Guan K L, Figueroa C, Brtva T R, Zhu T, Taylor J, Barber T D, Vojtek A B
Department of Biological Chemistry and the Institute of Gerontology, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 2000 Sep 1;275(35):27354-9. doi: 10.1074/jbc.M004371200.
B-Raf contains multiple Akt consensus sites located within its amino-terminal regulatory domain. One site, Ser(364), is conserved with c-Raf but two additional sites, Ser(428) and Thr(439), are unique to B-Raf. We have investigated the role of both the conserved and unique phosphorylation sites in the regulation of B-Raf activity in vitro and in vivo. We show that phosphorylation of B-Raf by Akt occurs at multiple residues within its amino-terminal regulatory domain, at both the conserved and unique phosphorylation sites. The alteration of the serine residues within the Akt consensus sites to alanines results in a progressive increase in enzymatic activity in vitro and in vivo. Furthermore, expression of Akt inhibits epidermal growth factor-induced B-Raf activity and inhibition of Akt with LY294002 up-regulates B-Raf activity, suggesting that Akt negatively regulates B-Raf in vivo. Our results demonstrate that B-Raf activity can be negatively regulated by Akt through phosphorylation in the amino-terminal regulatory domain of B-Raf. This cross-talk between the B-Raf and Akt serine/threonine kinases is likely to play an important role in modulating the signaling specificity of the Ras/Raf pathway and in promoting biological outcome.
B-Raf在其氨基末端调节域内含有多个Akt共有位点。一个位点Ser(364)与c-Raf保守，但另外两个位点Ser(428)和Thr(439)是B-Raf特有的。我们已经研究了保守和独特的磷酸化位点在体外和体内对B-Raf活性调节中的作用。我们发现，Akt对B-Raf的磷酸化发生在其氨基末端调节域内的多个残基上，包括保守和独特的磷酸化位点。将Akt共有位点内的丝氨酸残基替换为丙氨酸会导致体外和体内酶活性逐渐增加。此外，Akt的表达抑制表皮生长因子诱导的B-Raf活性，而用LY294002抑制Akt则上调B-Raf活性，这表明Akt在体内对B-Raf起负调节作用。我们的结果表明，Akt可通过对B-Raf氨基末端调节域的磷酸化对B-Raf活性进行负调节。B-Raf和Akt丝氨酸/苏氨酸激酶之间的这种相互作用可能在调节Ras/Raf途径的信号特异性以及促进生物学结果方面发挥重要作用。
