Regulation of RAF Activity by 14-3-3 Proteins
Mammalian 14-3-3 proteins play a crucial role in the activation process of RAF kinases. However, little is known about the selectivity of the mammalian 14-3-3 isoforms with respect to RAF association and activation. Using mass spectrometry, we analyzed the composition of the 14-3-3 isoforms attached to RAF kinases and found that B-RAF associates in vivo with 14-3-3 at much higher diversity than A- and C-RAF. We also examined in vitro binding of purified mammalian 14-3-3 proteins to RAF kinases using surface plasmon resonance techniques. While B- and C-RAF exhibited binding to all seven 14-3-3 isoforms, A-RAF bound with considerably lower affinities to ϵ, τ, and σ 14-3-3. These findings indicate that 14-3-3 proteins associate with RAF isoforms in a pronounced isoform-specific manner. Because 14-3-3 proteins appear in dimeric forms, we addressed the question of whether both homo- and heterodimeric forms of 14-3-3 proteins participate in RAF signaling. For that purpose, the budding yeast Saccharomyces cerevisiae, possessing only two 14-3-3 isoforms (BMH1 and BMH2), served as testing system. By deletion of the single BMH2 gene, we found that both homo- and heterodimeric forms of 14-3-3 can participate in RAF activation. Furthermore, we show that A-, B-, and C-RAF activity is differentially regulated by its C-terminal and internal 14-3-3 binding domain. Finally, prohibitin, a scaffold protein that affects C-RAF activation in a stimulatory manner, proved to interfere with the internal 14-3-3 binding site in C-RAF. Together, our results shed more light on the complex mechanism of RAF activation, particularly with respect to activation steps that are mediated by 14-3-3 proteins and prohibitin. Mammalian 14-3-3 proteins play a crucial role in the activation process of RAF kinases. However, little is known about the selectivity of the mammalian 14-3-3 isoforms with respect to RAF association and activation. Using mass spectrometry, we analyzed the composition of the 14-3-3 isoforms attached to RAF kinases and found that B-RAF associates in vivo with 14-3-3 at much higher diversity than A- and C-RAF. We also examined in vitro binding of purified mammalian 14-3-3 proteins to RAF kinases using surface plasmon resonance techniques. While B- and C-RAF exhibited binding to all seven 14-3-3 isoforms, A-RAF bound with considerably lower affinities to ϵ, τ, and σ 14-3-3. These findings indicate that 14-3-3 proteins associate with RAF isoforms in a pronounced isoform-specific manner. Because 14-3-3 proteins appear in dimeric forms, we addressed the question of whether both homo- and heterodimeric forms of 14-3-3 proteins participate in RAF signaling. For that purpose, the budding yeast Saccharomyces cerevisiae, possessing only two 14-3-3 isoforms (BMH1 and BMH2), served as testing system. By deletion of the single BMH2 gene, we found that both homo- and heterodimeric forms of 14-3-3 can participate in RAF activation. Furthermore, we show that A-, B-, and C-RAF activity is differentially regulated by its C-terminal and internal 14-3-3 binding domain. Finally, prohibitin, a scaffold protein that affects C-RAF activation in a stimulatory manner, proved to interfere with the internal 14-3-3 binding site in C-RAF. Together, our results shed more light on the complex mechanism of RAF activation, particularly with respect to activation steps that are mediated by 14-3-3 proteins and prohibitin. The serine/threonine-specific RAF kinases play a central role in several normal and pathologic cellular processes including proliferation, differentiation, cell cycle progression, senescence, and apoptosis (1Wellbrock C. Karasarides M. Marais R. Nat. Rev. Mol. Cell Biol... 2004; 5: 875-885Google Scholar, 2Rapp U.R. Gotz R. Albert S. Cancer Cell.. 2006; 9: 9-12Google