, for example a downstream target such as MEK or a oligopeptide substrate containing a target sequence, such as an oligopeptide consisting of a target sequence of MEK that includes an amino acid that is subject to phosphorylation by KSR. The inventors further determined that KSR can be a bona fide kinase whose activity can be required for activation of MEK. 
         [0034]    Human KSR L591F of the present teachings can have amino acid sequence 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                 (SEQ ID NO. 8) 
                   
               
             
          
           
               
                 1 
                 mneakvketl rrcgasgdec grlqyaltcl rkvtglggeh kedsswssld arresgsgps 
                   
               
               
                   
               
               
                 61 
                 tdtlsaaslp wppgssqlgr agnsaqgprs isvsalpasd sptpsfsegl sdtciplhas 
               
               
                   
               
               
                 121 
                 grltpralhs fitppttpql rrhtklkppr tppppsrkvf qllpsfptlt rskshesqlg 
               
               
                   
               
               
                 181 
                 nriddvssmr fdlshgspqm vrrdiglsvt hrfstkswls qvchvcqksm ifgvkckhcr 
               
               
                   
               
               
                 241 
                 lkchnkctke apacrisflp ltrlrrtesv psdinnpvdr aaephfgtlp kaltkkehpp 
               
               
                   
               
               
                 301 
                 amnhldsssn pssttsstps spapfptssn pssattppnp spgqrdsrfn fpaayfihhr 
               
               
                   
               
               
                 361 
                 qqfifpvpsa ghcwkcllia eslkenafni safahaaplp eaadgtrldd qpkadvleah 
               
               
                   
               
               
                 421 
                 eaeaeepeag kseaeddede vddlpssrrp wrgpisrkas qtsvylqewd ipfeqvelge 
               
               
                   
               
               
                 481 
                 pigqgnvgrv hrgrwhgeva irl   f   emdghn qdhlklfkke vmnyrqtrhe nvvlfmgacm 
               
               
                   
               
               
                 541 
                 npphlaiits fckgrtlhsf vrdpktsldi nktrqiaqei ikgmgylhak givhkdlksk 
               
               
                   
               
               
                 601 
                 nvfydngkvv itdfglfgis gvvregrren qlklshdwlc ylapeivrem tpgkdedqlp 
               
               
                   
               
               
                 661 
                 fskaadvyaf gtvwyelqar dwplknqaae asiwqigsge gmkrvltsvs lgkevseils 
               
               
                   
               
               
                 721 
                 acwafdlqer psfsllmdml eklpklnrrl shpghfwksa el. 
               
             
          
         
       
     
         [0035]    In some embodiments, the present teachings include assays for detecting dimerization of kinases as well as methods of screening compounds as activators or inhibitors of kinase activation. In some configurations, an inhibitor of kinase dimerization can be effective as an anti-cancer therapeutic. 
         [0036]    In various configurations, a dimerization assay of these embodiments can comprise expressing in a cell a first fusion polypeptide comprising a sequence of a kinase such as KSR or a dimerization domain thereof, and an amino terminal portion of an enzyme, as well as a expressing in the cell a second fusion polypeptide comprising a sequence of a binding partner of the kinase, and a carboxy terminal portion of the enzyme. The binding partner can be the same kinase, another kinase, or a dimerization domain thereof. Upon expression of both fusion constructs and in the absence of an inhibitor, the amino terminal and carboxy terminal portions of the enzymes interact to provide a functioning enzyme which can have enzyme activity which can be detected by methods well known to skilled artisans. In various configurations, the enzyme can be, without limitation a luciferase or a beta-galactosidase. In various configurations the luciferase can be a Ranilla luciferase. In various embodiments, a screen for an inhibitor of dimerization can comprise contacting a cell expressing both fusion polypeptides with a candidate inhibitor, and performing an assay for the enzyme. A reduction in enzyme activity compared to a control can indicate that the candidate inhibitor can inhibit dimerization of the kinase of the first fusion polypeptide with its binding partner comprised by the second fusion polypeptide. For example and without limitation, a kinase and binding partner of fusion polypeptides can be, respectively KSR/KSR, KSR/BRAF, KSR/CRAF, BRAF/BRAF, BRAF/CRAF, or CRAF/CRAF, or mutants thereof. 
         [0037]    In some embodiments, the present teachings includes nucleic acids that encode the first fusion polypeptide operably linked to a promoter, and nucleic acids that encode the second fusion polypeptide operably linked to a promoter. In each case, the nucleic acid can be comprised by a vector such as a plasmid or virus. 
         [0038]    In some embodiments, the present teachings include cells and cell lines comprising the nucleic acids. In various configurations, these cell cells lines can be stable transformations or transient transformations. A cell of these embodiments can be any suitable host, such as, without limitation, HeLa, A375, HEK293, mouse embryonic fibroblast, or CHO. 
         [0039]    In some embodiments, the present teachings include cell lines that are resistant to BRAF inhibition but dependent on KSR. In some embodiments, these cells can be stably transfected cells expressing BRAF V600E and NRASV12 or KRASV12. In some embodiments, these cells can be stably transfected cells expressing BRAF V600E and TPL2/COT. In various configurations, the cells can be, without limitation, HeLa transformed with BRAF V600E as well as NRASV12, KRASV12 or TPL2/COT, or a tumor-derived cell line comprising a BRAF V600E mutation and transformed with NRASV12, KRASV12 or TPL2/COT. In various configurations, a tumor-derived cell line comprising a BRAF V600E mutation can be a melanoma cell line such as, without limitation, A375, MALME-3M, Colo829, Colo38, SK-MEL28, SK-MELS, HT144, LOX, A2058, or a breast cancer cell line such as, without limitation, MDA-MB-435. 
         [0040]    The present teachings include the following aspects.
   1. A mutant kinase or pseudokinase, comprising an alanine-to-phenylalanine mutation of KSR A587F, or a homologous alanine-to-phenylalanine mutation in a homologue thereof.   2. A mutant kinase or pseudokinase in accordance with aspect I, comprising an alanine-to-phenylalanine mutation A587F of KSR.   3. A mutant kinase or pseudokinase in accordance with aspect 1, comprising an alanine-to-phenylalanine mutation of a Raf kinase at an alanine at a sequence homologous to KSR A487.   4. A mutant kinase or pseudokinase in accordance with aspect 3, comprising an alanine-to-phenylalanine mutation of a Raf kinase, selected from the group consisting of BRAF A481F and CRAF A373F.   5. A mutant kinase or pseudokinase in accordance with aspect 1, comprising an alanine-to-phenylalanine mutation of a mammalian kinase or pseudokinase, wherein the alanine is homologous to KSR A487.   6. A mutant kinase or pseudokinase in accordance with aspect 1, comprising an alanine-to-phenylalanine mutation of a human kinase or pseudokinase, wherein the alanine is homologous to KSR A487.   7. A mutant kinase or pseudokinase in accordance with aspect 1, comprising an alanine-to-phenylalanine mutation A70F of Protein Kinase A.   8. A mutant kinase or pseudokinase in accordance with aspect 1, comprising an alanine-to-phenylalanine mutation in a kinase at a sequence homologous to A70F of Protein Kinase A.   9. A nucleic acid encoding a mutant kinase or pseudokinase of any one of aspects 1-8.   10. A cell comprising a mutant kinase or pseudokinase of any one of aspects 1-8.   11. A cell in accordance with aspect 10, further comprising a dimerization partner of the mutant kinase or pseudokinase.   12. A cell comprising a nucleic acid encoding a mutant kinase or pseudokinase of any one of aspects 1-8.   13. A cell in accordance with aspect 12, further comprising a nucleic acid encoding a dimerization partner of the mutant kinase or pseudokinase.   14. A method of screening a compound for activity as a kinase inhibitor or agonist, comprising:   
 
