Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles
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Anja E. Eisenhardt1,2,3, Adrian Sprenger3,5,7, Michael Röring1,2,3,4, Ricarda Herr1,2,3, Florian Weinberg1,2,3, Martin Köhler1,2,3,4, Sandra Braun1,2,3, Joachim Orth5, Britta Diedrich3,6, Ulrike Lanner3, Natalja Tscherwinski2,3, Simon Schuster2,3, Nicolas Dumaz7, Enrico Schmidt2,3, Ralf Baumeister2,3,8,10, Andreas Schlosser2,3,9, Jörn Dengjel3,6,8,10,11,*, Tilman Brummer1,2,3,10,12,*
1Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, Albert-Ludwigs-University (ALU), Freiburg, Germany
2Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany
3Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany
4Spemann Graduate School of Biology and Medicine (SGBM), ALU, Freiburg, Germany
5Institute for Experimental and Clinical Pharmacology and Toxicology, ALU, Freiburg, Germany
6Department of Dermatology, University Medical Centre, ALU, Freiburg, Germany
7INSERM U976 and Université Paris Diderot, Sorbonne Paris Cité, Paris, France
8Freiburg Institute for Advanced Studies (FRIAS), ALU, Freiburg, Germany
9Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany
10Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany
11Department of Biology, University of Fribourg, Fribourg, Switzerland
12German Cancer Consortium (DKTK), Freiburg, Germany
*These authors have contributed equally to this work
Tilman Brummer, email: [email protected]
Jörn Dengjel, email: [email protected]
Keywords: BRAF, proteomics, phosphorylation, sorafenib, protein-protein interaction
Received: August 18, 2015 Accepted: March 07, 2016 Published: March 28, 2016
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.
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