Multifunctional roles for the PH domain of Dbs in regulating rho GTPase activation
Multifunctional roles for the PH domain of Dbs in regulating rho GTPase activation
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DOI：
10.1074/jbc.m300127200
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发表时间：
2003-05-16
影响因子：
4.8
通讯作者：
Sondek, J
中科院分区：
文献类型：
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作者：
Rossman, KL;Cheng, L;Sondek, J
Dbl家族成员是Rho鸟苷三磷酸酶（GTP酶）特异性的鸟嘌呤核苷酸交换因子，并且总是具有串联Dbl（DH）和普列克底物蛋白同源（PH）结构域。Dbs是Cdc42和RhoA特异性的Dbl家族成员，当在NIH 3T3小鼠成纤维细胞中过表达时表现出转化活性。在这项研究中，DB的PH结构域突变，选择性地损害鸟嘌呤核苷酸交换或磷酸肌醇结合在体外和由此产生的生理变化进行了评估。正如预期的那样，取代与GTP酶的界面整合的Db的PH结构域内的残基减少了核苷酸交换，并消除了Db转化NIH 3T3细胞的能力。更有趣的是，PH结构域内的取代阻止与磷酸肌醇的相互作用，但不改变GTP酶的体外活化，也不转化NIH 3T3细胞，并且尽管适当的亚细胞定位，也不能在体内活化RhoA。因此，Db的PH结构域在GTP酶的激活中起多种作用，不能被视为简单的膜锚定装置。特别是，这些数据表明，结合的磷酸肌醇的PH结构域内的膜表面的情况下，可以直接连接的DH和PH结构域的取向或构象，以调节GTP酶的激活。
Dbl family members are guanine nucleotide exchange factors specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. Dbs, a Dbl family member specific for Cdc42 and RhoA, exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. In this study, the PH domain of Dbs was mutated to impair selectively either guanine nucleotide exchange or phosphoinositide binding in vitro and resulting physiological alterations were assessed. As anticipated, substitution of residues within the PH domain of Dbs integral to the interface with GTPases reduced nucleotide exchange and eliminated the ability of Dbs to transform NIH 3T3 cells. More interestingly, substitutions within the PH domain that prevent interaction with phosphoinositides yet do not alter in vitro activation of GTPases also do not transform NIH 3T3 cell and fail to activate RhoA in vivo despite proper subcellular localization. Therefore, the PH domain of Dbs serves multiple roles in the activation of GTPases and cannot be viewed as a simple membrane-anchoring device. In particular, the data suggest that binding of phosphoinositides to the PH domain within the context of membrane surfaces may direct orientations or conformations of the linked DH and PH domains to regulate GTPases activation.