Zheng Y, Bagrodia S, Cerione R A
Department of Pharmacology, Cornell University, Ithaca, New York 14853.
J Biol Chem. 1994 Jul 22;269(29):18727-30.
The Ras-like GTPase Cdc42 is essential for cell polarity and bud site assembly in Saccharomyces cerevisiae by regulating cell cycle-dependent reorganization of cortical cytoskeletal elements. However, its role in mammalian cells is unknown. To identify potential effectors of Cdc42Hs, we incubated lysates from NIH 3T3 fibroblasts or PC12 cells with immobilized glutathione S-transferase (GST)-Cdc42Hs fusion proteins bound to different guanine nucleotides and observed a specific association between the 85-kDa subunit (p85) of phosphatidylinositol 3-kinase (PI 3-kinase) and GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-bound GST-Cdc42Hs. Recombinant p85 formed a complex with GTP gamma S-bound GST-Cdc42Hs and with a GTPase-defective GTP-bound GST-Cdc42Hs-Q61L mutant, but not with a GTP gamma S-bound, effector domain GST-Cdc42HsT35A mutant. Both the Rho-GAP homology domain of p85 and the Cdc42Hs-GAP competitively inhibited the binding of recombinant p85 to Cdc42Hs. In addition, PI 3-kinase activity immunoprecipitated from cell lysates with anti-p85 antibody was stimulated 2-4-fold by GST-Cdc42-GTP gamma S. Similar interactions were observed between p85 and GST-Rac1-GTP gamma S but not between p85 and GST-RhoA-GTP gamma S. These findings suggest that PI 3-kinase, through the Rho-GAP homology domain of p85, can couple to the effector domain of Cdc42Hs and that p85 may be a target for the GTP-bound forms of Cdc42Hs and Rac1.
类Ras GTP酶Cdc42通过调节皮质细胞骨架元件的细胞周期依赖性重组，对酿酒酵母中的细胞极性和芽位点组装至关重要。然而，其在哺乳动物细胞中的作用尚不清楚。为了鉴定Cdc42Hs的潜在效应器，我们将来自NIH 3T3成纤维细胞或PC12细胞的裂解物与结合了不同鸟嘌呤核苷酸的固定化谷胱甘肽S-转移酶（GST）-Cdc42Hs融合蛋白一起孵育，并观察到磷脂酰肌醇3-激酶（PI 3-激酶）的85-kDa亚基（p85）与GTPγS（鸟苷5'-3-O-（硫代）三磷酸）结合的GST-Cdc42Hs之间存在特异性结合。重组p85与GTPγS结合的GST-Cdc42Hs以及与GTP酶缺陷型GTP结合的GST-Cdc42Hs-Q61L突变体形成复合物，但不与GTPγS结合的效应器结构域GST-Cdc42HsT35A突变体形成复合物。p85的Rho-GAP同源结构域和Cdc42Hs-GAP均竞争性抑制重组p85与CdcHs42的结合。此外，用抗p85抗体从细胞裂解物中免疫沉淀的PI 3-激酶活性被GST-Cdc42-GTPγS刺激2至4倍。在p85与GST-Rac1-GTPγS之间观察到类似的相互作用，但在p85与GST-RhoA-GTPγS之间未观察到。这些发现表明，PI 3-激酶通过p85的Rho-GAP同源结构域，可以与Cdc42Hs的效应器结构域偶联，并且p8可能是Cdc42Hs和Rac1的GTP结合形式的靶标。
