Brummer T, Martin P, Herzog S, Misawa Y, Daly R J, Reth M
Cancer Research Program, The Garvan Institute of Medical Research, Sydney, NSW, Australia.
Oncogene. 2006 Oct 12;25(47):6262-76. doi: 10.1038/sj.onc.1209640. Epub 2006 May 15.
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.
BRAF（V600E）突变存在于约6%的人类癌症中，模拟激酶结构域激活片段的磷酸化。在野生型B-Raf（B-Raf（wt））中，激活片段磷酸化被认为与N区域内的负电荷协同作用以实现完全激活。与Raf-1不同，由于存在D447/D448残基和S446的磷酸化，B-Raf的N区域持续带负电荷。因此，有人提出这一特征使B-Raf易于发生致癌激活。在本研究中，我们证明这些残基（特别是S446和S447）的中和突变，或B-Raf与鸟嘌呤5'-三磷酸（GTP）-Ras的解偶联，在PC12细胞分化试验中强烈降低B-Raf的生物学活性。我们还证实S365是一个14-3-3结合位点，并确定该残基的突变挽救了具有中和N区域的B-Raf蛋白受损的生物学活性，表明N区域对抗14-3-3介导的向无活性构象的转变。然而，对于B-Raf（V600E），尽管完全的N区域中和导致体外激酶活性降低2.5倍，但这种癌蛋白强烈诱导PC12分化或转化以及MCF-10A细胞的上皮-间质转化，而不管其N区域电荷如何。此外，B-Raf（V600E）的生物学活性与其结合Ras-GTP的能力无关。我们的分析确定了B-Raf（wt）和B-Raf（V600E）之间重要的调控差异，并表明B-Raf（V600E）不能被旨在阻断S446磷酸化或Ras激活的策略所抑制。
