B-Raf and Raf-1 Are Regulated by Distinct Autoregulatory Mechanisms

B-Raf is a key regulator of the ERK pathway and is mutationally activated in two-thirds of human melanomas. In this work, we have investigated the activation mechanism of B-Raf and characterized the roles of Ras and of B-Raf phosphorylation in this regulation. Raf-1 is regulated by an N-terminal autoinhibitory domain whose actions are blocked by interaction with Ras and subsequent phosphorylation of Ser338. We observed that B-Raf also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active H-Ras. However, unlike Raf-1, the phosphorylation of B-Raf at Ser445 was constitutive and was only moderately increased by expression of constitutively active H-Ras or constitutively active PAK1. Ser445 phosphorylation is important to the B-Raf activation mechanism, however, because mutation of this site to alanine increased the affinity of the regulatory domain for the catalytic domain and increased autoinhibition. Similarly, expression of constitutively active PAK1 also decreased auto-inhibition. B-Raf autoinhibition was negatively regulated by acidic substitutions at phosphorylation sites within the activation loop of B-Raf and by the oncogenic substitution V599E. However, these substitutions did not affect the ability of the regulatory domain to co-immunoprecipitate with the catalytic domain. These data demonstrate that B-Raf activity is autoregulated, that constitutive phosphorylation of Ser445 primes B-Raf for activation, and that a key feature of phosphorylation within the activation loop or of oncogenic mutations within this region is to block autoinhibition. B-Raf is a key regulator of the ERK pathway and is mutationally activated in two-thirds of human melanomas. In this work, we have investigated the activation mechanism of B-Raf and characterized the roles of Ras and of B-Raf phosphorylation in this regulation. Raf-1 is regulated by an N-terminal autoinhibitory domain whose actions are blocked by interaction with Ras and subsequent phosphorylation of Ser338. We observed that B-Raf also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active H-Ras. However, unlike Raf-1, the phosphorylation of B-Raf at Ser445 was constitutive and was only moderately increased by expression of constitutively active H-Ras or constitutively active PAK1. Ser445 phosphorylation is important to the B-Raf activation mechanism, however, because mutation of this site to alanine increased the affinity of the regulatory domain for the catalytic domain and increased autoinhibition. Similarly, expression of constitutively active PAK1 also decreased auto-inhibition. B-Raf autoinhibition was negatively regulated by acidic substitutions at phosphorylation sites within the activation loop of B-Raf and by the oncogenic substitution V599E. However, these substitutions did not affect the ability of the regulatory domain to co-immunoprecipitate with the catalytic domain. These data demonstrate that B-Raf activity is autoregulated, that constitutive phosphorylation of Ser445 primes B-Raf for activation, and that a key feature of phosphorylation within the activation loop or of oncogenic mutations within this region is to block autoinhibition. Raf proteins are Ras-regulated serine/threonine protein kinases that control the activation of the ERK 1The abbreviations used are: ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; CR, conserved region; RBD, Ras-binding domain; CRD, cysteine-rich domain; PAK1, p21-activated kinase-1; HEK, human embryonic kidney; HA, hemagglutinin; MBP, myelin basic protein; EGF, epidermal growth factor. 1The abbreviations used are: ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; CR, conserved region; RBD, Ras-binding domain; CRD, cysteine-rich domain; PAK1, p21-activated kinase-1; HEK, human embryonic kidney; HA, hemagglutinin; MBP, myelin basic protein; EGF, epidermal growth factor./MAPK cascade and, as such, control a basic mechanism by which cells respond to extracellular ligands (1Hagemann C. 