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

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

doi:10.18632/oncotarget.8427

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Journal article

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

Eisenhardt, Anja E. 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
Sprenger, Adrian Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany - Institute for Experimental and Clinical Pharmacology and Toxicology, ALU, Freiburg, Germany - INSERM U976 and Université Paris Diderot, Sorbonne Paris Cité, Paris, France
Röring, Michael 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
Herr, Ricarda 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
Weinberg, Florian 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
Köhler, Martin 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
Braun, Sandra 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
Orth, Joachim Institute for Experimental and Clinical Pharmacology and Toxicology, ALU, Freiburg, Germany
Diedrich, Britta Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany - Department of Dermatology, University Medical Centre, ALU, Freiburg, Germany
Lanner, Ulrike Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany
Tscherwinski, Natalja Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany - Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany
Schuster, Simon Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany - Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany
Dumaz, Nicolas INSERM U976 and Université Paris Diderot, Sorbonne Paris Cité, Paris, France
Schmidt, Enrico Institute of Biology III, Faculty of Biology, ALU, Freiburg, Germany - Centre for Biological Systems Analysis (ZBSA), Freiburg, Germany
Baumeister, Ralf 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
Schlosser, Andreas 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
Dengjel, Jörn 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
Brummer, Tilman 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

29.03.2016

Published in:

Oncotarget. - 2016, vol. 7, no. 18, p. 26628–26652

English 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.

Faculty

Faculté des sciences et de médecine

Department

Département de Biologie

Language