Alberts A S, Bouquin N, Johnston L H, Treisman R
Transcription Laboratory, Imperial Cancer Research Fund Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
J Biol Chem. 1998 Apr 10;273(15):8616-22. doi: 10.1074/jbc.273.15.8616.
To identify potential RhoA effector proteins, we conducted a two-hybrid screen for cDNAs encoding proteins that interact with a Gal4-RhoA.V14 fusion protein. In addition to the RhoA effector ROCK-I we identified cDNAs encoding Kinectin, mDia2 (a p140 mDia-related protein), and the guanine nucleotide exchange factor, mNET1. ROCK-I, Kinectin, and mDia2 can bind the wild type forms of both RhoA and Cdc42 in a GTP-dependent manner in vitro. Comparison of the ROCK-I and Kinectin sequences revealed a short region of sequence homology that is both required for interaction in the two-hybrid assay and sufficient for weak interaction in vitro. Sequences related to the ROCK-I/Kinectin sequence homology are present in heterotrimeric G protein beta subunits and in the Saccharomyces cerevisiae Skn7 protein. We show that beta2 and Skn7 can interact with mammalian RhoA and Cdc42 and yeast Rho1, both in vivo and in vitro. Functional assays in yeast suggest that the Skn7 ROCK-I/Kinectin homology region is required for its function in vivo.
为了鉴定潜在的RhoA效应蛋白，我们进行了一项双杂交筛选，以寻找编码与Gal4-RhoA.V14融合蛋白相互作用的蛋白的cDNA。除了RhoA效应蛋白ROCK-I外，我们还鉴定出了编码驱动蛋白（Kinectin）、mDia2（一种与p140 mDia相关的蛋白）以及鸟嘌呤核苷酸交换因子mNET1的cDNA。ROCK-I、驱动蛋白和mDia2在体外能够以GTP依赖的方式结合RhoA和Cdc42的野生型形式。对ROCK-I和驱动蛋白序列的比较揭示了一个短的序列同源区域，该区域在双杂交试验中是相互作用所必需的，并且在体外足以产生弱相互作用。与ROCK-I/驱动蛋白序列同源性相关的序列存在于异源三聚体G蛋白β亚基和酿酒酵母Skn7蛋白中。我们发现，β2和Skn7在体内和体外都能与哺乳动物的RhoA和Cdc42以及酵母的Rho1相互作用。酵母中的功能试验表明，Skn7的ROCK-I/驱动蛋白同源区域在其体内功能中是必需的。
