Abstract
B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs in trans within a Raf dimer. Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase.
Highlights
The Ras/Raf/mitogen-activated/extracellular-regulated kinase (MEK)/extracellular signal regulated kinase (ERK) pathway plays a pivotal role in controlling proliferation, survival and differentiation of metazoan cells
Using an antibody recognizing phosphorylated T401, we show that this residue was already phosphorylated in murine embryonic fibroblasts (MEFs) in the absence of 4-HT and that induction of oncogenic Ras signaling did not lead to an increase in T401 phosphorylation
We obtained detailed new insights into the interaction repertoire of B-Raf and the spectrum of its phosphorylation sites. In respect to the former, we could identify a series of novel interaction partners and supply information regarding the dynamics of their recruitment
Summary
The Ras/Raf/mitogen-activated/extracellular-regulated kinase (MEK)/extracellular signal regulated kinase (ERK) pathway plays a pivotal role in controlling proliferation, survival and differentiation of metazoan cells. The multi-kinase inhibitor sorafenib, originally developed to block Raf-1 in tumor cells with aberrant Ras signaling [12], targets B-Raf, its efficacy in B-Raf driven melanoma has been disappointing [11]. The use of existing Raf-inhibitors is restricted to tumor cells with BRAFV600E/K mutations as the binding of these compounds to wildtype B-Raf provokes the paradoxical activation of the MEK/ERK pathway. This phenomenon, which causes therapy resistance and side effects including secondary neoplasms, involves the presence of active Ras and heterodimerization between a drug-bound and a drug-free Raf protomer [14, 16,17,18,19]. Alternative strategies for the inhibition of Raf-kinases in the context of aberrant Ras signaling are urgently needed and might emerge from studies of basic principles underlying B-Raf regulation
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