         [0055]    providing a mutant kinase or pseudokinase of any one of aspects 1-8; 
         [0056]    forming a mixture comprising the kinase or pseudokinase and a candidate inhibitor or agonist; and 
         [0057]    measuring kinase activity in the mixture.
   15. A method of screening a compound for activity as an inhibitor of kinase dimerization, comprising:   
 
         [0059]    providing a cell of any one of aspects 10-13; 
         [0060]    contacting the cell with a candidate inhibitor of kinase dimerization; and 
         [0061]    measuring dimer formation or stability in the cell.
   16. A method of screening a compound for activity as an inhibitor of kinase dimerization, comprising:   
 
         [0063]    providing a mutant kinase or pseudokinase of any one of aspects 1-8: 
         [0064]    forming a mixture comprising the kinase or pseudokinase, a dimerization partner of the kinase or pseudokinase, and a candidate inhibitor of kinase dimerization; and 
         [0065]    measuring dimer formation or stability in the mixture.
   17. A method of screening a compound for activity as an inhibitor of kinase dimerization in accordance with aspect 16, wherein the measuring dimer formation comprises using a complementation assay.   18. A method of screening a compound for activity as an inhibitor of kinase dimerization in accordance with aspect 16, wherein the measuring dimer formation comprises using a luciferase complementation assay.   19. A method of screening a compound for activity as an inhibitor of kinase dimerization in accordance with aspect 16, wherein the measuring dimer formation comprises using a fluorescent protein complementation assay.   20. A method for selecting a candidate drug which interferes with an activity of a kinase or pseudokinase, the method comprising: (a) providing a three-dimensional structure of the kinase or pseudokinase mutant of any one of aspects 1-8 in complex with an dimerization partner: and (b) designing a compound predicted to bind the complex.   21. A method for designing a compound which interferes with an activity of a kinase or pseudokinase, the method comprising: (a) providing on a digital computer a three-dimensional structure of a complex comprising a kinase or pseudokinase mutant of any one of aspects 1-8 and a dimerization partner; and (b) using software comprised by the digital computer to design a compound which is predicted to bind to the complex.   22. A method according to aspect 21, further comprising: (c) synthesizing the compound; and (d) evaluating the compound for an ability to interfere with dimerization of the kinase or pseudokinase mutant.   23. A crystal comprising a kinase or pseudokinase mutant of any one of aspects 1-8 and a dimerization partner.   24. A computer image of a comples comprising a mutant kinase or pseudokinase of any one of aspects 1-8 and a dimerization partner thereof.   25. A mutant kinase or pseudokinase, comprising a leucine-to-phenylalanine mutation of KSR L591F, or a homologous leucine-to-phenylalanine mutation in a homologue thereof.   26. A mutant kinase or pseudokinase in accordance with aspect 25, comprising a leucine-to-phenylalanine mutation L591 F of KSR.   27. A mutant kinase or pseudokinase in accordance with aspect 25, comprising a leucine-to-phenylalanine mutation of a Raf kinase at a leucine at a sequence homologous to KSR L591.   28. A mutant kinase or pseudokinase in accordance with aspect 25, comprising a leucine-to-phenylalanine mutation of a mammalian kinase or pseudokinase, wherein the leucine is homologous to KSR L591.   30. A mutant kinase or pseudokinase in accordance with aspect 25, comprising a leucine-to-phenylalanine mutation of a human kinase or pseudokinase, wherein the leucine is homologous to KSR L591.   31. A nucleic acid encoding a mutant kinase or pseudokinase of any one of aspects 25-30.   32. A cell comprising a mutant kinase or pseudokinase of any one of aspects 25-30.   33. A cell in accordance with aspect 32, further comprising a dimerization partner of the mutant kinase or pseudokinase encoded by a nucleic acid heterologous to the cell.   34. A cell comprising a nucleic acid encoding a mutant kinase or pseudokinase of any one of aspects 25-30.   35. A cell in accordance with aspect 9, further comprising a nucleic acid heterologous to the cell, said nucleic acid encoding a dimerization partner of the mutant kinase or pseudokinase encoded by a nucleic acid heterologous to the cell.   36. A method of screening a compound for activity as a kinase inhibitor or agonist, comprising:   
 
