Oncotarget

Research Papers:

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

Anja E. Eisenhardt _, Adrian Sprenger, Michael Röring, Ricarda Herr, Florian Weinberg, Martin Köhler, Sandra Braun, Joachim Orth, Britta Diedrich, Ulrike Lanner, Natalja Tscherwinski, Simon Tscherwinski, Simon Schuster, Nicolas Dumaz, Enrico Schmidt, Ralf Baumeister, Andreas Schlosser, Jörn Dengjel and Tilman Brummer

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Oncotarget. 2016; 7:26628-26652. https://doi.org/10.18632/oncotarget.8427

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Abstract

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

Correspondence to:

Tilman Brummer, email: tilman.brummer@zbsa.de

Jörn 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.


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