Vogel U S, Dixon R A, Schaber M D, Diehl R E, Marshall M S, Scolnick E M, Sigal I S, Gibbs J B
Department of Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Nature. 1988 Sep 1;335(6185):90-3. doi: 10.1038/335090a0.
The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.
相对分子质量为21,000的质膜结合哺乳动物ras蛋白（ras p21）与其他鸟嘌呤核苷酸结合调节蛋白（G蛋白）具有生化和结构特性。致癌性ras p21变体源于特定位置的氨基酸取代，导致p21在体内主要与GTP结合。最近，发现了一种具有胞质活性的GTP酶激活蛋白（GAP），它能刺激正常而非致癌性ras p21的GTP酶活性。GAP可能是在调节途径中作用于上游的负调节因子，也可能是ras p21的下游靶点。我们从牛脑中鉴定出一种表观相对分子质量为125,000的具有GAP活性的蛋白。在此，我们在动力学竞争试验中使用纯GAP表明，与无活性的GDP复合物相比，GAP优先与正常和致癌性哈维（Ha）ras p21的活性GTP复合物相互作用。我们还报告了牛GAP互补DNA的克隆和测序。GAP的区域与酿酒酵母腺苷酸环化酶的非催化结构域以及磷脂酶C - 148、crk癌基因产物和非受体酪氨酸激酶之间保守的区域具有氨基酸相似性。
