title: Discrete cytosolic macromolecular BRAF complexes exhibit distinct activities and composition.
abstract: As a central element within the RAS/ERK pathway, the serine/threonine kinase BRAF plays a key role in development and homeostasis and represents the most frequently mutated kinase in tumors. Consequently, it has emerged as an important therapeutic target in various malignancies. Nevertheless, the BRAF activation cycle still raises many mechanistic questions as illustrated by the paradoxical action and side effects of RAF inhibitors. By applying SEC-PCP-SILAC, we analyzed protein-protein interactions of hyperactive BRAFV600Eand wild-type BRAF (BRAFWT). We identified two macromolecular, cytosolic BRAF complexes of distinct molecular composition and phosphorylation status. Hyperactive BRAFV600Eresides in large complexes of higher molecular mass and activity, while BRAFWTis confined to smaller, slightly less active complexes. However, expression of oncogenic K-RasG12V, either by itself or in combination with RAF dimer promoting inhibitors, induces the incorporation of BRAFWTinto large, active complexes, whereas pharmacological inhibition of BRAFV600Ehas the opposite effect. Thus, the quaternary structure of BRAF complexes is shaped by its activation status, the conformation of its kinase domain, and clinically relevant inhibitors.
authors.affiliation: Department of Dermatology, Medical Center - University of Freiburg, Freiburg, Germany.|ZBSA Center for Biological Systems Analysis, University of Freiburg, Freiburg, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Diedrich
authors.affiliation: Department of Dermatology, Medical Center - University of Freiburg, Freiburg, Germany.|ZBSA Center for Biological Systems Analysis, University of Freiburg, Freiburg, Germany.
authors.forename: Kristoffer Tg
authors.initials: KT
authors.lastname: Rigbolt
authors.affiliation: Faculty of Medicine, Institute of Molecular Medicine and Cell Research (IMMZ), University of Freiburg, Freiburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Röring
authors.affiliation: Faculty of Medicine, Institute of Molecular Medicine and Cell Research (IMMZ), University of Freiburg, Freiburg, Germany.
authors.forename: Ricarda
authors.initials: R
authors.lastname: Herr
authors.affiliation: Department of Biology, University of Fribourg, Fribourg, Switzerland.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kaeser-Pebernard
authors.affiliation: Department of Dermatology, Medical Center - University of Freiburg, Freiburg, Germany.|ZBSA Center for Biological Systems Analysis, University of Freiburg, Freiburg, Germany.|Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Gretzmeier
authors.affiliation: Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany.|Computational Biology Department and Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA.
authors.forename: Robert F
authors.identifier: 0000-0003-0358-901X
authors.initials: RF
authors.lastname: Murphy
authors.affiliation: ZBSA Center for Biological Systems Analysis, University of Freiburg, Freiburg, Germany tilman.brummer@zbsa.uni-freiburg.de joern.dengjel@unifr.ch.|Faculty of Medicine, Institute of Molecular Medicine and Cell Research (IMMZ), University of Freiburg, Freiburg, Germany.|Centre for Biological Signalling Studies BIOSS, University of Freiburg, Freiburg, Germany.|Comprehensive Cancer Centre, Freiburg, Germany.|German Cancer Consortium (DKTK), partner site Freiburg, and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Tilman
authors.identifier: 0000-0003-4387-7905
authors.initials: T
authors.lastname: Brummer
authors.affiliation: Department of Dermatology, Medical Center - University of Freiburg, Freiburg, Germany tilman.brummer@zbsa.uni-freiburg.de joern.dengjel@unifr.ch.|ZBSA Center for Biological Systems Analysis, University of Freiburg, Freiburg, Germany.|Department of Biology, University of Fribourg, Fribourg, Switzerland.|Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany.|Centre for Biological Signalling Studies BIOSS, University of Freiburg, Freiburg, Germany.
authors.forename: Jörn
authors.identifier: 0000-0002-9453-4614
authors.initials: J
authors.lastname: Dengjel
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002850:Chromatography, Gel; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D010766:Phosphorylation; D011487:Protein Conformation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
keywords: 14‐3‐3 proteins; HSP90 CDC37 complex; geldanamycin; sorafenib; vemurafenib