REQUIREMENT FOR RAS IN RAF ACTIVATION IS OVERCOME BY TARGETING RAF TO THE PLASMA-MEMBRANE
REQUIREMENT FOR RAS IN RAF ACTIVATION IS OVERCOME BY TARGETING RAF TO THE PLASMA-MEMBRANE
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DOI：
10.1038/369411a0
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发表时间：
1994-06-02
期刊：
影响因子：
64.8
通讯作者：
MARSHALL, CJ
中科院分区：
文献类型：
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作者：
LEEVERS, SJ;PATERSON, HF;MARSHALL, CJ
最近已鉴定出一条保守的酪氨酸激酶激活信号转导通路，其中包含质膜结合的小鸟嘌呤核苷酸结合蛋白 Ras 和蛋白激酶 Raf、MAP 激酶激酶和 MAP 激酶(1,2)。 GTP 结合的 Ras 直接与 Raf 的氨基末端调节结构域相互作用 (3-8)，但尽管 Ras 和 Raf 可以从配体刺激的细胞中免疫共沉淀 (9,10)，但 Ras-GTP 不会在体外刺激 Raf 的激酶活性 (6)。此外，我们未能在活性去污剂溶解的 Raf 制剂中检测到 Ras，这表明一旦被激活，Raf 就不再需要 Ras。 Raf 通常位于细胞质中，而在表达活性 Ras 的细胞中，Raf 与质膜相关。这促使我们研究 Ras 是否需要将 Raf 定位到质膜才能激活 Raf。我们将 K-Ras(4B) 的膜定位信号融合到 Raf 的羧基末端。该蛋白具有组成型活性，可被表皮生长因子进一步激活，与 Ras 无关。我们的结果表明 Ras 充当 Raf 的受调节膜结合锚，并且其他信号也有助于 Raf 激活。
A CONSERVED tyrosine kinase-activated signal transduction pathway has recently been identified that comprises the plasma membrane-bound small guanine-nucleotide-binding protein Ras and the protein kinases Raf, MAP-kinase kinase and MAP kinase(1,2). GTP-bound Ras interacts directly with the amino-terminal regulatory domain of Raf(3-8), but although Ras and Raf can be coimmunoprecipitated from ligand-stimulated cells(9,10), Ras-GTP does not stimulate the kinase activity of Raf in vitro(6). Furthermore, we have failed to detect Ras in preparations of active detergent-solubilized Raf, demonstrating that once it is activated, Raf does not require Ras. Whereas Raf is normally cytosolic, in cells expressing active Ras, Raf is associated with the plasma membrane. This led us to investigate whether Ras is required to localize Raf to the plasma membrane in order for Raf to become activated. We fused the membrane localization signal of K-Ras(4B) to the carboxy terminus of Raf. This protein is constitutively active and can be further activated by epidermal growth factor, independently of Ras. Our results indicate that Ras functions as a regulated, membrane-bound anchor for Raf, and that other signal(s) also contribute to Raf activation.