Russo C, Gao Y, Mancini P, Vanni C, Porotto M, Falasca M, Torrisi M R, Zheng Y, Eva A
Laboratorio di Biologia Molecolare, Istituto G. Gaslini, Largo G. Gaslini 5, 16147 Genova, Italy.
J Biol Chem. 2001 Jun 1;276(22):19524-31. doi: 10.1074/jbc.M009742200. Epub 2001 Feb 26.
The Dbl family guanine nucleotide exchange factors (GEFs) contain a region of sequence similarity consisting of a catalytic Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. PH domains are involved in the regulated targeting of signaling molecules to plasma membranes by protein-protein and/or protein-lipid interactions. Here we show that Dbl PH domain binding to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate results in the inhibition of Dbl GEF activity on Rho family GTPase Cdc42. Phosphatidylinositol 4,5-bisphosphate binding to the PH domain significantly inhibits the Cdc42 interactive activity of the DH domain suggesting that the DH domain is subjected to the PH domain modulation under the influence of phosphoinositides (PIPs). We generated Dbl mutants unable to interact with PIPs. These mutants retained GEF activity on Cdc42 in the presence of PIPs and showed a markedly enhanced activating potential for both Cdc42 and RhoA in vivo while displaying decreased cellular transforming activity. Immunofluorescence analysis of NIH3T3 transfectants revealed that whereas the PH domain localizes to actin stress fibers and plasma membrane, the PH mutants are no longer detectable on the plasma membrane. These results suggest that modulation of PIPs in both the GEF catalytic activity and the targeting to plasma membrane determines the outcome of the biologic activity of Dbl.
Dbl家族鸟嘌呤核苷酸交换因子（GEFs）包含一个序列相似区域，该区域由一个催化性的Dbl同源（DH）结构域与一个普列克底物蛋白同源（PH）结构域串联组成。PH结构域通过蛋白质-蛋白质和/或蛋白质-脂质相互作用参与信号分子向质膜的靶向调控。在此我们表明，Dbl的PH结构域与磷脂酰肌醇4,5-二磷酸和磷脂酰肌醇3,4,5-三磷酸结合会抑制Dbl对Rho家族小GTP酶Cdc42的GEF活性。磷脂酰肌醇4,5-二磷酸与PH结构域结合显著抑制DH结构域与Cdc42的相互作用活性，这表明在磷酸肌醇（PIPs）的影响下，DH结构域受到PH结构域的调节。我们构建了无法与PIPs相互作用的Dbl突变体。这些突变体在存在PIPs的情况下保留了对Cdc42的GEF活性，并且在体内对Cdc42和RhoA均显示出明显增强的激活潜力，同时细胞转化活性降低。对NIH3T3转染细胞的免疫荧光分析显示，虽然PH结构域定位于肌动蛋白应力纤维和质膜，但PH突变体在质膜上不再可检测到。这些结果表明，PIPs对GEF催化活性和质膜靶向的调节决定了Dbl生物学活性的结果。
