Activation loop phosphorylation regulates B-Raf in vivo and transformation by B-Raf mutants
| Autor: | Franziska M. Uhl, Sandra Braun, Lisa C. Schmitt, Robert Zeiser, Björn Schorch, Martin Köhler, Wolfgang W. A. Schamel, Michael Röring, Katharina Heilmann, Sebastian Herzog, Florian Weinberg, Gina J. Fiala, Thorsten Kaltenbacher, Hassan Jumaa, Sophia Ehrenfeld, Natalie Stickel, Tilman Brummer |
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| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
MAPK/ERK pathway Male Proto-Oncogene Proteins B-raf MAP Kinase Signaling System Mutant Biology Models Biological General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Mice Animals News & Views Phosphorylation Molecular Biology Cells Cultured Cell Proliferation Phenocopy Mitogen-Activated Protein Kinase Kinases General Immunology and Microbiology General Neuroscience Gene targeting Articles Molecular biology Fusion protein Enzyme Activation 030104 developmental biology Mutation Female Signal transduction Immediate early gene Signal Transduction |
| Zdroj: | The EMBO journal. 35(2) |
| ISSN: | 1460-2075 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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