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Functional analysis of the regulatory requirements of B-Raf and B-RafV600E oncoprotein

MPG-Autoren
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Brummer,  T.
Research Group and Chair of Molecular Immunology of the University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Martin,  P.
Emeritus Group: Cellular Immunology, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Herzog,  S.
Research Group and Chair of Molecular Immunology of the University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Misawa,  Y.
Research Group and Chair of Molecular Immunology of the University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Reth,  M.
Research Group and Chair of Molecular Immunology of the University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Zitation

Brummer, T., Martin, P., Herzog, S., Misawa, Y., Daly, R. J., & Reth, M. (2006). Functional analysis of the regulatory requirements of B-Raf and B-RafV600E oncoprotein. Oncogene, 25, 6262-6276.


Zitierlink: https://hdl.handle.net/11858/00-001M-0000-002B-927A-5
Zusammenfassung
The BRAFV600E mutation is found in approximately 6% of human cancers and mimics the phosphorylation of the kinase domain activation segment. In wild-type B-Raf (B-Rafwt), activation segment phosphorylation is thought to cooperate with negative charges within the N-region for full activation. In contrast to Raf-1, the N-region of B-Raf is constitutively negatively charged owing to the presence of residues D447/D448 and the phosphorylation of S446. Therefore, it has been suggested that this hallmark predisposes B-Raf for oncogenic activation. In this study, we demonstrate that neutralizing mutations of these residues (in particular S446 and S447), or uncoupling of B-Raf from Ras-guanine 5'-triphosphate (GTP), strongly reduce the biological activity of B-Raf in a PC12 cell differentiation assay. We also confirm that S365 is a 14-3-3 binding site, and determine that mutation of this residue rescues the impaired biological activity of B-Raf proteins with a neutralized N-region, suggesting that the N-region opposes a 14-3-3-mediated transition into an inactive conformation. However, in the case of B-RafV600E, although complete N-region neutralization resulted in a 2.5-fold reduction in kinase activity in vitro, this oncoprotein strongly induced PC12 differentiation or transformation and epithelial-mesenchymal transition of MCF-10A cells regardless of its N-region charge. Furthermore, the biological activity of B-RafV600E was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Rafwt and B-RafV600E and suggests that B-RafV600E cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation.