Kuroda S, Fukata M, Kobayashi K, Nakafuku M, Nomura N, Iwamatsu A, Kaibuchi K
Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-01, Japan.
J Biol Chem. 1996 Sep 20;271(38):23363-7. doi: 10.1074/jbc.271.38.23363.
Cdc42 and Rac1 have been implicated in the regulation of various cell functions such as cell morphology, polarity, and cell proliferation. We have partially purified a Cdc42- and Rac1-associated protein with molecular mass of about 170 kDa (p170) from bovine brain cytosol. This protein interacted with guanosine 5'-(3-O-thio)triphosphate (GTPgammaS).glutathione S-transferase (GST)-Cdc42 and GTPgammaS++.GST-Rac1 but not with the GDP.GST-Cdc42, GDP.GST-Rac1, or GTPgammaS.GST-RhoA). We identified p170 as an IQGAP, which is originally identified as a putative Ras GTPase-activating protein. Recombinant IQGAP specifically interacted with GTPgammaS.Cdc42 and GTPgammaS.Rac1. The C-terminal fragment of IQGAP was responsible for their interactions. IQGAP was specifically immunoprecipitated with dominant-active Cdc42(Val12) or Rac1(Val12) from the COS7 cells expressing Cdc42(Val12) or Rac1(Val12), respectively. Immunofluorescence analysis revealed that IQGAP was accumulated at insulin- or Rac1-induced membrane ruffling areas. This accumulation of IQGAP was blocked by the microinjection of the dominant-negative Rac1(Asn17) or Cdc42(Asn17). Moreover, IQGAP was accumulated at the cell-cell junction in MDCK cells, where alpha-catenin and ZO-1 were localized. These results suggest that IQGAP is a novel target molecule for Cdc42 and Rac1.
Cdc42和Rac1参与调控多种细胞功能，如细胞形态、极性和细胞增殖。我们已从牛脑细胞质中部分纯化出一种分子量约为170 kDa（p170）的与Cdc42和Rac1相关的蛋白。该蛋白与鸟苷5'-(3-O-硫代)三磷酸（GTPγS）、谷胱甘肽S-转移酶（GST）-Cdc42以及GTPγS-GST-Rac1相互作用，但不与GDP-GST-Cdc42、GDP-GST-Rac1或GTPγS-GST-RhoA相互作用。我们鉴定p170为一种IQGAP，它最初被鉴定为一种假定的Ras GTP酶激活蛋白。重组IQGAP特异性地与GTPγS-Cdc42和GTPγS-Rac1相互作用。IQGAP的C末端片段负责它们之间的相互作用。IQGAP分别从表达Cdc42(Val12)或Rac1(Val12)的COS7细胞中被显性激活的Cdc42(Val12)或Rac1(Val12)特异性免疫沉淀。免疫荧光分析显示，IQGAP聚集在胰岛素或Rac1诱导的膜皱襞区域。IQGAP的这种聚集被显性负性的Rac1(Asn17)或Cdc42(Asn17)显微注射所阻断。此外，IQGAP聚集在MDCK细胞的细胞-细胞连接处，α-连环蛋白和ZO-1也定位于此处。这些结果表明，IQGAP是Cdc42和Rac1的一种新的靶分子。