         [0085]    providing a mutant kinase or pseudokinase of any one of aspects 25-30: 
         [0086]    forming a mixture comprising the kinase or pseudokinase and a candidate inhibitor or agonist; and 
         [0087]    measuring kinase activity in the mixture.
   37. A method of screening a compound for activity as an inhibitor of kinase dimerization, comprising:   
 
         [0089]    providing a cell of any one of aspects 32-35; 
         [0090]    contacting the cell with a candidate inhibitor of kinase dimerization; and 
         [0091]    measuring dimer formation or stability in the cell.
   38. A method of screening a compound for activity as an inhibitor of kinase dimerization, comprising:   
 
         [0093]    providing a mutant kinase or pseudokinase of any one of aspects 32-35; 
         [0094]    forming a mixture comprising the kinase or pseudokinase, a dimerization partner of the kinase or pseudokinase, and a candidate inhibitor of kinase dimerization; and 
         [0095]    measuring dimer formation or stability in the mixture.
   39. A method of screening a compound for activity as an inhibitor of kinase dimerization in accordance with aspect 38, wherein the measuring dimer formation comprises a complementation assay.   40. A method of screening a compound for activity as an inhibitor of kinase dimerization in accordance with aspect 38, wherein the measuring dimer formation comprises a luciferase complementation assay.   41. A method of screening a compound for activity as an inhibitor of kinase dimerization in accordance with aspect 38, wherein the measuring dimer formation comprises using a fluorescent protein complementation assay.   42. A method for selecting a candidate drug which interferes with an activity of a kinase or pseudokinase, the method comprising: (a) providing a three-dimensional structure of the kinase or pseudokinase mutant of any one of aspects 25-30 in complex with a dimerization partner; and (b) designing a compound predicted to bind the complex.   43. A method for designing a compound which interferes with an activity of a kinase or pseudokinase, the method comprising: (a) providing on a digital computer a three-dimensional structure of a complex comprising a kinase or pseudokinase mutant of any one of aspects 25-30 and a dimerization partner; and (b) using software comprised by the digital computer to design a compound which is predicted to bind to the complex.   44. A method according to aspect 43, further comprising: (c) synthesizing the compound: and (d) evaluating the compound for an ability to interfere with dimerization of the kinase or pseudokinase mutant.   45. A crystal comprising a kinase or pseudokinase mutant of any one of aspects 25-30 and a dimerization partner.   46. A computer image of a comples comprising a mutant kinase or pseudokinase of any one of aspects 25-30 and a dimerization partner thereof.   47. A fusion polypeptide comprising:   
 
         [0105]    A first inactive portion of an enzyme; and 
         [0106]    a KSR dimerization sequence, wherein the portion can be activated by complementation.
   48. A fusion polypeptide in accordance with aspect 47, wherein the first inactive portion of the enzyme is selected from the group consisting of an amino terminal portion of the enzyme and a carboxy terminal portion of the enzyme.   49. A polypeptide in accordance with aspect 47, wherein the KSR dimerization domain comprises an A587F mutation.   50. A polypeptide in accordance with aspect 47, wherein the enzyme is a luciferase.   51. A polypeptide in accordance with aspect 47, wherein the enzyme is a Ranilla luciferase.   52. A polypeptide in accordance with aspect 47, wherein the KSR dimerization sequence is comprised by a sequence of a full length KSR.   53. A fusion polypeptide comprising:   
 
         [0113]    a second inactive enzyme portion complementary to the first inactive portion of an enzyme of aspect 47; and
   a polypeptide sequence of a KSR kinase binding partner or a KSR-binding domain thereof.   54. A fusion polypeptide in accordance with aspect 53, wherein the polypeptide sequence of a KSR kinase binding partner or a KSR-binding domain thereof is a polypeptide sequence selected from the group consisting of the polypeptide sequence of KSR, the polypeptide sequence of BRAF, the polypeptide sequence of CRAF, the polypeptide sequence of a KSR dimerization domain, the polypeptide sequence of a BRAF dimerization domain and the polypeptide sequence of a CRAF dimerization domain.   55. A cell in vitro comprising:   
 
         [0117]    the polypeptide of any one of aspects 47-52; and 
         [0118]    the polypeptide of any one of aspects 53-54, 
         [0000]    whereby in the absence of an inhibitor, the first polypeptide and the second polypeptide form a complex, thereby activating activity of the enzyme.
   56. A cell in accordance with aspect 55, wherein the the polypeptide of any one of aspects 47-52 comprises the amino terminal portion of the enzyme and a KSR dimerization sequence, and the polypeptide of any one of aspects 53-54 comprises the carboxy terminal portion of the enzyme and a dimerization domain of a KSR dimerization partner.   57. A cell in accordance with aspect 55, wherein the the polypeptide of any one of aspects 47-52 comprises the carboxy terminal portion of the enzyme and a KSR dimerization sequence, and the polypeptide of any one of aspects 53-54 comprises the amino terminal portion of the enzyme and a dimerization domain of a KSR dimerization partner.   58. A cell in accordance with any one of aspects 55-57, wherein the enzyme is a luciferase.   59. A cell in accordance with any one of aspects 55-58, wherein the enzyme is a Ranilla luciferase.   60. A cell in accordance with any one of aspects 55-59, wherein the cell is a eukaryotic cell.   61. A cell in accordance with aspect 60, wherein the eukaryotic cell is a mammalian cell.   62. A cell in accordance with aspect 61, wherein the mammalian cell is selected from the group consisting of a human cell, a murine cell, and rat cell.   63. A method of screening for an inhibitor of KSR dimerization, comprising:   
 
         [0127]    providing a culture comprising a cell in accordance with any one of aspects 55-59; 
         [0128]    contacting the culture with a candidate inhibitor of KSR dimerization; and 
         [0129]    detecting the presence, absence, or quantity of activity of the enzyme, whereby a decrease in activity compared to a control indicates that the candidate inhibitor has activity as a KSR dimerization inhibitor. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0130]      FIG. 1  illustrates that RAF inhibitors can induce dimer formation between KSR and RAF, and activate KSR by CRAF. 
           [0131]      FIG. 2  illustrates that the ability of KSR to bind ATP is required for the function of KSR. 
           [0132]      FIG. 3  illustrates modeling the structural effects of the alanine-to-phenylalanine change in CRAF and BRAF. 
           [0133]      FIG. 4  illustrates that an A-to-F mutation in RAF can induce dimer formation and can activate ERK signaling. 
       