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We that the phosphorylation of B-Raf at Ser445 is that expression of constitutively active H-Ras or constitutively active PAK1 the phosphorylation of this We have also that B-Raf contains an N-terminal autoinhibitory domain and that the interaction of this domain with the B-Raf catalytic domain is negatively regulated by binding to active is to the phosphorylation of Ser445 because mutation of this site to alanine increased the affinity of the autoinhibitory domain for the catalytic domain and increased the of the the ability of the B-Raf autoinhibitory domain to block activation was negatively regulated by acidic substitutions at the phosphorylation sites and as well as by the oncogenic substitution V599E. These data demonstrate that B-Raf is also regulated by an autoinhibitory mechanism and that the of this autoinhibitory domain is by phosphorylation of also that B-Raf autoinhibition is a key feature of oncogenic mutations that within the activation loop of and embryonic cells in with and by Xu Cobb M.H. Mol. Cell. Biol. PubMed Scopus Google or to the B-Raf was in A-Raf, and with and was by as a The B-Raf catalytic domain was with and by to and by and to was in Xu Cobb M.H. Mol. Cell. Biol. PubMed Scopus Google Scholar). active H-Ras was in Xu Cobb M.H. Mol. Cell. Biol. PubMed Scopus Google Scholar). active PAK1 was in A. Cobb M.H. Biol. PubMed Scopus Google Scholar). and in cells and or affinity as A. Cobb M.H. Biol. PubMed Scopus Google Scholar). and in cells with with or the or N-terminal expression by The the was to and the cells for Cell then in and and was by at for at at for was of and of a of protein three with of high and and with of The of which was used to kinase activity and the to the in the by of myelin basic protein as an in a at for in kinase and of then by and by and proteins by was by of the protein to proteins by and to was was by Ras expression was Ras Raf and the phosphorylation of A-Raf, B-Raf, and Raf-1 at the phosphorylation sites and cells and then with human epidermal growth for different of with and was by at for at A-Raf or B-Raf protein was with or and protein with three with of high and with of in by and to of or Ser445 was by Raf-1 then with or to the of Raf phosphorylation of Raf-1 at cells in and of in for Raf-1 protein was of was by with or and with or B-Raf N-terminal was cells In constitutively active PAK1 or constitutively active H-Ras was also for to in and by at for at proteins and protein and three with of and and with of proteins by to and with or of of A-Raf, B-Raf, and Raf-1 at and activation is regulated in by phosphorylation of and substitution of this site with alanine Raf-1 activation by A. 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R. 1999; PubMed Scopus Google we the phosphorylation of A-Raf or B-Raf at the in with for different of and and the A-Raf and B-Raf proteins the phosphorylation of A-Raf and B-Raf at and Ser445 was by of Raf-1 at was by in of a phosphorylation of Raf-1 at as as and well with the activation of and Raf-1 and also with the phosphorylation of Raf-1 at in cells A. A. Mol. Cell. Biol. PubMed Scopus Google Scholar, A. Mason C.S. R. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). we that the phosphorylation of A-Raf at was also by EGF, the of phosphorylation of this site was for by with Raf-1, phosphorylation of this site an important in the A-Raf activation the B-Raf was at Ser445 in the of EGF, and the of Ser445 phosphorylation was not of a to B-Raf and Raf-1 at Ser445 and not C.S. G. R. 1999; PubMed Scopus Google Scholar). these data well with that B-Raf is constitutively in cells C.S. G. R. 1999; PubMed Scopus Google Scholar). We then the phosphorylation of Ser445 increased by expression of constitutively active Ras or constitutively active PAK1 because Ras B-Raf activity and because PAK1 Raf-1 at the site Biol. 2003; PubMed Scopus Google Scholar, H. W. PubMed Scopus Google Scholar, M. C.A. A. R. Biol. 2001; PubMed Scopus Google Scholar, M. C. Biol. 2002; PubMed Scopus Google Scholar). cells with B-Raf and of constitutively active Ras or constitutively active PAK1. B-Raf was and for phosphorylation of Ser445 by in B-Raf a high degree of Ser445 to B-Raf However, expression of active Ras did a in this phosphorylation with the constitutively active PAK1 was at Ser445 phosphorylation and that the phosphorylation of B-Raf at Ser445 in cells is not and that Ras the phosphorylation of this also that PAK1 is a B-Raf The B-Raf an Raf-1 contains an autoinhibitory domain that encompasses and that the and of the We have that of this domain with the catalytic domain of Raf-1 ability to activation Biol. 2003; PubMed Scopus Google Scholar). we B-Raf also contains an autoinhibitory domain in We expression of the B-Raf catalytic domain cells with of the cells and was and for kinase activity as a well in these and of as little as of the a activation of as by kinase activity and by for active and The three are in We then of the B-Raf activation by these of which activation of ERK2. in the ability of to activity was