Scholar). The first RAF kinase was originally discovered as the oncogenic product of mouse sarcoma virus 3611 (3Rapp U.R. Goldsborough M.D. Mark G.E. Bonner T.I. Groffen J. Reynolds Jr., F.H. Stephenson J.R. Proc. Natl. Acad. Sci. U. S. A... 1983; 80: 4218-4222Google Scholar). Although invertebrates encode only a single RAF kinase, vertebrates express three isoforms, designated as A-, B-, and C-RAF. The C-RAF gene encodes a protein of 648 amino acids that is expressed as a 74-kDa polypeptide (4Bonner T.I. Kerby S.B. Sutrave P. Gunnell M.A. Mark G. Rapp U.R. Mol. Cell. Biol... 1985; 5: 1400-1407Google Scholar). A-RAF is a 68-kDa protein showing 60% homology to C-RAF (5Huleihel M. Goldsborough M. Cleveland J. Gunnell M. Bonner T. Rapp U.R. Mol. Cell. Biol... 1986; 6: 2655-2662Google Scholar). B-RAF is expressed as a full-length protein of 95 kDa or as smaller splice variants (6Stephens R.M. Sithanandam G. Copeland T.D. Kaplan D.R. Rapp U.R. Morrison D.K. Mol. Cell. Biol... 1992; 12: 3733-3742Google Scholar). All RAF proteins share a similar structure and possess three conserved regions, CR1, CR2, and CR3, that are embedded between variable segments. The CR1 and CR2 domains are part of the regulatory N-terminal half of the RAF proteins, whereas CR3 represents the C-terminal kinase domain. CR1 contains a Ras binding domain and a zinc binding domain, also called cysteine-rich domain. Although all RAF isoforms share a high degree of sequence similarity, they are obviously under different regulation and may have individual functions, mediated by isoform-specific protein-protein interactions (1Wellbrock C. Karasarides M. Marais R. Nat. Rev. Mol. Cell Biol... 2004; 5: 875-885Google Scholar, 7Fischer A. Hekman M. J. S. Rapp U.R. J. Scholar, A. T. Hekman M. Rapp U.R. J. Scholar). are in RAF activation process and are to Although several are RAF of the of RAF the of of C-RAF D.K. Kaplan D.R. Rapp U. Proc. Natl. Acad. Sci. U. S. A... Scholar). are three of regulatory 14-3-3 proteins Cell.. A. R. and S. Marais R. Cell.. 2004; Scholar). Morrison D.K. G. Rapp U.R. Copeland T.D. J. three in that the at and of and are in binding of 14-3-3 proteins to C-RAF. of to C-RAF activation, as the of to in a RAF protein that by G. J. Scholar, M. S. R. Albert S. J. J. M. Rapp U.R. J. 2004; Scholar). of by or in of kinase that of is D.K. G. Rapp U.R. Copeland T.D. J. Scholar, S. Scholar, S. M. Scholar, M. S. M. J. Scholar, M. M. M. M. J. Scholar). Furthermore, the binding of 14-3-3 proteins to the C-terminal conserved site found to differentially and regulated M. S. R. Albert S. J. J. M. Rapp U.R. J. 2004; Scholar). by A. Mol. Biol... and by and Morrison Morrison D.K. J. Cell 2004; 14-3-3 proteins a of in the have to participate in the regulation of crucial cellular processes as cell cycle protein and The regulation of cellular processes by 14-3-3 several different protein protein protein and protein mammalian isoforms of 14-3-3 proteins have A. Mol. Biol... Scholar, S. A. Scholar, M. M. A. Scholar). The association of 14-3-3 with proteins high two of and are conserved and by all 14-3-3 binding only a the is 14-3-3 interactions are of A. Mol. Biol... Scholar). of 14-3-3 protein binding may of proteins the All of the 14-3-3 proteins that with proteins including protein kinase RAF kinase of Ras kinase of mass surface plasmon protein high and protein G. J. J. Scholar). The of the two of the mammalian isoforms, and by M. M. A. Scholar). found to in also with with and M. M. A. Scholar). The of the 14-3-3 and of the in vivo may have of 14-3-3 proteins as a or However, little is known about the of the the budding yeast Saccharomyces contains only two 14-3-3 and as with mammalian and that express isoforms, a of 14-3-3 interactions with by of mass spectrometry, a of in vivo 14-3-3 interactions of the yeast 14-3-3 proteins at