Dent P, Reardon D B, Morrison D K, Sturgill T W
Howard Hughes Medical Institute, University of Virginia, Charlottesville 22908, USA.
Mol Cell Biol. 1995 Aug;15(8):4125-35. doi: 10.1128/MCB.15.8.4125.
The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type (His)6- and FLAG-Raf-1 were activated in a Ras- and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and SrcY527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a membrane cofactor.
丝氨酸/苏氨酸激酶Raf-1在Ras下游发挥作用，激活丝裂原活化蛋白激酶激酶，但Raf-1的激活机制尚未完全明确。为剖析这些机制，我们在一个体外系统中研究了野生型和突变型Raf-1蛋白，该系统使用了来自v-Ras和v-Src转化细胞的纯化质膜（转化膜）。野生型（His）6-和FLAG-Raf-1被转化膜以Ras和ATP依赖的方式激活；然而，激酶缺陷型（K375M）、缺乏体内调节性酪氨酸位点（YY340/341FF）或组成型丝氨酸磷酸化位点（S621A）、不结合Ras（R89L）或缺乏完整锌指（CC165/168SS）的Raf-1蛋白则未被激活。缺乏未知激酶（S259A）或蛋白激酶C（S499A）的推定调节位点的Raf-1蛋白被激活，但效率明显降低。负责由Ras或Src激活的激酶可能存在于质膜中，因为来自静止的NIH 3T3细胞的质膜（亲本质膜）的GTP加载诱导了激活Raf-1的从头能力。仅具有基础活性的野生型Raf-1在没有GTP加载的情况下未被亲本质膜激活。相反，具有显著活性的Raf-1 Y340D令人惊讶地以不依赖Ras的方式被亲本质膜刺激。结果表明，通过磷酸化激活Raf-1可能允许另一种膜因子（如脂质）进行进一步调节。用甲醇 - 氯仿从转化膜或共表达Ras和SrcY527F的Sf9细胞的膜中提取的一种因子显著增强了Raf-1 Y340D或活性Raf-1的活性，但未增强无活性Raf-1的活性。我们的数据表明了一种Raf-1激活模型，其中（i）Raf-1与Ras-GTP结合，（ii）Raf-1通过酪氨酸和/或丝氨酸磷酸化被激活，并且（iii）Raf-1活性通过膜辅因子进一步增加。