inhibited by of of the B-Raf The are in activation by is of these data that the B-Raf contains an autoinhibitory domain that the activity of the catalytic to In the of these we that the degree of autoinhibition observed of of of and was not was because was at and because did not as as for the autoinhibition of activation by we to only of this we then the region the autoinhibitory domain of In Raf-1, the autoinhibitory domain encompasses the and a of the CRD, not CR2 Biol. 2003; PubMed Scopus Google Scholar). we a of of the B-Raf to the region for autoinhibition in of the B-Raf regulatory domain to and CR2 and did not affect autoinhibition. However, of and the of the blocked autoinhibition. to also at activation not these data demonstrate that to the CRD, not CR2, are for The B-Raf with a followed by the RBD, which at We observed that of the of the did not affect the ability of the to block activation because and as as at activation However, of and the autoinhibition not was not that the Raf-1 autoinhibitory domain also N-terminal to Biol. 2003; PubMed Scopus Google Scholar). these data demonstrate that the B-Raf autoinhibitory domain is and region encompasses the and as well as to the the autoinhibitory domain of B-Raf that of Raf-1 because the domain in Raf-1 did not the or to the The B-Raf a with the in in the Raf-1 regulatory domain a with the catalytic domain such that with the catalytic domain H. Guan K.L. Biol. 2003; PubMed Scopus Google Scholar). we the B-Raf autoinhibitory domain with the B-Raf catalytic domain and this binding with the ability of this domain to cells with and B-Raf N-terminal was then and for of the N-terminal by in the and N-terminal with the well with ability to block activation by the the N-terminal which did not block activation by also did not with These data demonstrate that the B-Raf autoinhibitory domain with the catalytic domain in cells and that this interaction for autoinhibition. The Raf-1 with the B-Raf and Raf-1 have to a in the that an for R. Cell. PubMed Scopus Google Scholar, Rapp U.R. Res. 2001; Google Scholar). the B-Raf and Raf-1 autoinhibitory a high degree of we the Raf-1 autoinhibitory domain with the B-Raf catalytic domain and block cells with and of the B-Raf or Raf-1 regulatory and the activity of was in of blocked activation by was by an in the of as ability to activate was The of the protein to phosphorylation by which to the of B-Raf H. M. Y. 2003; 22: PubMed Scopus Google Scholar). the Raf-1 regulatory domain also blocked with with the B-Raf regulatory domain. these data demonstrate that the Raf-1 regulatory domain homology with the B-Raf regulatory domain to B-Raf catalytic We then the Raf-1 regulatory domain a with cells with with or the B-Raf or Raf-1 regulatory domain. was then and for the of regulatory domain. in the Raf-1 regulatory domain also with the B-Raf catalytic with affinity with the B-Raf regulatory domain. these data demonstrate that the Raf-1 regulatory domain with the B-Raf catalytic with and that B-Raf and Raf-1 regulate the activity of through autoinhibitory of the B-Raf to the by of then the interaction the B-Raf autoinhibitory and catalytic is regulated by In the of Raf-1, this interaction is blocked by of constitutively active Ras H. Guan K.L. Biol. 2003; PubMed Scopus Google Scholar). cells with and with or constitutively active H-Ras was then and for of the B-Raf with the B-Raf catalytic and this was inhibited by the expression of and the ability of Ras to with because a of binding to Ras with in the of active H-Ras and the protein was at and expression of active Ras did not this that a in the phosphorylation of this site is not for Ras to block the interaction the regulatory and catalytic we an alanine substitution at Ser445 is to by cells with or with or and The proteins then and for of the B-Raf was at was not expression of active Ras the the B-Raf regulatory domain and the and these data demonstrate that Ras the interaction the regulatory and catalytic by binding to the B-Raf and of the of to with with and that phosphorylation of Ser445 the binding of the regulatory domain to the catalytic domain. of B-Raf at Ser445 the of the for the that phosphorylation of at Ser445 negatively the affinity of the regulatory domain for the catalytic domain. with the of a binding affinity of the autoinhibitory domain for the catalytic Mason C.S. G. R. 1999; PubMed Scopus Google have that mutation of Ser445 and to the and of we the of Ser445 in the degree of autoinhibition by the cells with and or in the or of of the B-Raf regulatory domain. was