Jacques Kerry M, Nie Zhongzhen, Stauffer Stacey, Hirsch Dianne S, Chen Ling-Xin, Stanley Katherine T, Randazzo Paul A
Laboratory of Cellular Oncology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2002 Dec 6;277(49):47235-41. doi: 10.1074/jbc.M208875200. Epub 2002 Oct 9.
The effectors of monomeric GTP-binding proteins can influence interactions with GTPase-activating proteins (GAPs) in two ways. In one case, effector and GAP binding to the GTP-binding protein is mutually exclusive. In another case, the GTP-binding protein bound to an effector is the substrate for the GTPase-activating protein. Here predictions for these two mechanisms were tested for the Arf1 effector GGA and ASAP family Arf GAPs. GGA inhibited Arf GAP activity of ASAP1, AGAP1, ARAP1, and Arf GAP1 and inhibited binding of Arf1.GTPgammaS to AGAP1 with K(i) values correlating with the K(d) for the GGA.Arf1 complex. ASAP1 blocked Arf1.GTPgammaS binding to GGA with a K(i) similar to the K(d) for the ASAP.Arf1.GTPgammaS complex. No interaction of GGA with ASAP1 was detected. Consistent with GGA sequestering Arf from GAPs, overexpression of GGA slowed the rate of Arf dissociation from the Golgi apparatus following treatment with brefeldin A. Mutational analysis revealed the amino-terminal alpha-helix and switch I of Arf1 contributed to interaction with both GGA and GAPs. These data exclude the mechanism previously documented for Arf GAP1/coatomer in which Arf1 is inactivated in a tripartite complex. Instead, termination of Arf1 signals mediated through GGA require that Arf1.GTP dissociates from GGA prior to interaction with GAP and consequent hydrolysis of GTP.
单体GTP结合蛋白的效应器可通过两种方式影响与GTP酶激活蛋白（GAP）的相互作用。在一种情况下，效应器与GAP对GTP结合蛋白的结合是相互排斥的。在另一种情况下，与效应器结合的GTP结合蛋白是GTP酶激活蛋白的底物。在此，针对Arf1效应器GGA和ASAP家族Arf GAP对这两种机制的预测进行了测试。GGA抑制了ASAP1、AGAP1、ARAP1和Arf GAP1的Arf GAP活性，并抑制了Arf1.GTPγS与AGAP1的结合，其抑制常数（K(i)）值与GGA.Arf1复合物的解离常数（K(d)）相关。ASAP1以与ASAP.Arf1.GTPγS复合物的K(d)相似的K(i)阻断Arf1.GTPγS与GGA的结合。未检测到GGA与ASAP1之间的相互作用。与GGA将Arf从GAP中隔离一致，GGA的过表达减缓了用布雷菲德菌素A处理后Arf从高尔基体解离的速率。突变分析表明，Arf1的氨基末端α螺旋和开关I有助于与GGA和GAP的相互作用。这些数据排除了先前记录的Arf GAP1/包被蛋白的机制，在该机制中Arf1在三方复合物中失活。相反，通过GGA介导的Arf1信号的终止要求Arf1.GTP在与GAP相互作用并随后水解GTP之前从GGA解离。
