Feldmann P, Eicher E N, Leevers S J, Hafen E, Hughes D A
Cancer Research Campaign Center for Cell and Molecular Biology, The Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB, United Kingdom.
Mol Cell Biol. 1999 Mar;19(3):1928-37. doi: 10.1128/MCB.19.3.1928.
Mammalian Ras GTPase-activating protein (GAP), p120 Ras-GAP, has been implicated as both a downregulator and effector of Ras proteins, but its precise role in Ras-mediated signal transduction pathways is unclear. To begin a genetic analysis of the role of p120 Ras-GAP we identified a homolog from the fruit fly Drosophila melanogaster through its ability to complement the sterility of a Schizosaccharomyces pombe (fission yeast) gap1 mutant strain. Like its mammalian homolog, Drosophila RasGAP stimulated the intrinsic GTPase activity of normal mammalian H-Ras but not that of the oncogenic Val12 mutant. RasGAP was tyrosine phosphorylated in embryos and its Src homology 2 (SH2) domains could bind in vitro to a small number of tyrosine-phosphorylated proteins expressed at various developmental stages. Ectopic expression of RasGAP in the wing imaginal disc reduced the size of the adult wing by up to 45% and suppressed ectopic wing vein formation caused by expression of activated forms of Breathless and Heartless, two Drosophila receptor tyrosine kinases of the fibroblast growth factor receptor family. The in vivo effects of RasGAP overexpression required intact SH2 domains, indicating that intracellular localization of RasGAP through SH2-phosphotyrosine interactions is important for its activity. These results show that RasGAP can function as an inhibitor of signaling pathways mediated by Ras and receptor tyrosine kinases in vivo. Genetic interactions, however, suggested a Ras-independent role for RasGAP in the regulation of growth. The system described here should enable genetic screens to be performed to identify regulators and effectors of p120 Ras-GAP.
哺乳动物Ras GTP酶激活蛋白（GAP），即p120 Ras - GAP，被认为既是Ras蛋白的下调因子又是效应器，但其在Ras介导的信号转导途径中的精确作用尚不清楚。为了开始对p120 Ras - GAP的作用进行遗传分析，我们通过其能够互补粟酒裂殖酵母（裂殖酵母）gap1突变菌株的不育性，从果蝇黑腹果蝇中鉴定出了一个同源物。与它的哺乳动物同源物一样，果蝇RasGAP刺激正常哺乳动物H - Ras的内在GTP酶活性，但不刺激致癌性Val12突变体的活性。RasGAP在胚胎中被酪氨酸磷酸化，其Src同源2（SH2）结构域在体外可与在不同发育阶段表达的少量酪氨酸磷酸化蛋白结合。RasGAP在翅成虫盘的异位表达使成虫翅的大小减少了多达45%，并抑制了由成纤维细胞生长因子受体家族的两种果蝇受体酪氨酸激酶—— Breathless和Heartless的活化形式表达所引起的异位翅脉形成。RasGAP过表达的体内效应需要完整的SH2结构域，这表明通过SH2 - 磷酸酪氨酸相互作用实现的RasGAP细胞内定位对其活性很重要。这些结果表明，RasGAP在体内可作为Ras和受体酪氨酸激酶介导的信号通路的抑制剂。然而，遗传相互作用表明RasGAP在生长调节中具有不依赖Ras的作用。这里描述的系统应该能够进行遗传筛选，以鉴定p120 Ras - GAP的调节因子和效应器。
