CRITICAL TYROSINE RESIDUES REGULATE THE ENZYMATIC AND BIOLOGICAL-ACTIVITY OF RAF-1 KINASE
CRITICAL TYROSINE RESIDUES REGULATE THE ENZYMATIC AND BIOLOGICAL-ACTIVITY OF RAF-1 KINASE
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DOI：
10.1128/mcb.13.11.7170
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发表时间：
1993-11-01
影响因子：
5.3
通讯作者：
MORRISON, DK
中科院分区：
文献类型：
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作者：
FABIAN, JR;DAAR, IO;MORRISON, DK
Raf-1 原癌基因产物的丝氨酸/苏氨酸激酶活性受到许多酪氨酸激酶（包括生长因子受体和 pp60v-src）的激活的刺激。最近对生长因子信号转导途径的研究表明，Raf-1 在激活的酪氨酸激酶和 p21ras 的下游以及丝裂原激活的蛋白激酶的上游发挥作用。然而，为了在杆状病毒-Sf9 表达系统中最大程度地激活 Raf-1，需要激活的酪氨酸激酶和 p21ras 共表达。在这项研究中，我们研究了酪氨酸激酶和酪氨酸磷酸化在调节 Raf-I 活性中的作用。使用杆状病毒-Sf9表达系统，我们确定Tyr-340和Tyr-341是Raf-1与激活的酪氨酸激酶共表达时的主要酪氨酸磷酸化位点。在这些位点引入可能模拟磷酸化作用的带负电残基，激活了 Raf-1 的催化活性，并产生了可以转化 BALB/3T3 细胞并诱导非洲爪蟾卵母细胞减数分裂成熟的蛋白质。相比之下，无法磷酸化的不带电残基的取代产生了不能被酪氨酸激酶酶促激活的蛋白质，并且可以阻止受体酪氨酸激酶途径的成分诱导的卵母细胞减数分裂成熟。这些发现表明酪氨酸磷酸化位点的突变可以显着改变 Raf-1 的功能。此外，这是首次报道可通过单个氨基酸取代产生转化Raf-1蛋白。
The serine/threonine kinase activity of the Raf-1 proto-oncogene product is stimulated by the activation of many tyrosine kinases, including growth factor receptors and pp60v-src. Recent studies of growth factor signal transduction pathways demonstrate that Raf-1 functions downstream of activated tyrosine kinases and p21ras and upstream of mitogen-activated protein kinase. However, coexpression of both activated tyrosine kinases and p21ras is required for maximal activation of Raf-1 in the baculovirus-Sf9 expression system. In this study, we investigated the role of tyrosine kinases and tyrosine phosphorylation in the regulation of Raf-I activity. Using the baculovirus-Sf9 expression system, we identified Tyr-340 and Tyr-341 as the major tyrosine phosphorylation sites of Raf-1 when coexpressed with activated tyrosine kinases. Introduction of a negatively charged residue that may mimic the effect of phosphorylation at these sites activated the catalytic activity of Raf-1 and generated proteins that could transform BALB/3T3 cells and induce the meiotic maturation of Xenopus oocytes. In contrast, substitution of noncharged residues that were unable to be phosphorylated produced a protein that could not be enzymatically activated by tyrosine kinases and that could block the meiotic maturation of oocytes induced by components of the receptor tyrosine kinase pathway. These findings demonstrate that mutation of the tyrosine phosphorylation sites can dramatically alter the function of Raf-1. In addition, this is the first report that a transforming Raf-1 protein can be generated by a single amino acid substitution.