title: Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles.
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.
authors.affiliation: Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, Albert-Ludwigs-University (ALU), Freiburg, Germany.|Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany.|Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.
authors.forename: Anja E
authors.initials: AE
authors.lastname: Eisenhardt
authors.affiliation: Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.|Institute for Experimental and Clinical Pharmacology and Toxicology, ALU, Freiburg, Germany.|INSERM U976 and Universitéi Paris Diderot, Sorbonne Paris Cité, Paris, France.
authors.forename: Adrian
authors.initials: A
authors.lastname: Sprenger
authors.affiliation: Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, Albert-Ludwigs-University (ALU), Freiburg, Germany.|Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany.|Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.|Spemann Graduate School of Biology and Medicine (SGBM), ALU, Freiburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Röring
authors.affiliation: Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, Albert-Ludwigs-University (ALU), Freiburg, Germany.|Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany.|Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.
authors.forename: Ricarda
authors.initials: R
authors.lastname: Herr
authors.affiliation: Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, Albert-Ludwigs-University (ALU), Freiburg, Germany.|Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany.|Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Weinberg
authors.affiliation: Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, Albert-Ludwigs-University (ALU), Freiburg, Germany.|Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany.|Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.|Spemann Graduate School of Biology and Medicine (SGBM), ALU, Freiburg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Köhler
authors.affiliation: Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, Albert-Ludwigs-University (ALU), Freiburg, Germany.|Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany.|Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Braun
authors.affiliation: Institute for Experimental and Clinical Pharmacology and Toxicology, ALU, Freiburg, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Orth
authors.affiliation: Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.|Department of Dermatology, University Medical Centre, ALU, Freiburg, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Diedrich
authors.affiliation: Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Lanner
authors.affiliation: Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany.|Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.
authors.forename: Natalja
authors.initials: N
authors.lastname: Tscherwinski
authors.affiliation: Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany.|Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Schuster
authors.affiliation: INSERM U976 and Universitéi Paris Diderot, Sorbonne Paris Cité, Paris, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Dumaz
authors.affiliation: Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany.|Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.
authors.forename: Enrico
authors.initials: E
authors.lastname: Schmidt
authors.affiliation: Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany.|Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.|Freiburg Institute for Advanced Studies (FRIAS), ALU, Freiburg, Germany.|Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Baumeister
authors.affiliation: Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany.|Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.|Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schlosser
authors.affiliation: Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.|Department of Dermatology, University Medical Centre, ALU, Freiburg, Germany.|Freiburg Institute for Advanced Studies (FRIAS), ALU, Freiburg, Germany.|Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany.|Department of Biology, University of Fribourg, Fribourg, Switzerland.
authors.forename: Jörn
authors.initials: J
authors.lastname: Dengjel
authors.affiliation: Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, Albert-Ludwigs-University (ALU), Freiburg, Germany.|Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany.|Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany.|Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany.|German Cancer Consortium (DKTK), Freiburg, Germany.
authors.forename: Tilman
authors.initials: T
authors.lastname: Brummer
mesh_terms: D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000737:chemistry / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D040901:Proteomics; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D048493:Proto-Oncogene Proteins B-raf
keywords: BRAF; phosphorylation; protein-protein interaction; proteomics; sorafenib