Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase.
Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase.
复制标题
p21ras 和 pp60v-src 都是激活 Raf-1 激酶所必需的，但单独使用两者都不足以激活 Raf-1 激酶。
DOI：
10.1073/pnas.89.7.2922
复制
发表时间：
1992
影响因子：
11.1
通讯作者：
Li,P
中科院分区：
文献类型：
--
作者：
Williams,NG;Roberts,TM;Li,P
raf基因编码具有内在蛋白丝氨酸/苏氨酸激酶活性的胞质蛋白家族。c-raf基因是鼠肉瘤病毒3611的转化基因v-raf的细胞同源物。v-Raf蛋白的组成性激酶活性与转化和有丝分裂有关。Raf-1是c-raf基因的蛋白产物，其活性通常受到N端调控结构域的抑制。各种酪氨酸激酶生长因子受体的激活导致Raf-1的激活及其过度磷酸化。此外，Raf-1已被证明是下游或独立的p21 ras蛋白，如涉及抗Ras抗体的显微注射实验所示。为了研究p21 ras在酪氨酸激酶激活Raf-1中的潜在作用，我们使用杆状病毒/Sf 9细胞系统过量产生各种野生型和突变型pp 60 src、p21 ras和Raf-1蛋白。我们表明pp 60 v-SRC或p21 c-ras都可以独立激活Raf-1的自激酶活性，但程度有限。令人惊讶的是，pp 60 v-src和p21 c-ras都需要完全激活Raf-1。Raf-1自身激酶活性的体外分析表明，Raf-1自身磷酸化位点均匀分布在丝氨酸和苏氨酸残基上。当Raf-1的免疫印迹分析，如以前报道的哺乳动物细胞实验，Raf-1的表观分子量的显着增加被视为只有当它与pp 60 v-src和p21 ras共表达。
The raf genes encode a family of cytoplasmic proteins with intrinsic protein-serine/threonine kinase activity. The c-raf gene is the cellular homolog of v-raf, the transforming gene of murine sarcoma virus 3611. The constitutive kinase activity of the v-Raf protein has been implicated in transformation and mitogenesis. The activity of Raf-1, the protein product of the c-raf gene, is normally suppressed by a regulatory N-terminal domain. Activation of various tyrosine-kinase growth factor receptors results in activation of Raf-1 and its hyperphosphorylation. Further, Raf-1 has been shown to act either downstream or independently of the p21ras protein, as indicated by experiments involving microinjection of anti-Ras antibodies. To investigate the potential role of p21ras in the activation of Raf-1 by tyrosine kinases, we have used the baculovirus/Sf9 cell system to overproduce various wild-type and mutant forms of pp60src, p21ras, and Raf-1 proteins. We show that either pp60v-src or p21c-ras can independently activate the autokinase activity of Raf-1, but only to a limited extent. Surprisingly, both pp60v-src and p21c-ras are required to fully activate Raf-1. Analysis of the Raf-1 autokinase activity in vitro shows that Raf-1 autophosphorylation sites are distributed equally on serine and threonine residues. When Raf-1 is analyzed by immunoblotting, as previously reported for mammalian cell experiments, a marked increase in the apparent molecular weight of Raf-1 is seen only when it is coexpressed with both pp60v-src and p21ras.