association M. M. T. T. A. T. T. Scholar). These findings the and the complex role of the 14-3-3 proteins in the All three RAF kinases possess two 14-3-3 binding and in B-, and Although the C-terminal 14-3-3 protein binding of RAF kinases is the sequence in B-RAF the 14-3-3 binding in A- and C-RAF 14-3-3 binding site in C-RAF also Marais R. J. Scholar). Furthermore, 14-3-3 binding site at the C-terminal part of domain and to Ras binding domain J. Scholar). the of 14-3-3 binding a domain of Ras proteins J. Scholar, R. J. Mol. Biol... 2004; Scholar). the of with domain to by 14-3-3 Although is is that the N-terminal regulatory part of RAF in the with the domain a of the with 14-3-3 proteins may G. J. Rapp U.R. Scholar). on more Rapp U.R. Gotz R. Albert S. Cancer Cell.. 2006; 9: 9-12Google a in 14-3-3 proteins are the as as the of that RAF association with called represents the in the RAF activation a the association of RAF with and 14-3-3 RAF A. Hekman M. J. S. Rapp U.R. J. Scholar, M. J. J. Rapp U.R. J. Scholar). of 14-3-3 proteins RAF to of the internal 14-3-3 binding site by by several M. M. M. M. J. Scholar, C. Mol. Cell. Biol... Scholar, S. M. T.D. Morrison D.K. Biol... Scholar). a RAF activation in a and 14-3-3 C. Hekman M. C. Rapp U.R. T. Nat. Cell Biol... Scholar). the steps with respect to C-RAF activation that B- and C-RAF A. Hekman M. J. S. Rapp U.R. J. Scholar, J.R. Rapp U.R. Cancer Scholar, S. Marais R. Mol. Cell.. Scholar, Mol. Cell. Biol... 2006; Scholar). by our J.R. Rapp U.R. Cancer of the to in the C-terminal 14-3-3 binding of C-RAF considerably the of the that 14-3-3 proteins our M. S. R. Albert S. J. J. M. Rapp U.R. J. 2004; 14-3-3 association with RAF we with the of the interactions between and C-RAF. We examined the of C-RAF association with 14-3-3 proteins by surface plasmon resonance and found that the 14-3-3 binding domain represents the high and the binding of C-RAF activation in mammalian between 14-3-3 binding we the association of all seven mammalian 14-3-3 isoforms with RAF in vivo and in By of mass spectrometry, we analyzed the composition of the attached 14-3-3 isoforms to A-, B-, and C-RAF proteins and found that B-RAF associates in vivo with a much higher of 14-3-3 in binding of 14-3-3 proteins to RAF isoforms have using the By deletion of the gene in S. cerevisiae, we found that both homo- and heterodimeric forms of yeast 14-3-3 proteins participate in RAF activation. Furthermore, we show that the of the C-terminal 14-3-3 binding site by results in a of activity of all three RAF the of the internal 14-3-3 binding site in of B- and C-RAF The binding affinities of the single 14-3-3 binding domains in A-, B-, and C-RAF have using Finally, we show that prohibitin, a scaffold protein C-RAF activation in a stimulatory C. Hekman M. C. Rapp U.R. T. Nat. Cell Biol... the 14-3-3 binding at the internal 14-3-3 binding and was was and was B-, and C-RAF and protein and the was and of C-RAF Cell M. S. R. Albert S. J. J. M. Rapp U.R. J. 2004; Scholar). was the 14-3-3 binding domains of A-, B-, and C-RAF amino acids purified by high and the was by mass of RAF of A-, B-, and C-RAF full-length proteins mammalian cell a was For RAF proteins have to the sequence of of was gene was with and in with of the the by with and RAF proteins expressed by by amino acids including the on of RAF kinases in vivo or in regulation of A-, B-, and C-RAF kinase by 14-3-3 proteins, RAF and RAF possessing to the internal C-terminal 14-3-3 binding also and as in A. T. Hekman M. Rapp U.R. J. Scholar). The the 14-3-3 binding domains using to the by of and of using The The to by of and of as on the The by cell and the of the was by and and the with the The with These in in the of and of RAF and 14-3-3 and the of RAF the with the at a of of and at The with and at and of the and RAF kinases