1. ¹Cancer Research Program, The Garvan Institute of Medical Research, Sydney, NSW, Australia
2. ²Department of Molecular Immunology, Institute for Biology III, Albert-Ludwigs-University of Freiburg and Max-Planck-Institut for Immunobiology, Freiburg, Germany
3. ³Department of Microbiology, University of Virginia Health System, University of Virginia, Charlottesville, VA, USA

Correspondence: Dr T Brummer, Cancer Research Program, The Garvan Institute of Medical Research, 384 Victoria Street, Sydney, NSW 2010, Australia. E-mail: t.brummer@garvan.org.au

Received 16 September 2005; Revised 20 March 2006; Accepted 21 March 2006; Published online 15 May 2006.

Abstract

The BRAF^V600E 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-Raf^wt), 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-Raf^V600E, 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-Raf^V600E was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Raf^wt and B-Raf^V600E and suggests that B-Raf^V600E cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation.