title: Activation loop phosphorylation regulates B-Raf in vivo and transformation by B-Raf mutants.
abstract: Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B-Raf signaling in vivo. Here, we generated a conditional knock-in mouse allowing the expression of the B-Raf(AVKA) mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase-impaired protein, the Braf(AVKA) allele does not phenocopy the lethality of Braf-knockout or paradoxically acting knock-in alleles. However, Braf(AVKA) mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B-Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B-Raf(AVKA). Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non-V600E B-Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP-competitive inhibitors.
authors.affiliation: Faculty of Medicine, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University (ALU), Freiburg, Germany Centre for Biological Systems Analysis ZBSA, ALU, Freiburg, Germany Spemann Graduate School for Biology and Medicine, ALU, Freiburg, Germany Faculty of Biology, ALU, Freiburg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Köhler
authors.affiliation: Faculty of Medicine, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University (ALU), Freiburg, Germany Centre for Biological Systems Analysis ZBSA, ALU, Freiburg, Germany Spemann Graduate School for Biology and Medicine, ALU, Freiburg, Germany Faculty of Biology, ALU, Freiburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Röring
authors.affiliation: Centre for Biological Systems Analysis ZBSA, ALU, Freiburg, Germany Spemann Graduate School for Biology and Medicine, ALU, Freiburg, Germany Faculty of Biology, ALU, Freiburg, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Schorch
authors.affiliation: Centre for Biological Systems Analysis ZBSA, ALU, Freiburg, Germany Faculty of Biology, ALU, Freiburg, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Heilmann
authors.affiliation: Spemann Graduate School for Biology and Medicine, ALU, Freiburg, Germany Faculty of Biology, ALU, Freiburg, Germany Department of Hematology and Oncology, University Medical Center ALU, Freiburg, Germany.
authors.forename: Natalie
authors.initials: N
authors.lastname: Stickel
authors.affiliation: Spemann Graduate School for Biology and Medicine, ALU, Freiburg, Germany Faculty of Biology, ALU, Freiburg, Germany Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany Department of Molecular Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Gina J
authors.initials: GJ
authors.lastname: Fiala
authors.affiliation: Faculty of Medicine, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University (ALU), Freiburg, Germany Centre for Biological Systems Analysis ZBSA, ALU, Freiburg, Germany Faculty of Biology, ALU, Freiburg, Germany.
authors.forename: Lisa C
authors.initials: LC
authors.lastname: Schmitt
authors.affiliation: Faculty of Medicine, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University (ALU), Freiburg, Germany Centre for Biological Systems Analysis ZBSA, ALU, Freiburg, Germany Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Braun
authors.affiliation: Faculty of Medicine, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University (ALU), Freiburg, Germany Faculty of Biology, ALU, Freiburg, Germany.
authors.forename: Sophia
authors.initials: S
authors.lastname: Ehrenfeld
authors.affiliation: Faculty of Medicine, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University (ALU), Freiburg, Germany Faculty of Biology, ALU, Freiburg, Germany.
authors.forename: Franziska M
authors.initials: FM
authors.lastname: Uhl
authors.affiliation: Faculty of Medicine, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University (ALU), Freiburg, Germany Faculty of Biology, ALU, Freiburg, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Kaltenbacher
authors.affiliation: Faculty of Medicine, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University (ALU), Freiburg, Germany Centre for Biological Systems Analysis ZBSA, ALU, Freiburg, Germany Faculty of Biology, ALU, Freiburg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Weinberg
authors.affiliation: Centre for Biological Systems Analysis ZBSA, ALU, Freiburg, Germany Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany Department of Molecular Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Herzog
authors.affiliation: Department of Hematology and Oncology, University Medical Center ALU, Freiburg, Germany Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany Comprehensive Cancer Centre, Freiburg, Germany German Consortium for Translational Cancer Research DKTK, Standort Freiburg, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Zeiser
authors.affiliation: Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany Department of Molecular Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany Center for Chronic Immunodeficiency CCI, University Medical Center, Freiburg, Germany.
authors.forename: Wolfgang W
authors.initials: WW
authors.lastname: Schamel
authors.affiliation: Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany Department of Molecular Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany Institute of Immunology, University Hospital Ulm, Ulm, Germany.
authors.forename: Hassan
authors.initials: H
authors.lastname: Jumaa
authors.affiliation: Faculty of Medicine, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University (ALU), Freiburg, Germany Centre for Biological Systems Analysis ZBSA, ALU, Freiburg, Germany Faculty of Biology, ALU, Freiburg, Germany Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany Comprehensive Cancer Centre, Freiburg, Germany German Consortium for Translational Cancer Research DKTK, Standort Freiburg, Germany tilman.brummer@zbsa.de.
authors.forename: Tilman
authors.initials: T
authors.lastname: Brummer
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000235:genetics / Q000502:physiology; D005260:Female; D020935:MAP Kinase Signaling System / Q000235:genetics / Q000528:radiation effects; D008297:Male; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
chemical_list: D048493:Proto-Oncogene Proteins B-raf; D020929:Mitogen-Activated Protein Kinase Kinases
keywords: BRAF fusion; Cre/loxP system; MAPK pathway; Ras; gene targeting