    
    
     DETAILED DESCRIPTION 
       [0134]    The present inventors demonstrate that the activity of both kinase-active and -inactive BRAF mutants require KSR for their function. In various configurations, KSR is required when BRAF is directly phosphorylating MEK (V600E) or when BRAF is activating MEK through activation of CRAF (kinase-dead BRAF). KSR can function to bring both MEK and BRAF to CRAF. Since MEK and BRAF binding to KSR are constitutive (McKay, M. M., Proc. Nat&#39;l. Acad. Sci. USA 106, 11022-11027, 2009), activation of the pathway can involve the induced recruitment of CRAF. 
         [0135]    The present inventors demonstrate that MEK phosphorylation can be mediated by KSR catalytic activity. 
         [0136]    By mutating the conserved Ala in the catalytic spine to Phe of KSR, CRAF and BRAF, the present inventors created an adenine mimetic that can stabilize the closed conformation of the kinase core that includes the dimer interface but renders the kinase inactive. These pseudokinases that were generated assume a conformation that resembles the active kinase but because they can&#39;t bind ATP, they are unambiguously catalytically dead. All previous known strategies to inactivate kinase activity results in a dynamic kinase with impaired scaffolding function. 
         [0137]    Because some of the scaffolding functions of kinases require the active conformation, the present inventors demonstrate that the alanine to phenylalanine mutant is unique because it can stabilize the scaffolding function. The mutants can be used to separate the scaffolding properties of BRAF, CRAF and KSR from their catalytic activity. In the case of BRAF, the A481 F mutant, can constitutively activate MEK and ERK in a manner that is kinase independent, RAS independent but KSR dependent. The RAS independence is similar to the V600E mutant and both the V600E and A481 F mutations can uncouple the inhibitory amino-terminal domain from the kinase domain. Since the AF mutant lacks catalytic activity, the scaffolding and not the kinase function of the BRAF V600E mutant can be sufficient to account for its transformation activity. 
         [0138]    The A587F mutant of KSR can still retain scaffolding function as it can dimerize with BRAF and CRAF and still bind to MEK. The inventors&#39; findings establish at least two functions of KSR: it not only has the scaffolding function; ATP binding and kinase activity are also functional properties of KSR. 
         [0139]    A mutant of the present teachings that induces the closed, active conformation but is catalytically active can be used to separate the two different functions of kinases. While BRAF could function as a scaffold alone, the requirement for both CRAF and KSR to bind to ATP for downstream activation of MEK and ERK demonstrate that both can function as kinases and can have distinct functions from BRAF. 
         [0140]    By mutating the Leu to the catalytic spine Phe of KSR, the present inventors created a mimetic that can stabilize the closed conformation of the kinase core that excludes the dimer interface and therefore cannot bind to CRAF or BRAF. This leucine-to-phenylalanine mutant maintains a constitutively active kinase conformation but is independent of BRAF and CRAF. Furthermore, KSR L591F can bind ATP. 
       Methods 
       [0141]    The methods and compositions described herein utilize laboratory techniques well known to skilled artisans, and can be found in laboratory manuals such as Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Spector, D. L. et al., Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; Ausubel, F. M., et al., ed., Current Protocols in Molecular Biology, Wiley Interscience, 2003. These and all other publications cited in this disclosure are incorporated herein by reference, each in its entirety. As used in the description and any appended claims, the singular forms “a”. “an” and “the” are intended to include the plural forms as well, unless the context indicates otherwise. 
         [0142]    Some Examples set forth infra may include the following materials and methods. 
         [0143]    Chemicals: PLX4720 and GDC0879 were purchased from Selleck Chemicals. 
         [0144]    Antibodies: Phospho-ERK (T202-Y204) and phospho-MEK (S217/S221) antibodies were purchased from Cell Signaling. 
         [0145]    Dimerization experiments: For most experiments, constructs for wild-type and mutated BRAF, CRAF and KSR1were appended with epitope tags (FLAG, 6× His, Myc, GFP) and were expressed by transient transfection into 293T cells. Cells were lysed in a buffer containing 1% NP40 and 0.1% deoxycholate. Cells were pretreated with drugs for 1 hour prior to lysis. Immunoprecipitates were analyzed by gel electrophoresis and immunoblotted after transfer to nitrocellulose membranes using standard methods. 
         [0146]    ATP binding assay: WT and mutated KSR1 constructs epitope tagged with 6× His were expressed in 293T cells and purified using Ni-NTA agarose. ATP binding was assessed by incubating the samples with 100 uM biotin-azido-ATP (2-azidoadeosine, 5′ triphosphate [γ] - - - 5-biotinpentylamine, Affinity Probes) in a buffer containing 20 mM Na 2 HPO 4 /NaH 2 PO 4  (pH 7.2) and 10mM MgCl 2 . After incubation on ice for 5 min, samples were irradiated by UV for 2 min. The ATP-crosslinked KSR or mutants in samples were examined by SDS-PAGE and Western blotting with strepavidin-HRP. 
         [0147]    Kinase reactions: Cells transfected with various constructs were treated or not with PLX4720 for 1-2 hours. Cells were lysed with 1% NP40 and immunoprecipitates prepared. In vitro kinase reactions were performed in a standard buffer with 10 mM MgCl 2 , with 1 ug of kinase dead MEK, and 100 uM cold ATP. In some experiments, to inhibit contaminating Raf activity, 50 uM PLX4720 was preincubated with the reactions. 
       EXAMPLES  
