Functional analysis of the regulatory requirements of B-Raf and the B-Raf(V600E) oncoprotein.
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.
Brummer
T
T
Cancer Research Program, The Garvan Institute of Medical Research, Sydney, NSW, Australia. t.brummer@garvan.org.au
Martin
P
P
Herzog
S
S
Misawa
Y
Y
Daly
R J
RJ
Reth
M
M
D048948:14-3-3 Proteins / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line / Q000166:cytology / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D002645:Chickens; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000166:cytology; D025461:Feedback, Physiological; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System; D008648:Mesoderm / Q000166:cytology; D051379:Mice; D008954:Models, Biological; D020125:Mutation, Missense; D041681:NIH 3T3 Cells; D015689:Oncogene Protein p21(ras) / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D017354:Point Mutation; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology; D013329:Structure-Activity Relationship; D014162:Transfection
D048948:14-3-3 Proteins; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D015689:Oncogene Protein p21(ras)