Che Magnus M, Boja Emily S, Yoon Hye-Young, Gruschus James, Jaffe Howard, Stauffer Stacey, Schuck Peter, Fales Henry M, Randazzo Paul A
Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, United States.
Cell Signal. 2005 Oct;17(10):1276-88. doi: 10.1016/j.cellsig.2005.01.007. Epub 2005 Feb 25.
ASAP1 is an Arf GAP with a PH domain immediately N-terminal to the catalytic Arf GAP domain. PH domains are thought to regulate enzymes by binding to specific phosphoinositide lipids in membranes, thereby recruiting the enzyme to a site of action. Here, we have examined the functional relationship between the PH and Arf GAP domains. We found that GAP activity requires the cognate PH domain of ASAP1, leading us to hypothesize that the Arf GAP and PH domains directly interact to form the substrate binding site. This hypothesis was supported by the combined results of protection and hydrodynamic studies. We then examined the role of the PH domain in the regulation of Arf GAP activity. The results of saturation kinetics, limited proteolysis, FRET and fluorescence spectrometry support a model in which regulation of the GAP activity of ASAP1 involves a conformational change coincident with recruitment to a membrane surface, and a second conformational change following the specific binding of phosphatidylinositol 4,5-bisphosphate.
ASAP1是一种具有PH结构域的Arf GAP，该PH结构域位于催化性Arf GAP结构域的紧邻N端。人们认为PH结构域通过与膜中的特定磷酸肌醇脂质结合来调节酶，从而将酶招募到作用位点。在此，我们研究了PH结构域与Arf GAP结构域之间的功能关系。我们发现GAP活性需要ASAP1的同源PH结构域，这使我们推测Arf GAP结构域和PH结构域直接相互作用以形成底物结合位点。保护和流体动力学研究的综合结果支持了这一假设。然后，我们研究了PH结构域在调节Arf GAP活性中的作用。饱和动力学、有限蛋白酶解、荧光共振能量转移和荧光光谱分析的结果支持了一个模型，其中ASAP1的GAP活性调节涉及与募集到膜表面同时发生的构象变化，以及在磷脂酰肌醇4,5-二磷酸特异性结合后的第二次构象变化。
