Fuentes Ernesto J, Karnoub Antoine E, Booden Michelle A, Der Channing J, Campbell Sharon L
Department of Biochemistry and Biophysics, University of North Carolina, Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2003 Jun 6;278(23):21188-96. doi: 10.1074/jbc.M211792200. Epub 2003 Mar 13.
Dbl family proteins act as guanine nucleotide exchange factors and positive regulators of Rho GTPase function by stimulating formation of the active, GTP-bound state. All Dbl family Rho guanine nucleotide exchange factors possess an invariant tandem domain structure consisting of a Dbl homology (DH) catalytic domain followed by a pleckstrin homology (PH) regulatory domain. We determined previously that the PH domain of Dbs was critical for the intrinsic catalytic activity of the DH domain in vitro and for Dbs transformation in vivo. In this study, we evaluated the role of phosphoinositide binding to the PH domain in regulating the DH domain function of Dbs in vitro and in vivo. We determined that mutation of basic amino acids located within the beta1-beta2 and beta3-beta4 loops of the PH domain resulted in impaired phospholipid binding in vitro, yet full guanine nucleotide exchange activity in vitro was retained for RhoA and Cdc42. Surprisingly, these mutants were compromised in their ability to activate Rho GTPases in vivo and to cause transformation of NIH 3T3 cells. However, Dbs subcellular localization was impaired by these PH domain mutations, supporting a role for phospholipid interactions in facilitating membrane association. Despite the importance of phospholipid binding for Dbs function in vivo, we found that Dbs signaling and transforming activity was not stimulated by phosphatidylinositol 3-kinase activation. We suggest that the PH domain of Dbs facilitates two distinct roles in the regulation of DH domain function, one critical for GTPase association and activation in vitro and one critical for phosphoinositide binding and GTPase interaction in vivo, that together promote Dbs association with membranes.
Dbl家族蛋白作为鸟嘌呤核苷酸交换因子，通过刺激活性GTP结合状态的形成，成为Rho GTP酶功能的正调节因子。所有Dbl家族的Rho鸟嘌呤核苷酸交换因子都具有不变的串联结构域，由一个Dbl同源（DH）催化结构域和一个普列克底物蛋白同源（PH）调节结构域组成。我们先前确定，Dbs的PH结构域对于DH结构域在体外的内在催化活性以及在体内的Dbs转化至关重要。在本研究中，我们评估了磷酸肌醇与PH结构域结合在体外和体内调节Dbs的DH结构域功能中的作用。我们确定，位于PH结构域β1-β2和β3-β4环内的碱性氨基酸突变导致体外磷脂结合受损，但RhoA和Cdc42在体外仍保留了完整的鸟嘌呤核苷酸交换活性。令人惊讶的是，这些突变体在体内激活Rho GTP酶以及导致NIH 3T3细胞转化的能力上受到损害。然而，这些PH结构域突变损害了Dbs的亚细胞定位，支持了磷脂相互作用在促进膜结合中的作用。尽管磷脂结合对Dbs在体内的功能很重要，但我们发现磷脂酰肌醇3激酶激活并不能刺激Dbs信号传导和转化活性。我们认为，Dbs的PH结构域在调节DH结构域功能中发挥了两个不同的作用：一个对体外GTP酶的结合和激活至关重要，另一个对体内磷酸肌醇结合和GTP酶相互作用至关重要，两者共同促进Dbs与膜的结合。