was as in Hekman M. S. R. Albert S. J. J. M. Rapp U.R. J. 2004; Scholar). For the of the RAF kinases the as was 14-3-3 proteins with in the of All seven mammalian 14-3-3 isoforms expressed in as proteins using and purified by the heterodimeric forms of 14-3-3 proteins, as the system. For that the with both and The 14-3-3 was by as The forms of and purified as in the as with whether 14-3-3 proteins purified in dimeric we using For that purpose, 14-3-3 by to a and by and All of the 14-3-3 protein at forms of 14-3-3 was purified as C. Hekman M. C. Rapp U.R. T. Nat. Cell Biol... Scholar). The of RAF prohibitin, and 14-3-3 proteins was by and with For the to and with or with A-, B-, and C-RAF and as in the to the the the with and by or proteins of in of with at and as A. T. Hekman M. Rapp U.R. J. Scholar). of 14-3-3 with and and with The in with and C-RAF was using with RAF as in Hekman M. S. R. Albert S. J. J. M. Rapp U.R. J. 2004; using and as the interactions between the purified RAF and different 14-3-3 isoforms, the was on a or at For that the was first with using and 14-3-3 proteins at a of in a of the purified RAF proteins as binding of 14-3-3 proteins to RAF also first C-RAF was by and purified 14-3-3 proteins the between RAF and 14-3-3 using was first by RAF proteins in the and of of the as in and the between RAF and 14-3-3 binding was by using to and the and to A. M. M. Scholar). with of and in the in a of in to and at The with of and to was The on a The using with a of The on a using a to The was to mass and three of the in the The the using the and the using the as of N-terminal as variable and and only and proteins with at two different with that Mammalian 14-3-3 with RAF in and processes are regulated by 14-3-3 A. Mol. Biol... Scholar, Morrison D.K. J. Cell 2004; Scholar, Cancer Biol... 2006; Scholar, Cancer Biol... 2006; the 14-3-3 and are on 14-3-3 isoforms in RAF signaling. whether mammalian 14-3-3 proteins associate with RAF we mass of RAF purified and several 14-3-3 isoforms attached to in both B- and C-RAF with and B-RAF found to with 14-3-3 isoforms τ, and that B-RAF may possess more in cell with C-RAF. to A-RAF was only by two 14-3-3 isoforms and Furthermore, we several proteins and attached to RAF isoforms The also in However, as also in the we to a RAF to B- and A-RAF with The results in 14-3-3 association with RAF about the and of the attached 14-3-3 proteins, binding of both homo- and heterodimeric forms are that single 14-3-3 isoforms, of in vivo appear as a as a is to M. M. A. Scholar). However, the composition of 14-3-3 the of RAF activation the the of purified 14-3-3 proteins and RAF kinases the of and of the single mammalian 14-3-3 proteins we our M. S. R. Albert S. J. J. M. Rapp U.R. J. 2004; and binding of A-, B-, and C-RAF kinases to all seven mammalian 14-3-3 isoforms ϵ, τ, and For that 14-3-3 isoforms purified on the the purified and RAF proteins the interactions between the 14-3-3 proteins and RAF kinases in binding of 14-3-3 isoforms to A-, B-, and C-RAF and Although B- and C-RAF exhibited binding to all of the seven 14-3-3 isoforms, A-RAF in vitro binding to ϵ, τ, and σ 14-3-3. The association of 14-3-3 binding to B- and C-RAF are in the A-RAF binding was The all of the RAF isoforms was the that bound with and with the seven 14-3-3 we that a much higher B-RAF with A- and C-RAF and we show that mammalian 14-3-3 proteins associate with RAF in a pronounced isoform-specific manner. The that in to associates with 14-3-3 isoforms that B-RAF in the of The binding in and have with 14-3-3 binding of the heterodimeric forms of 14-3-3 proteins association with RAF we a 14-3-3 of and as binding the C-RAF was to the and purified homo- and heterodimeric 14-3-3 and binding affinities to We also the yeast 14-3-3 that and purified in the only association with that