       [0148]    The present teachings including descriptions provided in the Examples that are not intended to limit the scope of any claim or aspect. Unless specifically presented in the past tense, an example can be a prophetic or an actual example. The following non-limiting examples are provided to further illustrate the present teachings. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings. 
       Example 1 
       [0149]    This example demonstrates that BRAF inhibitors can induce KSR/RAF dimers. 
         [0150]    In these experiments, two different RAF inhibitors, GDC0879 and PLX4720 were used. While the drugs are structurally unrelated, both drugs were selected for their ability to inhibit a constitutively active form of BRAF (V600E) but also bind with lower affinities to all of the wild-type RAF isoforms (Hatzivassiliou, G., et al., Nature 464, 431-435, 2010; Therrien, M., et al., Cell 83. 879-888, 1995). Crystallography studies show that both drugs are Type I inhibitors that induce formation of the closed conformation of the kinase (Hatzivassiliou, G., et al., Nature 464, 431-435, 201; Therrien, M., et al., Cell 83, 879-888, 1995). While previous reports showed that most RAF inhibitors induce the formation of BRAF/CRAF dimers supporting this as a potential mechanism for RAF activation (Hatzivassiliou, G., et al., Nature 464, 431-435, 2010; Heidorn, S. J., et al., Cell 140, 209-221, 2010; Poulikakos, P. I., et al., Nature 464, 427-430, 2010), this mechanism is not supported by the fact that PLX4720 cannot induce dimers between BRAF and CRAF and by the fact that drug induced ERK stimulation does not require BRAF (Hatzivassiliou, G., et al., Nature 464, 431-435, 2010; Heidorn, S. J., et al., Cell 140, 209-221, 2010; Poulikakos, P. I., et al., Nature 464, 427-430, 2010). 
         [0151]    Since KSR can also form complexes with BRAF and with CRAF (McKay. M. M., et al., Proc. Nat&#39;l. Acad. Sci. USA 106, 11022-11027, 2009; Rajakulendran, T., et al., Nature 461, 542-545, 2009), we tested whether RAF inhibitors could enhance dimer formation between RAF and KSR. Cells grown in serum, expressing combinations of KSR, BRAF and CRAF, were treated with both drugs. Co-immunoprecipitations were then performed to examine dimer formation. 
         [0152]      FIG. 1 : RAF inhibitors induce dimer formation between KSR and RAF, and activate KSR by CRAF.  FIG. 1A . GDC0879 but not PLX4720 induces dimers between BRAF and CRAF. Cells overexpressing myc-CRAF and BRAF were treated with drug for 1 hour and CRAF immunoprecipitates were immunoblotted for BRAF and CRAF (myc).  FIG. 1B . GDC0879 but not PLX4720 can induce dimer formation between FLAG-KSR and BRAF. KSR immunoprecipitates were prepared from cells overexpressing FLAG-KSR and BRAF after treatment with the indicated drug for 1 hour and immunoblotted using antibodies to BRAF.  FIG. 1C . Both GDC0879 and PLX4720 induce dimer formation between KSR and CRAF. FLAG-KSR immunoprecipitates were prepared from cells overexpressing FLAG-KSR and myc-CRAF after treatment with the indicated drug for 1 hour and immunoblotted for CRAF using myc antibodies. 
         [0153]    As reported previously (Hatzivassiliou, G., et al., Nature 464, 431-435, 2010; Heidorn, S. J., et al., Cell 140, 209-221, 2010; Poulikakos, P. I., et al., Nature 464, 427-430, 2010), GDC0879 but not PLX4720 induced BRAF/CRAF dimer formation ( FIG. 1A ). However, both drugs induce d dimers between KSR and CRAF and enhanced dimer formation between KSR and BRAF (FIG.  1 B/C). This suggested that KSR complexes induced by the drug might explain the positive effects of the BRAF inhibitors. 
       Example 2  
       [0154]    This Example illustrates that BRAF inhibitor-induced ERK activation requires KSR. 
         [0155]    In these experiments, we used KSR deficient cells (Nguyen, A., et al., Mol. Cell Biol. 22, 3035-3045, 2002) to determine whether KSR was required for the ability of the drugs to induce ERK activation. Cells transduced with constitutively active RAS (V12) or grown in serum were treated with various doses of each drug and activation was assessed by immunoblotting cell lysates with an antibody that detects active ERK. As reported previously, treatment of wild-type cells with either drug strongly induced ERK activation at low to intermediate doses but inhibited ERK activation at higher doses (Hatzivassiliou, G., et al., Nature 464, 431-435, 2010; Heidorn, S. J., et al., Cell 140, 209-221, 2010; Poulikakos, P. I., et al., Nature 464, 427-430, 2010) (FIG.  1 D/E). Similar results were obtained with cells expressing constitutively active RAS (FIG.  1 D/E) or after serum treatment (data not shown). Strikingly, ERK activation was almost undetectable in KSR deficient cells after drug treatment with either drug (FIG.  1 D/E).  FIG. 1D-E : Lysates, obtained from wild-type and KSR-deficient fibroblasts transfected with RasV12 and treated with the indicated doses of either GDC-0879 ( FIG. 1D ) or PLX4720 ( FIG. 1E ) for 1 hour, were immunoblotted for phospho-ERK1 and 2, ERK2 and RasV12. 
         [0156]    Our data demonstrate that the ability of RAF inhibitors to activate ERK requires the presence of KSR. Given previous reports, demonstrating that CRAF and not BRAF is required for the positive effect of the drugs on ERK activation (Hatzivassiliou, G., et al., Nature 464, 431-435, 2010; Poulikakos, P. I., et al., Nature 464, 427-430, 2010), our data suggest that drug induced CRAF/KSR dimers may be the relevant complex. 
       Example 3 
       [0157]    This example illustrates that KSR is a MEK kinase activated by CRAF. 
         [0158]    In these experiments, we tested the function of the CRAF/KSR dimer by co-expressing both proteins and using PLX4720 ( FIG. 1F ) or GDC0879 (data not shown) to induce dimer formation between the two proteins. Because drug treatment is expected to induce activation of MEK and ERK, we treated cells with a saturating dose that would be expected to induce dimers but also inhibit CRAF activity. Under these conditions, we found that MEK was still activated suggesting that the presence of KSR might be effecting the function of the drugs ( FIG. 1F ). Importantly, a mutated form of CRAF (CRAF™) that is unable to bind to the drug, did not result in phosphorylation of MEK. This result suggested that induction of the CRAF/KSR dimer might function to activate kinase activity towards MEK.  FIG. 1F : KSR and CRAF cooperate to activate MEK. Cells expressing the indicated constructs were treated with a saturating dose of PLX for 2 hours before cell lysates were prepared and analyzed for pMEK by immunoblotting. CRAF™ refers to the T421M gatekeeper mutant that cannot bind to the drug (Heidorn, S. J., et al., Cell 140, 209-221, 2010). 
