Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles

// Anja E. Eisenhardt 1, 2, 3 , Adrian Sprenger 3, 5, 7 , Michael Roring 1, 2, 3, 4 , Ricarda Herr 1, 2, 3 , Florian Weinberg 1, 2, 3 , Martin Kohler 1, 2, 3, 4 , Sandra Braun 1, 2, 3 , Joachim Orth 5 , Britta Diedrich 3, 6 , Ulrike Lanner 3 , Natalja Tscherwinski 2, 3 , Simon Schuster 2, 3 , Nicolas Dumaz 7 , Enrico Schmidt 2, 3 , Ralf Baumeister 2, 3, 8, 10 , Andreas Schlosser 2, 3, 9 , Jorn Dengjel 3, 6, 8, 10, 11, * , Tilman Brummer 1, 2, 3, 10, 12, * 1 Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, Albert-Ludwigs-University (ALU), Freiburg, Germany 2 Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany 3 Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany 4 Spemann Graduate School of Biology and Medicine (SGBM), ALU, Freiburg, Germany 5 Institute for Experimental and Clinical Pharmacology and Toxicology, ALU, Freiburg, Germany 6 Department of Dermatology, University Medical Centre, ALU, Freiburg, Germany 7 INSERM U976 and Universite Paris Diderot, Sorbonne Paris Cite, Paris, France 8 Freiburg Institute for Advanced Studies (FRIAS), ALU, Freiburg, Germany 9 Rudolf Virchow Center for Experimental Biomedicine, University of Wurzburg, Wurzburg, Germany 10 Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany 11 Department of Biology, University of Fribourg, Fribourg, Switzerland 12 German Cancer Consortium (DKTK), Freiburg, Germany * These authors have contributed equally to this work Correspondence to: Tilman Brummer, email: tilman.brummer@zbsa.de Jorn Dengjel, email: joern.dengjel@unifr.ch Keywords: BRAF , proteomics, phosphorylation, sorafenib, protein-protein interaction Received: August 18, 2015     Accepted: March 07, 2016     Published: March 28, 2016 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.