both of 14-3-3 with RAF and also in vivo the of the may indicate a binding to C-RAF as complex G. J. Rapp U.R. Scholar). exhibited C-RAF with in the budding yeast 14-3-3 protein bound with similar to C-RAF with whether the 14-3-3 in the binding in in dimeric we using All 14-3-3 proteins in at Together, results indicate that RAF activation, the between 14-3-3 RAF binding may the in the composition of the 14-3-3 and the structure of the 14-3-3 binding domains in the proteins may the and of 14-3-3 RAF in with and of 14-3-3 in that C-RAF associates in vitro with both homo- and heterodimeric forms of 14-3-3 also the yeast 14-3-3 as C-RAF to whether RAF kinases associate in vivo with homo- and heterodimeric forms of 14-3-3 proteins, we the budding yeast S. These only two 14-3-3 and a of 14-3-3 We also of a of S. the BMH2 BMH2 a high degree of homology with mammalian of and yeast with B- and we the association of yeast 14-3-3 proteins with RAF and the its kinase we also RAF variants that at the single 14-3-3 binding and RAF in both 14-3-3 domains and in that B- and C-RAF expressed in yeast with both and forms of yeast 14-3-3 The of kinase with of whether only or complex was in the yeast of at the internal or C-terminal binding domain considerably the activation of to of activation of C-RAF The results using yeast as a our in vitro in and and the and of binding with 14-3-3 of RAF in in and that mammalian 14-3-3 isoforms differentially to RAF However, about the binding of 14-3-3 to the individual binding the binding affinities of 14-3-3 the C-terminal and internal binding domains in we in we purified full-length protein and 14-3-3 and as the The the C-terminal and internal 14-3-3 binding domains of A-, B-, and C-RAF. The association was by as Because found to with similar affinities with all three RAF isoforms, we For that purpose, protein was first by and RAF association was in the of as 14-3-3 binding domains a of between and with RAF proteins, and the degree of was in to the C-terminal 14-3-3 binding of A-, B-, and C-RAF considerably higher than the internal binding with of in that the C-terminal 14-3-3 binding domain in RAF represents the high binding site and the binding These are in with findings M. S. R. Albert S. J. J. M. Rapp U.R. J. 2004; Scholar). C-terminal to the internal binding exhibited in the to the B-RAF internal binding site the the by and A-RAF These results that B-RAF may 14-3-3 with the binding These are in with mass of RAF by and C-terminal 14-3-3 A-, B-, and respect to RAF activation 14-3-3 proteins have found to RAF activation G. J. Scholar, S. A. J. A. J. Mol. Cell. Biol... Scholar). the also that 14-3-3 are RAF M. Scholar). Although the C-terminal 14-3-3 protein binding of RAF kinases is the sequence in B-RAF the 14-3-3 binding in A- and C-RAF also of 14-3-3 binding and the activation cycle of A-, B-, and we the regulatory the 14-3-3 binding domains in A-, B-, and C-RAF by and both and RAF with M. S. R. Albert S. J. J. M. Rapp U.R. J. 2004; the degree of in was between two was more pronounced in the expressed in the the degree of the was considerably by the of in kinase of C-RAF the of by in the activity the activity with D.K. G. Rapp U.R. Copeland T.D. J. Scholar, S. M. Scholar, M. M. M. M. J. Scholar, Rapp U.R. Cell.. Scholar). as in in in the of RAF that can by with and the of by the C-RAF activation with the C-RAF the at as 14-3-3 binding in RAF expressed in the These findings indicate that 14-3-3 association is regulation of RAF activation B-RAF in A- and C-RAF. high activity in the of cell to we between two 14-3-3 binding as the degree of and was of in or with by T. P. S. M. 2006; we a of kinase activity in and a in activity by of the internal 14-3-3 binding site The of and the kinase activity of the as by kinases the oncogenic of B-RAF to B-RAF The of by