Synergistic activation of dynamin GTPase by Grb2 and phosphoinositides
Synergistic activation of dynamin GTPase by Grb2 and phosphoinositides
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DOI：
10.1074/jbc.273.6.3791
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发表时间：
1998-02-06
影响因子：
4.8
通讯作者：
Albanesi, JP
中科院分区：
文献类型：
--
作者：
Barylko, B;Binns, D;Albanesi, JP
GTP被发动蛋白水解对于从质膜出芽网格蛋白包被的囊泡是必不可少的。动力蛋白的两个不同结构域与动力蛋白GT3激活剂的相互作用有关。微管和Grb 2结合到羧基末端脯氨酸/脯氨酸富集结构域（PRD），而磷酸肌醇结合到普列克底物蛋白同源（PH）结构域。在这项研究中，我们测试了不同的磷酸肌醇对动力蛋白GT3活性的影响，发现最好的激活剂是磷脂酰肌醇4，5-二磷酸，其次是1-O-（1，2-二-O-棕榈酰-sn-甘油-3-苄氧基磷酰基）-D-肌肌醇3，4，5-三磷酸。磷脂酰肌醇4-磷酸是一种弱激活剂，磷脂酰肌醇3，4-二磷酸根本不激活GT3。然后，我们解决了发动蛋白，PRD和PH，这两个域是否可以同时进行的问题，并确定双重占用发动蛋白GTdR活性的影响。我们发现，Grb 2和磷脂酰肌醇4，5-二磷酸一起增加发动蛋白GTdR活性高达4倍，高于这些激活剂单独测试获得的，也减少了发动蛋白浓度所需的半最大活动的3倍。这些结果表明，这两种刺激剂可以结合发动蛋白，同时导致发动蛋白GT3活性的超激活。我们建议，含SH 3的蛋白质，如Grb 2结合发动蛋白PRD，将其靶向网格蛋白包被的凹坑，并通过磷酸肌醇使其超活化。
Hydrolysis of GTP by dynamin is essential for budding clathrin-coated vesicles from the plasma membrane. Two distinct domains of dynamin are implicated in the interactions with dynamin GTPase activators. Microtubules and Grb2 bind to the carboxyl-terminal proline/arginine-rich domain (PRD), whereas phosphoinositides bind to the pleckstrin homology (PH) domain. In this study we tested the effect of different phosphoinositides on dynamin GTPase activity and found that the best activator is phosphatidylinositol 4,5-bisphosphate followed by 1-O-(1,2-di-O-palmitoyl-sn-glycerol-3-benzyloxyphosphoryl)-D-myo-inositol 3,4,5-triphosphate. Phosphatidylinositol 4-phosphate was a weak activator and phosphatidylinositol 3,4-bisphosphate did not activate GTPase at all. We then addressed the question of whether both domains of dynamin, PRD and PH, can be engaged simultaneously, and determined the effects of dual occupancy on dynamin GTPase activity. We found that Grb2 and phosphatidylinositol 4,5-bisphosphate together increased the dynamin GTPase activity up to 4-fold higher than that obtained by these activators tested separately, and also reduced the dynamin concentration required for half-maximal activities by 3-fold. These results indicate that both stimulators can bind to dynamin simultaneously resulting in superactivation of dynamin GTPase activity. We propose that SH3-containing proteins such as Grb2 bind to the dynamin PRD to target it to clathrin-coated pits and prime it for superactivation by phosphoinositides.