Phosphorylation of the C-Raf N Region Promotes Raf Dimerization

Abstract

Activation of Raf kinases by the small GTPase Ras requires two major sets of phosphorylations. One set lies within the activation loop and the other lies within the N-terminal acidic region (N-region). In the most abundant isoform of Raf, C-Raf, N-region phosphorylations occur on serine 338 (S338) and tyrosine 341 (Y341), and are thought to provide allosteric activation of the Raf dimer. We show that the phosphorylation of these N-region sites does not require C-Raf dimerization, but rather, they precede dimerization. One of these phosphorylations (phospho-Y341) is required for C-Raf dimerization and this action can be replicated by phosphomimetic mutants both and The role of phosphorylation of Y341 in promoting Raf dimerization is distinct from its well-known function to facilitate S338 phosphorylation.In Ras-mutant pancreatic cancer cell lines, phosphorylation and dimerization of C-Raf is basally elevated. Dimerization is thought to contribute to their elevated growth rate through their activation of the MAP kinase (ERK) signaling cascade. Blocking tyrosine phosphorylation of C-Raf with Src family inhibitors blocks the growth, basal dimerization, and ERK activation in these cells. We suggest that the kinases mediating C-Raf Y341 phosphorylation are potential candidate drug targets in selected Ras-dependent cancers.

SHOC2 complex-driven RAF dimerization selectively contributes to ERK pathway dynamics

“…On the other hand, we note that experimental constraints when analyzing endogenous proteins have not allowed us to measure homodimerization, and S259-independent CRAF homodimerization during the slow, sustained phase remains a distinct possibility. Reports of N-region CRAF phosphorylation promoting relief from autoinhibition and dimerization (70, 71) and of high levels of S338 phosphorylation activating CRAF in the presence of high levels of inhibitory phosphorylation at S43 and S259 (72) support this scenario. It is also worth noting that both SFK and PAK activators, such as RAC and CDC42, are palmitoylated and expected to travel with H/N-RAS during both endocytosis and acylation cycle scenarios of intracellular trafficking, which would thus facilitate N-region phosphorylation of the H/N-RAS bound CRAF at these compartments.…”

Inhibition of RAF dimers: it takes two to tango

“…The RKTR motif is correctly positioned for dimerisation after the alignment of the regulatory spine and adoption of an αC-helix-in conformation so RAF dimerisation is intrinsically linked to the adoption of an αC-helix-in conformation and RAF activation [ 59 ]. The positive charge of the RKTR motif interacts with the phosphorylated NtA region of the partner RAF protein after phosphorylation of the Tyr and Ser residue in ARAF [ 62 ] and CRAF [ 63 ], whilst BRAF contains an Asp in place of the Tyr and is constitutively phosphorylated at the Ser, forming a salt bridge that stabilises the dimer. The constitutive negative charge within the BRAF NtA region leaves the protein ‘primed’ for dimerisation, and able to be activated by single site phosphorylation or single point mutations [ 64 ]; this may explain why BRAF mutations are far more common in cancer than ARAF or CRAF mutations.…”

SRC and ERK cooperatively phosphorylate DLC1 and attenuate its Rho-GAP and tumor suppressor functions

“…SRC also positively regulates the RhoA GTPase, which is frequently activated in advanced cancer ( Ellenbroek and Collard, 2007 ). Many of the previously identified SRC and SFK substrates are pro-oncogenic proteins, such as FAK, p130Cas, Cortactin, and c-RAF, whose phosphorylation by SRC increases their activity, or substrates whose consequences for tumorigenesis have not been clearly established ( Reynolds et al, 2014 ; Sulzmaier et al, 2014 ; Takahashi et al, 2017 ). In addition, SRC phosphorylation of other targets, such as PP2A and caveolin-1, can attenuate their tumor suppressor activities.…”