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首页> 外文期刊>Molecular and Cellular Biology >Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues.
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Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues.

机译：Ras和Raf之间的关键结合和调节相互作用通过一个小的，稳定的Raf N末端结构域和特定的Ras效应子残基发生。

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Genetic and biochemical evidence suggests that the Ras protooncogene product regulates the activation of the Raf kinase pathway, leading to the proposal that Raf is a direct mitogenic effector of activated Ras. Here we report the use of a novel competition assay to measure in vitro the relative affinity of the c-Raf-1 regulatory region for Ras-GTP, Ras-GDP, and 10 oncogenic and effector mutant Ras proteins. c-Raf-1 associates with normal Ras and the oncogenic V12 and L61 forms of Ras with equal affinity. The moderately transforming mutant Ras[E30K31] also bound to the c-Raf-1 regulatory region with normal affinity. Transformation-defective Ras effector mutants Ras[N33], Ras[S35], and Ras[N38] bound poorly. In contrast, the transformation defective Ras[G26I27] and Ras[E45] mutants bound to the c-Raf-1 regulatory region with nearly wild-type affinity. A stable, high-affinity Ras-binding region of c-Raf-1 was mapped to a 99-amino-acid subfragment of the first 257 residues. The smallest Ras-binding region identified consisted of N-terminal residues 51 to 131, although stable expression of the domain and high-affinity binding were improved by the presence of residues 132 to 149. Deletion of the Raf zinc finger region did not reduce Ras-binding affinity, while removal of the first 50 amino acids greatly increased affinity. Phosphorylation of Raf[1-149] by protein kinase A on serine 43 resulted in significant inhibiton of Ras binding. demonstrating that the mechanism of cyclic AMP downregulation results through structural changes occurring exclusively in this small Ras-binding domain.
机译：遗传和生化证据表明，Ras原癌基因产物调节Raf激酶途径的活化，从而导致有人提出Raf是活化Ras的直接促有丝分裂效应子。在这里，我们报告使用一种新颖的竞争测定法来测量c-Raf-1调节区对Ras-GTP，Ras-GDP和10种致癌和效应突变Ras蛋白的相对亲和力。 c-Raf-1与正常Ras以及致癌的V12和L61形式的Ras具有相同的亲和力。中等转化的突变体Ras [E30K31]也以正常亲和力与c-Raf-1调节区结合。转化缺陷型Ras效应突变体Ras [N33]，Ras [S35]和Ras [N38]结合不良。相反，转化缺陷型Ras [G26I27]和Ras [E45]突变体几乎以野生型亲和力与c-Raf-1调控区结合。将c-Raf-1的稳定，高亲和力的Ras结合区域定位到前257个残基的99个氨基酸亚片段上。鉴定出的最小Ras结合区由N端残基51至131组成，尽管结构域的稳定表达和高亲和力结合由于残基132至149的存在而得以改善。Raf锌指区的缺失不会降低Ras -结合亲和力，同时去除前50个氨基酸大大增加了亲和力。蛋白激酶A在丝氨酸43上使Raf [1-149]磷酸化，从而显着抑制Ras结合。证明了环状AMP下调的机制是由仅在这个小的Ras结合域中发生的结构变化导致的。

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