Leevers S J, Paterson H F, Marshall C J
Section of Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, London, UK.
Nature. 1994 Jun 2;369(6479):411-4. doi: 10.1038/369411a0.
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. GTP-bound Ras interacts directly with the amino-terminal regulatory domain of Raf, but although Ras and Raf can be coimmunoprecipitated from ligand-stimulated cells, Ras-GTP does not stimulate the kinase activity of Raf in vitro. 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.
最近发现了一条保守的酪氨酸激酶激活的信号转导途径，它由质膜结合的小GTP核苷酸结合蛋白Ras以及蛋白激酶Raf、丝裂原活化蛋白激酶激酶（MAP-kinase kinase）和丝裂原活化蛋白激酶（MAP kinase）组成。结合GTP的Ras直接与Raf的氨基末端调节结构域相互作用，但是尽管Ras和Raf可以从配体刺激的细胞中共免疫沉淀，但Ras-GTP在体外并不刺激Raf的激酶活性。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Raf被激活，它就不再需要Ras。虽然Raf通常位于胞质溶胶中，但在表达活性Ras的细胞中，Raf与质膜相关联。这使我们研究Ras是否是Raf定位到质膜从而被激活所必需的。我们将K-Ras(4B)的膜定位信号融合到Raf的羧基末端。这种蛋白具有组成型活性，并且可以独立于Ras被表皮生长因子进一步激活。我们的结果表明，Ras作为Raf的一种受调控的膜结合锚定物发挥作用，并且其他信号也有助于Raf的激活。
