Activation loop phosphorylation regulates B‐Raf in vivo and transformation by B‐Raf mutants

Abstract

Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B-Raf signaling in vivo. Here, we generated a conditional knock-in mouse allowing the expression of the B-Raf AVKA mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase-impaired protein, the Braf AVKA allele does not phenocopy the lethality of Braf-knockout or paradoxically acting knock-in alleles. However, Braf AVKA mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B-Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B-Raf AVKA . Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non-V600E B-Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP-competitive inhibitors.

B-Raf activation loop phosphorylation revisited

“…This concept is supported by (pre) clinical observations showing that drug-bound or kinase-dead B-Raf provokes paradoxical ERK-pathway activation, a phenomenon that underlies therapy resistance, inhibitor promoted secondary neoplasms and restricts the application of clinically approved B-Raf inhibitors to BRAF V600E/K mutant tumors. 1,7 Interestingly, however, B-Raf AVKA did not provoke paradoxical ERK activation like the kinase-dead B-Raf D594A mutant, 3,4,7 suggesting that the inability of B-Raf AVKA to undergo AL phosphorylation precludes a proper DIF conformation in which it can trans-activate another protomer, e.g., Raf-1 (Fig. 1).…”

“…If Braf deficiency is introduced via the germline, Braf ¡/¡ mice display a lethal placental phenotype and, if deficiency is limited to the nervous system, a myelination defect restricts viability to the first 5 postnatal weeks (see Refs. 2,3 for discussion and references).…”

“…1,4 The relevance of TVKS-motif phosphorylation was demonstrated by experiments showing that substitution of T599/S602 by alanine (AVKA) and phosphomimetic (EVKD) residues impairs Ras-induced activity and confers transforming properties to B-Raf, respectively. 3,5 The dominance of V600 substitutions in tumor-associated BRAF mutations further underscores that AL phosphorylation mimicking mutations induce conformational changes that cut the B-Raf activation cycle short. Indeed, B-Raf V600E signals independently of RAS, 14-3-3 binding, critical phosphorylation sites and, although it forms particularly stable dimers in its normal state, an intact DIF.…”

“…Indeed, B-Raf V600E signals independently of RAS, 14-3-3 binding, critical phosphorylation sites and, although it forms particularly stable dimers in its normal state, an intact DIF. 3,4 Thus, various lines of evidence suggest that AL phosphorylation induces conformational changes in the B-Raf kinase domain, promoting both dimerization and kinase activity ( Fig. 1) and that, once this conformation is stabilized by V600E mutation-specific effects, 1,4 AL phosphorylation becomes redundant.…”

“…3 Although this Braf AVKA allele produces a kinase with significantly impaired activity, mice homozygous for this allele were surprisingly viable, fertile and had a normal life span. Nevertheless, Braf AVKA mice presented with mild abnormalities in the haematopoietic system, a distinct facial morphology, reduced MEK/ERK phosphorylation in the brain and slight gait abnormalities.…”