only of the kinase that C-terminal 14-3-3 binding site of its respect to regulation of kinase activity by internal 14-3-3 binding between and of by the kinase activity of only with are of with B-RAF expressed in yeast the of by its kinase Although A-RAF contains two 14-3-3 binding domains that are similar to the 14-3-3 domains in little is known about the binding and regulation of kinase by 14-3-3 We show that A-RAF is at in both and to the of in A-RAF by to a of a between two 14-3-3 binding The of the and in and to the activation of kinases. We whether the of the in A-RAF a similar The results in that A-RAF differentially with B- and C-RAF. The of the C-terminal 14-3-3 binding domain was only the role of the internal 14-3-3 binding site in we found that the kinase activity of the was in the of Ras and These results indicate that similar to B- and the activation of A-RAF is by the internal 14-3-3 binding in the may the that at the we show that the of the internal and C-terminal 14-3-3 binding both 14-3-3 binding 14-3-3 in C-RAF the 14-3-3 to of that a expressed protein S. Mol. in vivo RAF activity in a manner. we that the of in the cell association of 14-3-3 to that the of 14-3-3 in the of RAF activation C. Hekman M. C. Rapp U.R. T. Nat. Cell Biol... Scholar). results by of with However, whether 14-3-3 or by the whether as a C-RAF association with 14-3-3 proteins, we For that purpose, we first the purified and to the as under we binding of C-RAF to 14-3-3. whether may the association of C-RAF with 14-3-3 proteins, we C-RAF with of and the in binding to 14-3-3. in the of considerably the C-RAF binding to that and 14-3-3 C-RAF in to the association of the with 14-3-3 was only to degree by the of testing all seven mammalian 14-3-3 proteins, we pronounced to and by the binding of and to C-RAF was by The degree of was with the that by the of a high of the also the in vivo between C-RAF and we C-RAF and 14-3-3 binding with with the in vitro in the of with C-RAF was considerably in the of the that is in 14-3-3 binding at the internal site results the that activation of C-RAF at by 14-3-3 the internal 14-3-3 binding Although the of 14-3-3 proteins in RAF in by Rapp U. J. J. to our are on and of mammalian 14-3-3 isoforms in RAF process association of 14-3-3 proteins with the internal and C-terminal 14-3-3 binding of C-RAF M. S. R. Albert S. J. J. M. Rapp U.R. J. 2004; to the and C-RAF the question of whether mammalian 14-3-3 isoforms in association with RAF we binding using purified of all seven mammalian 14-3-3 isoforms and RAF B-, and by both binding and and by that 14-3-3 proteins with RAF and in isoform-specific manner. the mammalian 14-3-3 isoforms between individual isoforms, we of mass to 14-3-3 B-RAF with a much higher of 14-3-3 isoforms with A- and C-RAF The was in that 14-3-3 proteins appear in heterodimeric showing that A-, B-, and C-RAF in vivo composition of 14-3-3 proteins with findings that RAF isoforms different in the cell to Although C-RAF at the B-RAF in the A. Hekman M. J. S. Rapp U.R. J. the high of attached 14-3-3 The that A-RAF associates with and the that A-RAF at the are with a of RAF the RAF of kinases. S. M. and Rapp in using the scaffold protein with the kinase of the found to also with a higher of mammalian 14-3-3 isoforms S. M. T.D. Morrison D.K. Biol... Scholar). three isoforms and in a in vitro binding by our with purified and 14-3-3 proteins three isoforms as high binding Although a conserved kinase domain, the to associate with C-RAF in a Biol... Scholar). is to that the high of 14-3-3 proteins may to with its as C-RAF. to 14-3-3 isoforms attached to B-RAF may have different is in that 14-3-3 proteins are crucial B-RAF the of by the C-terminal 14-3-3 binding site the activation of However, as B-RAF to heterodimeric with C-RAF with A-RAF as in a and J.R. Rapp