         [0159]    We tested the possibility that KSR might have kinase activity by performing KSR in vitro kinase reactions. Consistent with previous reports, when KSR was expressed alone, we failed to detect KSR kinase activity in vitro against purified RAF (data not shown) or MEK ( FIG. 1G ). To test whether KSR might be activated by CRAF, we co-expressed KSR and CRAF and induced dimerization of CRAF with KSR by adding a low dose (10 μM) of PLX4720. KSR immunoprecipitates were then prepared and tested for kinase activity in vitro. To inhibit any contaminating RAF kinase activity co-precipitating with KSR, we pre-incubated the immunoprecipitates with an inhibitory dose of PLX4720 (50 μM). Treatment of cells with PLX4720 induced kinase activity towards MEK in the KSR immunoprecipitates and only occurred when KSR and CRAF were co-expressed together ( FIG. 1G ). This suggests that dimerization of KSR and CRAF activates KSR allowing it to phosphorylate MEK.  FIG. 1G : KSR in vitro kinase reactions. Cells were co-transfected with WT or ATP binding deficient KSR and CRAF and immunoprecipitates prepared after cells were treated with an activating dose of PLX (10 μM) for one hour. Immunoprecipitates were prepared, pre-treated with 50 μM PLX to inhibit co-precipitating RAF activity and then tested for kinase activity using purified MEK. MEK phosphorylation was detected using a phosphospecific antibody that recognizes active MEK. 
       Example 4 
       [0160]    This Example illustrates that ATP binding to KSR is required for its function. 
         [0161]    In these experiments, to confirm the ability of KSR to function as a kinase, we were interested to generate a kinase-inactive mutant. Typically, substitution of the catalytic lysine with arginine or methionine can be used to ablate catalytic activity in most kinases (Gibbs, C. S., et al., J. Biol. Chem. 267, 4806-4814, 1992). Mammalian KSR lacks the catalytic lysine, partly explaining why it has always been considered to be an inactive pseudokinase. Recently several kinases lacking the catalytic lysine have been found to have kinase activity suggesting that new mutagenesis strategies might be needed to ablate kinase activity (Taylor, S. S. et al., Proc. Nat&#39;l. Acad. Sci. USA 107, 8047-8048, 2010). Because mutations that disrupt catalytic activity still allow binding of ATP to the kinase (Iyer, G. H., et at., J. Mol. Biol. 351, 1110-1122, 2005), we sought to generate a KSR mutant that could not bind ATP and thus could not possess any catalytic activity. Based on the conserved structure of protein kinases, we reasoned that substituting the highly conserved alanine (A587) residue located in the back of the ATP binding pocket with a bulky hydrophobic residue might block ATP binding. Testing mutants for ATP binding using a biotin-ATP analog ( FIG. 2A ) demonstrated that substitution of A587 with phenylalanine, but not valine disrupted ATP binding.  FIG. 2  illustrates that the ability of KSR to bind ATP is required for the function of KSR.  FIG. 2A : mutagenesis was performed to substitute phenylalanine or valine for A587 of mouse 6× His-KSR1. Each mutant was expressed in cells, purified using Ni 2+  agarose and tested for ATP binding using a biotinylated-ATP analog after UV cross-linking and immunoblotting for the presence of biotin. 
         [0162]    The function of the ATP binding deficient (A587F) KSR mutant was tested by reconstituting KSR deficient cells with either wild-type or one of the two KSR mutants, A587F or A587V. Because expression levels can affect the function of KSR, we used cell sorting of KSR-YFP fusion proteins to isolate stable cell lines with equivalent levels of KSR expression. EGF mediated ERK activation was then tested ( FIG. 2B ). While wild-type KSR and the ATP binding A587V mutant were both able to rescue ERK activation, the ATP binding deficient A587F mutant did not hilly rescue ERK activation in KSR deficient cell lines.  FIG. 2B : KSR deficient fibroblasts were reconstituted with YFP-fused to wild-type or mutated KSR and sorted to generate cell lines with similar expression levels. Cells were stimulated with EGF for the indicated times and cell lysates were immunoblotted with an antibody to phosphorylated ERK (pERK). 
         [0163]    We confirmed the inability of A587F to reconstitute KSR function using a Ras transformation assay ( FIG. 2C ). As cell transformation by RasV12 is dependent on KSR16, we transduced cell lines generated above with RasV12 and assessed cell transformation by focus-formation ( FIG. 2C ) or soft-agar assay (data not shown). While the wild-type and A587V mutants supported RasV12 transformation, the A587F mutant could not. Thus, replacement of alanine 587 of KSR with phenylalanine disrupts ATP binding and abrogates KSR function.  FIG. 2C : Stably transfected KSR deficient cell lines, described in  FIG. 2B , were transfected with an expression vector for RasV12 and assessed for transformed colony formation. 
