Functional analysis of the regulatory requirements of B-Raf and the B-Raf(V600E) oncoprotein

Tilman Brummer, P Martin, S Herzog, Y Misawa, Roger John Daly, M Reth

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50 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)6262 - 6276
Number of pages15
JournalOncogene
Volume25
Issue number47
DOIs
Publication statusPublished - 2006
Externally publishedYes

Cite this

Brummer, Tilman ; Martin, P ; Herzog, S ; Misawa, Y ; Daly, Roger John ; Reth, M. / Functional analysis of the regulatory requirements of B-Raf and the B-Raf(V600E) oncoprotein. In: Oncogene. 2006 ; Vol. 25, No. 47. pp. 6262 - 6276.
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Brummer, T, Martin, P, Herzog, S, Misawa, Y, Daly, RJ & Reth, M 2006, 'Functional analysis of the regulatory requirements of B-Raf and the B-Raf(V600E) oncoprotein', Oncogene, vol. 25, no. 47, pp. 6262 - 6276. https://doi.org/10.1038/sj.onc.1209640

Functional analysis of the regulatory requirements of B-Raf and the B-Raf(V600E) oncoprotein. / Brummer, Tilman; Martin, P; Herzog, S; Misawa, Y; Daly, Roger John; Reth, M.

In: Oncogene, Vol. 25, No. 47, 2006, p. 6262 - 6276.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Reth, M

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