Regulation of Raf through Phosphorylation and N Terminus-C Terminus Interaction

Abstract

The Raf family of serine/threonine kinases is highly conserved in structure. There are three conserved regions (CR) 1 in Raf kinases, CR1, CR2, and CR3 (1). CR1 contains the Ras binding domain (RBD) and the cysteine-rich domain (CRD), and both bind the activated small GTPase Ras (2, 3). CR2 is serine/threonine-rich, and CR3 is the catalytic kinase domain and contains several activating phosphorylation sites. Activated Ras binds to CR1 and recruits Raf to the plasma membrane, which is necessary for Raf activation. Phosphorylation of activating sites and dephosphorylation of inhibitory sites must be coordinated for full Raf activation. Once Raf is activated, it can phosphorylate and activate MEK, which is then able to phosphorylate and activate MAPK/ERK, which phosphorylates cytosolic and nuclear proteins.Raf was first identified as a retroviral oncogene v-Raf, which has a deletion of the 5Ј end of the normal cellular gene encoding CR1 and CR2 (4). This deletion correlates to an increase in transforming activity independent of Ras, which leads to the hypothesis that the N-terminal domain of Raf proteins has a regulatory function (5-9). In defining the minimal region required for cell transformation, deletion of both CR1 and CR2 but an intact kinase domain was required for high transforming activity (6, 9). Deletion of the N-terminal region renders Raf kinase activity independent of Ras.The N-terminal domain serves the following two functions. 1) It binds to the activated form of the small GTPase Ras required for membrane recruitment. 2) It binds and suppresses the C-terminal kinase. Expression of the N terminus of c-Raf can inhibit Ras activation of the MAPK pathway in germinal vesicle breakdown (GVBD) in Xenopus oocytes by binding and sequestering activated Ras from endogenous c-Raf (10). The N-terminal half was also shown to co-immunoprecipitate and inhibit the C-terminal half of Raf. The mechanism of Raf activation may be similar to that of p21-activated kinase (PAK) (11). The Rac and Cdc42, members of the Rho family of small GTPases, directly bind the N terminus of PAK and relieve an auto-inhibited conformation held together by intramolecular interactions. The new conformation allows phosphorylation of activating sites, which are thought to help maintain an open conformation of PAK.Two different mechanisms have given rise to oncogenic forms of Raf kinases. v-Raf has a deletion of the entire 5Ј end of the cellular gene that encodes for the N-terminal regulatory domain. v-Raf has high transforming activity because of a loss of the auto-inhibitory domain. Recently, mutations in B-RAF were identified in 60% of human melanomas and identified B-RAF as an oncogene (12, 13). A common oncogenic mutation of B-RAF has a Val to Glu mutation in the activation loop of the kinase domain and results in high kinase activity. This mutation, V599E, lies between two conserved phosphorylation sites, Thr-598 and Ser-601, in the activation loop of B-Raf, previously identified by our lab (14,15). We have shown that acidic residue sub...

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

“…Our model (Figure 9a), similar to that suggested by Mercer and Pritchard (2003), is consistent with recent studies showing that the negatively charged N-region of Raf-1 relieves autoinhibition by the N-terminal moiety owing to its reduced affinity towards a CR3 with a charged N-region (Cutler et al, 1998;Tran and Frost, 2003). However, we accept that similar experiments led to the alternative conclusion that the charged N-region induces a conformational change that does not affect the interaction between N-and C-terminal moiety but renders the CR3 resistant to the autoinhibition imposed by the N-terminal moiety (Chong and Guan, 2003). Furthermore, while our manuscript was in preparation, Tran et al (2005) reported that introduction of the Regulation of B-Raf signalling T Brummer et al S446A mutation into the isolated CR3 increased the affinity of this region towards the isolated N-terminal domain of B-Raf (Tran et al, 2005).…”

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

“…338 , and substitution of this site with alanine precludes Raf-1 activation by physiologic stimuli (34 -39). Phosphorylation of this site contributes to Raf-1 activation by blocking the ability of the Nterminal autoinhibitory domain to negatively regulate Raf-1 catalytic activity (28,29). Because this residue is conserved among Raf family members (Fig.…”

“…In this respect, the activation requirements of B-Raf are similar to those of Raf-1 because Raf-1 must also bind to Ras and requires phosphorylation of sites within the activation loop of its catalytic domain (Thr 491 and Ser 494 ) (27). However, full activation of Raf-1 also requires phosphorylation of Ser 338 and Tyr 341 , which lie outside the catalytic domain and contribute to the Raf-1 activation mechanism by blocking the actions of the autoinhibitory do-main (28,29). Interestingly, the residues in B-Raf corresponding to Tyr 340 and Tyr 341 in Raf-1 are substituted with aspartic acids, and Ser 445 , which corresponds to Ser 338 in Raf-1, appears to be constitutively phosphorylated in COS and PC12 cells (30).…”

Caenorhabditis elegans CNK-1 promotes Raf activation but is not essential for Ras/Raf signaling

“…3). Phosphorylation of these activating sites by an unknown, membrane-localized kinase is thought to be a late step in Raf activation (43). Both LIN-45(AA) and LIN-45(ED) mutants can induce a Muv phenotype, when expressed as a transgene (32).…”

“…The analogous residues in mammalian Raf proteins serve as docking sites for the 14-3-3 chaperone, which inhibits translocation of Raf to the membrane (42). Dephosphorylation of these inhibitory sites by PP2A is thought to be an early step in Raf activation (43). The second form of activated LIN-45 Raf, LIN-45(ED), contains negatively charged residues that mimic activating phosphorylation within the activation loop of the kinase domain (32) (Fig.…”