title: Dimerization of the kinase ARAF promotes MAPK pathway activation and cell migration.
abstract: The RAF family of kinases mediates RAS signaling, and RAF inhibitors can be effective for treating tumors with BRAF(V600E) mutant protein. However, RAF inhibitors paradoxically accelerate metastasis in RAS-mutant tumors and become ineffective in BRAF(V600E) tumors because of reactivation of downstream mitogen-activated protein kinase (MAPK) signaling. We found that the RAF isoform ARAF has an obligatory role in promoting MAPK activity and cell migration in a cell type-dependent manner. Knocking down ARAF prevented the activation of MAPK kinase 1 (MEK1) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) and decreased the number of protrusions from tumor cell spheroids in three-dimensional culture that were induced by BRAF(V600E)-specific or BRAF/CRAF inhibitors (GDC-0879 and sorafenib, respectively). RAF inhibitors induced the homodimerization of ARAF and the heterodimerization of BRAF with CRAF and the scaffolding protein KSR1. In a purified protein solution, recombinant proteins of the three RAF isoforms competed for binding to MEK1. In cells in culture, overexpressing mutants of ARAF that could not homodimerize impaired the interaction between ARAF and endogenous MEK1 and thus prevented the subsequent activation of MEK1 and ERK1/2. Our findings reveal a new role for ARAF in directly activating the MAPK cascade and promoting tumor cell invasion and suggest a new therapeutic target for RAS- and RAF-mediated cancers.
authors.affiliation: Cell death signalling group, Institute of Biochemistry II, Goethe University Medical School, Frankfurt 60590, Germany. Institute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstraße 1, Building 708, Mainz 55131, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Mooz
authors.affiliation: Cell death signalling group, Institute of Biochemistry II, Goethe University Medical School, Frankfurt 60590, Germany.
authors.forename: Tripat Kaur
authors.initials: TK
authors.lastname: Oberoi-Khanuja
authors.affiliation: Stark Learning Center, Department of Biology and Health Sciences, Wilkes University, Wilkes-Barre, Pennsylvania, PA 18766, USA.
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Harms
authors.affiliation: Department of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Weiru
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Molecular Biology, Genentech Inc., South San Francisco, CA 94080, USA.
authors.forename: Bijay S
authors.initials: BS
authors.lastname: Jaiswal
authors.affiliation: Department of Molecular Biology, Genentech Inc., South San Francisco, CA 94080, USA.
authors.forename: Somasekar
authors.initials: S
authors.lastname: Seshagiri
authors.affiliation: Institute of Biochemistry, Medical Faculty, University of Giessen, Giessen 35392, Germany.
authors.forename: Ritva
authors.initials: R
authors.lastname: Tikkanen
authors.affiliation: Cell death signalling group, Institute of Biochemistry II, Goethe University Medical School, Frankfurt 60590, Germany. Institute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstraße 1, Building 708, Mainz 55131, Germany. krishna@biochem2.de.
authors.forename: Krishnaraj
authors.initials: K
authors.lastname: Rajalingam
mesh_terms: D000704:Analysis of Variance; D001667:Binding, Competitive; D015153:Blotting, Western; D002465:Cell Movement / Q000502:physiology*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000235:genetics; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D007192:Indenes / Q000494:pharmacology; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D009361:Neoplasm Invasiveness; D009536:Niacinamide / Q000031:analogs & derivatives / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000494:pharmacology; D048491:Proto-Oncogene Proteins A-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors; D011720:Pyrazoles / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D000077157:Sorafenib; D059008:Time-Lapse Imaging; D014407:Tumor Cells, Cultured
chemical_list: C540167:2-(4-(1-(hydroxyimino)-2,3-dihydro-1H-inden-5-yl)-3-(pyridin-4-yl)-1H-pyrazol-1-yl)ethan-1-ol; D007192:Indenes; D010671:Phenylurea Compounds; D011720:Pyrazoles; D034741:RNA, Small Interfering; D009536:Niacinamide; D000077157:Sorafenib; D048491:Proto-Oncogene Proteins A-raf; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1