U.R. Cancer Scholar, S. Marais R. Mol. Cell.. Scholar, Mol. Cell. Biol... 2006; we that at of the 14-3-3 isoforms are in the of B-RAF with a complex between B-RAF and also However, on B-RAF with are to C-RAF purified found to only with and the of two isoforms is activation of C-RAF is in with the that several invertebrates as and possess only two 14-3-3 isoforms that are regulation of the we addressed the of whether individual 14-3-3 the to RAF in the budding yeast S. cerevisiae, possessing only two 14-3-3 isoforms, served as the testing system. By deletion of the BMH2 gene we to show that the activation of both B- and C-RAF in vivo by a single 14-3-3 also on we that whereas a single of 14-3-3 is RAF activation, the of heterodimeric forms of 14-3-3 may to RAF isoforms or the of between RAF and 14-3-3 that RAF activity also heterodimeric forms of 14-3-3 proteins to the different and binding affinities of the internal C-terminal 14-3-3 binding regulation of RAF activity by 14-3-3 proteins, we show that the C-terminal 14-3-3 binding site is in RAF activation. the regulation of the RAF activity by the internal 14-3-3 binding site the RAF Although the of and by in C-RAF and B-RAF a of the kinase activity and the A-RAF a of activity with A-RAF by the degree of in interactions with 14-3-3 of A-RAF kinase its activation as regulation of kinase activity by A. T. Hekman M. Rapp U.R. J. and isoform-specific domain A. T. A. Hekman M. Rapp U.R. J. Scholar). findings by two by the 14-3-3 binding domains in C-RAF are of A. C. S. A. A. G. C. P. R. A. C. G. M. Nat. Scholar, M.A. T. M. R. M. M. M. M. J. T. R. Nat. Scholar). of the in B- and C-RAF that are with have found the kinase domain (1Wellbrock C. Karasarides M. Marais R. Nat. Rev. Mol. Cell Biol... 2004; 5: 875-885Google A. C. S. A. A. G. C. P. R. A. C. G. M. Nat. and M.A. T. M. R. M. M. M. M. J. T. R. Nat. the first on C-RAF the internal 14-3-3 binding site and in the of the C-terminal 14-3-3 binding domain. These to activation of C-RAF. The of are called and of the of the internal 14-3-3 binding domain. of the by the mechanism of in a C-RAF our results and by A. C. S. A. A. G. C. P. R. A. C. G. M. Nat. and M.A. T. M. R. M. M. M. M. J. T. R. Nat. the of 14-3-3 binding in C-RAF regulation of in the We also the of the of crucial the 14-3-3 binding domains of the oncogenic also of the by the activation of B-RAF to a B-RAF kinase that (1Wellbrock C. Karasarides M. Marais R. Nat. Rev. Mol. Cell Biol... 2004; 5: 875-885Google Scholar, S. Marais R. Cell.. 2004; Scholar). in about of the was C. P. S. S. J. R. S. R. J. A. C. A. C. S. S. R. C. J. G. G. A. A. A. Marais R. R. Scholar, R. A. P. R. Rapp U.R. J. 2004; Scholar). the that C-RAF activity was by the and is M. C. Marais R. Cancer Scholar). similar with as in in the of B-RAF the activity was only by to in These the of oncogenic B-RAF and of 14-3-3 proteins activation and of activity of similar have with B-RAF expressed in in the of by to B-RAF by a of the activation at the by kinase in the as in the S. Marais R. Cell.. 2004; Scholar). the of C-RAF activity by 14-3-3 as we that is a of C-RAF of in the cell of and results in 14-3-3 association and C. Hekman M. C. Rapp U.R. T. Nat. Cell Biol... Scholar). These findings that may the of 14-3-3 in the of RAF activation, regulatory However, whether 14-3-3 or by to we in vitro with purified and that with the binding of 14-3-3 to C-RAF The more pronounced by testing the that to RAF activation by of 14-3-3 the internal 14-3-3 binding in with in vitro in vivo that associates with a C-RAF in 14-3-3 binding These our in that the of 14-3-3 the internal binding site in C-RAF is by at the the C-RAF that is to as a complex with the dimeric of 14-3-3 proteins in the the activation in 14-3-3 may However, with B-RAF also We and We are to to the We also the with