         [0164]    We confirmed this function of the mutants in  Drosophila  cells because overexpression of  Drosophila  KSR (dKSR) with  Drosophila  RAF (dRAF) is able to activate MEK by itself (Rajakulendran, T., et al., Nature 461, 542-545, 2009). We generated the analogous mutations in dKSR (A703V and A703F) and expressed them in  Drosophila  S2 cells with dRAF ( FIG. 2D ). Consistent with our previous results, the A703V mutant was still able to activate MEK while the A703F mutant had no effect. Lastly, we tested the KSR A587F mutant for kinase activity as described in  FIG. 1G . In contrast to wild-type KSR that showed kinase activity towards MEK, no kinase activity was detected in KSR A587F immunoprecipitates. These results show that replacement of alanine 587 of KSR with phenylalanine disrupts ATP binding and that ATP binding is required for KSR function.  FIG. 2D :  Drosophila  KSR mutant analogues were generated by site-directed mutagenesis, and coexpressed with Pyo-dBRAF and GFP-MEK in S2 cells. Cell lysates were immunoblotted for pMEK, GFP-MEK, V5-KSR and Pyo-dBRAF. 
       Example 5 
       [0165]    This Example illustrates that KSR A587F mutant forms constitutive dimers with CRAF. 
         [0166]    Kinases have two different functions, catalytic and scaffold. Since the scaffold function of KSR requires its ability to dimerize with RAF (McKay, M. M., Proc. Nat&#39;l. Acad. Sci. USA 106, 11022-11027, 2009; Rajakulendran, T., et al., Nature 461, 542-545, 2009) and to bind MEK, we thus tested the KSR alanine to phenylalanine mutant for its ability to bind to RAF ( FIG. 2  E/F/G) as well as to MEK ( FIG. 2H ). First, using the A703F mutant of dKSR to assess dimer formation between dKSR and dBRAF, we found, surprisingly, that dKSR/dBRAF dimers were promoted by the A703F mutation. To confirm whether this also occurred in mammalian KSR, we coexpressed the A587F KSR mutant with CRAF or BRAF and assessed dimer formation by co-immunoprecipitation ( FIG. 2 ). While the A587F mutant now forms constitutive dimers with CRAF, it had little to no effect on dimer formation with BRAF ( FIG. 2 ). The lack of any effect may be related to the high constitutive levels of KSR/BRAF dimers found in most cells. Lastly, the A587F mutation did not effect KSR binding to MEK ( FIG. 2H ). Thus, the two known scaffold functions of KSR are preserved.  FIG. 2E : To access dimer formation, dBRAF immunoprecipitates were prepared from S2 cells that coexpressed dBRAF with either wild-type or mutated dKSR and immunoblotted for dKSR(V5) and dBRAF(pyo).  FIG. 2F : Constitutive dimer formation between A587F KSR and CRAF. FLAG-KSR immunoprecipitates were prepared from lysates from cells expressing A587F FLAG-KSR with myc-CRAF and immunoblotted for CRAF (myc).  FIG. 2G : No effect of A587F KSR mutation on binding to BRAF. Experiment was performed as described in F, except BRAF was used instead of CRAF.  FIG. 2H : A587F KSR mutation does not effect MEK binding. KSR immunoprecipitates from cell co-expressing GFP-MEK1 and WT or KSR mutants were immunoblotted for GFP-MEK and KSR (FLAG). 
       Example 6 
       [0167]    This Example illustrates that molecular modeling suggests that the A587F mutation induces the closed, active conformation of KSR. 
         [0168]    The ability of the A587F mutant of KSR to induce constitutive dimer formation suggested that the phenylalanine substitution might be affecting the conformation of the kinase domain of KSR. A recent study of features conserved in the structures of active kinases and not present in the structures of inactive kinases suggests that kinase activation involves the formation of two hydrophobic spines, the catalytic and regulatory hydrophobic spines (Taylor, S. S. et al., Trends Biochem. Sci., 2010) ( FIG. 3A ). The formation of these two hydrophobic spines during the process of kinase activation serves to generate a hydrophobic core that stablizes the active conformation of the kinase. In the catalytic hydrophobic spine of PKA, a conserved alanine (A70) from the upper lobe and a conserved leucine (L173) from the lower lobe interact with the top and bottom of the adenine ring from ATP to bring the two lobes of the kinase together. Alanine 587 of KSR corresponds to the conserved alanine residue from the upper lobe and the leucine in the lower lobe (173 of PKA) corresponds to phenylalanine (690) of KSR. 
         [0169]    We first analyzed the published structure of CRAF bound to GDC08792, a Type I inhibitor, and confirmed that drug binding induced the formation of both the catalytic and regulatory spines ( FIG. 3 ). In contrast, analysis of a structure of BRAF complexed with Sorafenib (Wan, P. T., et al. Cell 116, 855-867, 2004), a Type II inhibitor, was consistent with an inactive kinase without assembly of the hydrophobic spines ( FIG. 3 ). Using energy minimization modeling, the structure of CRAF with alanine replaced by phenylalanine was modeled. The results showed that the phenylalanine residue in CRAF position 573 can complete the catalytic hydrophobic spine by interacting with phenylalanine 690 in the lower lobe. This interaction induces the closed, active conformation of the kinase ( FIG. 3 ). This model suggested that the A587F mutant of KSR mimics ATP binding resulting truly in a pseudokinase that is conformationally active but catalytically inert because it can no longer bind to ATP. 
         [0170]      FIG. 3  illustrates modeling the structural effects of the alanine to phenylalanine change in CRAF and BRAF. The position of residues constituting the hydrophobic spines of CRAF crystallized with a Type I inhibitor (stabilizes the closed and ATP bound form of the kinase) are shown in  FIG. 3A  while the hydrophobic spine residues in BRAF bound to a Type II inhibitor (binds to the open conformation preventing closing of the cleft) is shown in  FIG. 3B . Components of the catalytic hydrophobic spine are indicated by thin arrows while components of the regulatory hydrophobic spine are indicated by thick arrows. Note the contiguous residues of induced by the Type I inhibitor indicated by asterisk in.  FIG. 3A  while the pattern of these residues is interrupted in  FIG. 3B , in which the inhibitor is also indicated by an asterisk. Note also how the drug molecule in  FIG. 3A  functions to connect components of the catalytic hydrophobic spine in the upper and lower lobes of the kinase. In  FIG. 3C , a simulated structure of CRAF where A373 is replaced with Phe is shown. Energy minimization was done using the program TINKER. 
