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首页> 外文期刊>Molecular and Cellular Biology >Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro.
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Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro.

机译：Raf-1和Raf-1突变体的体外Ras依赖性和Ras非依赖性机制的调控。

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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.
机译：丝氨酸/苏氨酸激酶Raf-1在Ras下游起作用以激活丝裂原活化的蛋白激酶激酶，但Raf-1激活的机制尚未完全了解。为了剖析这些机制，在体外系统中使用来自v-Ras-和v-Src转化细胞（转化膜）的纯化质膜研究了野生型和突变型Raf-1蛋白。野生型（His）6-和FLAG-Raf-1通过转化膜以Ras和ATP依赖性方式被激活。但是，Raf-1蛋白是激酶缺陷的（K375M），缺乏体内的调节酪氨酸（YY340 / 341FF）或组成型丝氨酸（S621A）磷酸化的位点，不与Ras（R89L）结合，或者缺少完整的锌指（CC165 / 168SS）的人没有。缺乏一个未知的激酶（S259A）或蛋白激酶C（S499A）的假定调控位点的Raf-1蛋白被激活，但效率明显降低。负责通过Ras或Src激活的激酶可能驻留在质膜中，因为来自静止的NIH 3T3细胞（亲代膜）的质膜的GTP负载诱导了从头激活Raf-1的能力。在没有GTP负载的情况下，仅具有基础活性的野生型Raf-1不会被亲代膜激活。相反，具有显着活性的Raf-1 Y340D令人惊讶地被亲本膜以与Ras无关的方式刺激。结果表明通过磷酸化激活Raf-1可能被另一种膜因子（例如脂质）进一步调节。用甲醇-氯仿从转化膜或共表达Ras和SrcY527F的Sf9细胞膜中提取的因子显着增强Raf-1 Y340D或活性Raf-1的活性，但不增强非活性Raf-1的活性。我们的发现提出了激活Raf-1的模型，其中（i）Raf-1与Ras-GTP缔合，（ii）Raf-1被酪氨酸和/或丝氨酸磷酸化激活，并且（iii）Raf-1活性为膜辅助因子进一步增加。

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