Eisenhardt, Anja E. Sprenger, Adrian Röring, Michael Herr, Ricarda Weinberg, Florian Köhler, Martin Braun, Sandra Orth, Joachim Diedrich, Britta Lanner, Ulrike Tscherwinski, Natalja Schuster, Simon Dumaz, Nicolas Schmidt, Enrico Baumeister, Ralf Schlosser, Andreas Dengjel, Jörn Brummer, Tilman 2016-03-29 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. application/pdf https://folia.unifr.ch/global/documents/305068 https://folia.unifr.ch/documents/305068/files/den_ppa.pdf eng info:eu-repo/semantics/altIdentifier/doi/10.18632/oncotarget.8427 info:eu-repo/semantics/openAccess License undefined Oncotarget. - 2016, vol. 7, no. 18, p. 26628–26652 BRAF proteomics phosphorylation sorafenib protein-protein interaction info:eu-repo/classification/udc/57 Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles http://purl.org/coar/resource_type/c_6501