       Example 7 
       [0171]    This Example illustrates that Analogous A to F mutations in BRAF and CRAF induce dimer formation. 
         [0172]    In these experiments, to test the generality of this hypothesis, we generated analogous mutations in BRAF and CRAF. Co-immunoprecipitation assays showed that BRAF A481 F formed constitutive dimers with CRAF and that CR AF A373F formed constitutive dimers with BRAF ( FIG. 4A ). The CRAF A373F mutant also formed constitutive dimers with KSR but the BRAF A481 F did not enhance basal dimer formation with KSR ( FIG. 4B ). 
         [0173]    Since the AF mutants appear to induce the closed, active conformation of all three kinases, we reasoned that we could use these mutants to distinguish between their functions as enzymes or as scaffolds. All three AF mutants (BRAF, CRAF and KSR) were over-expressed in cells and tested for their effects on endogenous ERK activation ( FIG. 4C ). Consistent with previous work showing that kinase-inactive forms of BRAF can stimulate the activation of MEK and ERK, overexpression of the BRAF A481 F mutant resulted in constitutive activation of ERK. However, co-expression of a dominant negative RAS (N17), showed that its ability to activate ERK was RAS independent ( FIG. 4C ). The ability of kinase dead BRAFs to activate ERK usually requires RAS activation (Heidorn, S. J., et al., Cell 140, 209-221, 2010; Wan, P. T., et al. Cell 116, 855-867, 2004) presumably because this is required to induce the active conformation of BRAF by releasing the inhibitory N-terminal domain. The RAS independence of A481 F BRAF supports the idea that the phenylalanine mutation is sufficient to induce the active conformation of the kinase domain but also results in displacement of the inhibitory N-terminal domain. The RAS independence of A481 F BRAF thus resembles the V600E mutant of BRAF and suggests provocatively that the greatly increased kinase activity of BRAF V600E need not be the only reason it is oncogenic. Rather, the scaffold function and not its kinase activity of BRAF is required. 
         [0174]    We tested whether ERK activation by BRAF A481 F or BRAF V600E required KSR by expressing each construct in the KSR deficient cell line (FIG.  4 D/E). The ability of both proteins to activate ERK was significantly compromised in the absence of KSR. This supports the idea that the mechanism of function of both A481F and V600E are similar and dependent on the presence of KSR. In contrast, overexpression of CRAF A373F or KSR A587F had no constitutive effects on ERK activation ( FIG. 4C ). As both mutants form constitutive dimers with each other, and as shown above ( FIG. 2 ), dimerization induces MEK phosphorylation, these results suggest that both proteins need to be enzymatically active. 
         [0175]      FIG. 4  illustrates that an A to F mutation in RAF can induce dimer formation and activate ERK signaling.  FIG. 4A  illustrates that phenylalanine substitutions in CRAF and BRAF allow for constitutive CRAF/BRAF dimers. The myc-CRAF A373F and the BRAF A481 F mutants were co-expressed with wild-type BRAF or wild-type myc-CRAF respectively and heterodimers assessed by co-immunoprecipitation.  FIG. 4B  illustrates that CRAF but not the BRAF phenylalanine substitution allows enhanced KSR dimer formation. In these experiments, the myc-CRAF A373F and the BRAF A481 F mutants were co-expressed with wild-type FLAG-KSR and heterodimers assessed by co-immunoprecipitations.  FIG. 4C  illustrates that expression of BRAF A481 F stimulates Ras independent ERK activation in cells. In these experiments, cells were transiently transfected with expression constructs for BRAF A481 F, myc-CRAF A373F or FLAG-KSR A587F mutants. Lysates were immunoblotted with antibodies to pERK after 18 hours. The effect of BRAF A481 F was not inhibited by co-expression of dominant negative Ras (N17).  FIG. 4D  illustrates ERK phosphorylation. In these experiments, WT and ksr −/−  MEFs were transfected with BRAF(A481 F). 24 hours later, cells were treated with or without 20 uM GDC0879 for 60 min, before lysis. ERK phosphorylation was assessed by immunoblotting. Immunoblotting for total ERK2 was used as a loading control.  FIG. 4E  illustrates WT and ksr −/—  MEFs transfected with BRAF(V600E) and prepared as described in  FIG. 4D . 
       Example 8 
       [0176]    This example illustrates an in vitro kinase assay that can be used to identify an inhibitor of KSR. 
         [0177]    HeLa cells can be transiently transfected with expression constructs for FLAG-KSR L591F. Cells can be treated with a candidate inhibitor of KSR kinase activity for 60 min before lysis. Cells then can be lysed with buffer containing 20 mM HEPES (pH 7.5). 50 mM GP, 100 M sodium vanadate, 2 mM magnesium chloride, 1 mM EGTA, 0.5% Triton X-100, 5 g/ml leupeptin, 21 g/ml aprotinin and 1 mM DTT. Protein concentration can be determined using the method of Bradford and KSR can be immunoprecipitated from 400 g of cell lysate with an anti-FLAG antibody. Immunoprecipitates can be washed and in vitro kinase assays can be carried out at 30° C. for 20 min in buffer containing 20 mM HEPES (pH 7.5), 50 mM β-glycerophosphate, 100 μM sodium vanadate, 20 mM magnesium chloride, 0.1 mM EGTA, 0.2 mM ATP, 10 μCi [γ −32 P]ATP (ICN Biologicals), 50 μg/ml IP-20 peptide and 80 μM of MEK peptide as a selective substrate for KSR L591F activity. The kinase reaction can be terminated by the addition of SDS sample buffer (0.31 M Tris pH 6.8, 11.5% SDS, 50 mM DTT, 50% glycerol), samples can be boiled, and then size fractionated by SDS-PAGE, and 32P-labeled MEK can be visualized by autoradiography. PhosphorImager analysis can be utilized to quantify the relative differences in MEK phosphorylation as a measure of KSR L591 F activity in the absence or presence of an inhibitor. 
         [0178]    As used in the description and the appended claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context indicates otherwise. 
         [0179]    All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.