then and for kinase In these and the was active in this the protein and Similarly, the and proteins by However, was to with such at the of regulatory domain the activity of the was these data that the of Ser445 negatively the ability of the to regulate the activity of the catalytic domain and that the constitutive phosphorylation of Ser445 primes B-Raf for We then of the Ser445 phosphorylation site the affinity of the regulatory domain for the catalytic domain. cells with the or of with or of was then and for the of the B-Raf by The B-Raf with to a degree with with with that only was at Ser445 These data that to to and that the phosphorylation of Ser445 the affinity of the autoinhibitory domain for the catalytic domain. expression of active PAK1 the phosphorylation of B-Raf at Ser445 we expression of active PAK1 the of B-Raf autoinhibition. of a of activation by to and However, of constitutively active PAK1 the degree of autoinhibition such that activation was to of and the was not of affinity to the phosphorylation of at this site with the of being in this we not demonstrate that PAK1 expression the phosphorylation of in this However, autoinhibition of was only by active PAK1 expression that PAK1 most autoinhibition through the phosphorylation of at PAK1 the phosphorylation of at Ser445 of and these we that the phosphorylation of Ser445 to the affinity of the B-Raf regulatory domain for the catalytic domain. in the B-Raf B-Raf activation phosphorylation of and which are within the activation loop of the catalytic domain. acidic substitutions at these sites a constitutively active of B-Raf that not Ras for activation Guan K.L. 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These that the mutation autoinhibition by the in which the autoinhibitory and catalytic binding B-Raf by the in substitution is the most oncogenic mutation in B-Raf and in a constitutively active protein with an kinase activity that is that by active Ras H. C. H. W. E. R. Y. K. R. A. C. A. C. R. H. C. K. G. G. A. A. A. H.F. Paterson H. R. R. 2002; PubMed Scopus Google Scholar, R. Cell. 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However, the phosphorylation of which to in Raf-1, is constitutive and not to autoinhibition. this phosphorylation to B-Raf for activation because substitution of Ser445 with alanine increased the affinity of the regulatory domain for the catalytic domain and autoinhibition. the phosphorylation of sites within the activation loop of B-Raf to with autoinhibition because acidic substitutions at the activation loop phosphorylation sites as well as the oncogenic mutation blocked the of the regulatory domain. mechanism that activation of Raf-1 because phosphorylation of sites within activation loop not affect autoinhibition Biol. 2003; PubMed Scopus Google Scholar). these demonstrate that B-Raf activity is also through the actions of an N-terminal autoinhibitory that the in which interaction with the catalytic domain is regulated is data that the autoinhibitory domain of B-Raf encompasses the and as well as a region of Raf the of the autoinhibitory domain of B-Raf that of Raf-1, which contains the RBD, only of the and not to the Biol. 2003; PubMed Scopus Google Scholar). the of these are within the and identity that we a for we observed that the Raf-1 regulatory domain is to block activation by the B-Raf catalytic with with the B-Raf regulatory domain Similarly, the Raf-1 regulatory domain with the B-Raf catalytic with a affinity these the for Raf-1 and important to the activation mechanism of each the that Raf-1 and B-Raf a in the Rapp U.R. 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R. 1999; PubMed Scopus Google observed that substitution of Ser445 and with the and of B-Raf, and that the phosphorylation of this site to the activity observed for B-Raf by autoinhibition. We then B-Raf autoinhibition is regulated by phosphorylation of sites within the activation loop of the catalytic domain. sites within this for B-Raf activation and and substitution of these sites with acidic a constitutively active of B-Raf that activated by Ras Guan K.L. PubMed Scopus Google Scholar, Guan K.L. Exp. Res. 2001; PubMed Scopus Google Scholar). we observed that the ability of to activity was by the of the B-Raf regulatory domain that these sites to or these sites are B-Raf activity In this the mechanism of B-Raf that of Raf-1, which is by acidic substitutions at the phosphorylation sites within activation loop Biol. 2003; PubMed Scopus Google Scholar). 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