Phosphorylation of Raf-1 by p21-activated Kinase 1 and Src Regulates Raf-1 Autoinhibition

Abstract

Exposure of cells to mitogens or growth factors stimulates Raf-1 activity through a complex mechanism that involves binding to active Ras, phosphorylation on multiple residues, and protein-protein interactions. Recently it was shown that the amino terminus of Raf-1 contains an autoregulatory domain that can inhibit its activity in Xenopus oocytes. In the present work we show that expression of the Raf-1 autoinhibitory domain blocks extracellular signal-regulated kinase 2 activation by the Raf-1 catalytic domain in mammalian cells. We also show that phosphorylation of Raf-1 on serine 338 by PAK1 and tyrosines 340 and 341 by Src relieves autoinhibition and that this occurs through a specific decrease in the binding of the Raf-1 regulatory domain to its catalytic domain. In addition, we demonstrate that phosphorylation of threonine 491 and serine 494, two phosphorylation sites in the catalytic domain that are required for Raf-1 activation, is unlikely to regulate autoinhibition. These results demonstrate that the autoinhibitory domain of Raf-1 is functional in mammalian cells and that its interaction with the Raf-1 catalytic domain is regulated by phosphorylation of serine 338 and tyrosines 340 and 341.Activation of the extracellular signal-regulated kinases (ERK) 1 1 and 2 is required for cellular proliferation in many cell types and is also a requisite event in neoplastic transformation (1-4). The activities of ERKs 1 and 2 are regulated through the activation of a sequentially acting protein kinase cascade, known generically as a mitogen-activated protein kinase module. At the top of this cascade is the serine/threonine protein kinase Raf-1. Once activated, Raf-1 phosphorylates and activates the mitogen and extracellular signal-regulated kinase kinases 1 and 2 (MEKs 1 and 2), which subsequently phosphorylate and activate ERKs 1 and 2. ERKs then phosphorylate a large number of nuclear and cytoplasmic substrates that ultimately regulate the ability of a cell to grow and divide (2).The Raf-1 activation mechanism is a complex process that involves binding to the small GTP binding protein Ras, phosphorylation on multiple residues, altered protein-protein interactions, and perhaps direct interaction with lipids (5-7). Binding to active Ras relocalizes Raf-1 from the cytosol to the plasma membrane. This interaction is initially mediated by a conserved domain in the Raf-1 amino terminus known as the Ras binding domain (RBD). Binding of Ras to the RBD then promotes contact with an adjacent domain known as the cysteine-rich domain (CRD), and binding of Ras to both of these domains is required for full activation of Raf-1 (5). Interaction between Ras and Raf-1 also stimulates the release of 14 -3-3 from its amino-terminal binding site, which is centered on serine 259, and the subsequent dephosphorylation of this site (8 -10). This is thought to contribute to the separation of the catalytic domain from the amino-terminal regulatory domain.Activation of Raf-1 by Ras is also accompanied by phosphorylation on multiple residues...

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

“…Our finding that the S365A mutation increases not only the biological activity of B-Raf wt but also rescues that of B-Raf with a neutralized N-region (Figure 3b and c) strongly supports a model in which a major function of the N-region is to oppose the transition into an inactive conformation stabilized by binding of a 14-3-3 dimer to S365 and S729. 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).…”

Spatial regulation of Raf kinase signaling by RKTG

“…interaction of Raf-1 with MEK (32,33). We found that RKTG overexpression inhibited Raf-1 Ser-338 phosphorylation in response to EGF (Fig.…”

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).…”