Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein β subunits and the yeast response regulator protein Skn7
Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein β subunits and the yeast response regulator protein Skn7
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DOI：
10.1074/jbc.273.15.8616
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发表时间：
1998-04-10
影响因子：
4.8
通讯作者：
Treisman, R
中科院分区：
文献类型：
--
作者：
Alberts, AS;Bouquin, N;Treisman, R
为了鉴定潜在的RhoA效应蛋白，我们对编码与Gal 4-RhoA.V14融合蛋白相互作用的蛋白质的cDNA进行了双杂交筛选。除了RhoA效应器ROCK-I，我们还鉴定了编码Kinectin、mDia 2（一种p140 mDia相关蛋白）和鸟嘌呤核苷酸交换因子mNET 1的cDNA。ROCK-I、Kinectin和mDia 2可以在体外以GTP依赖性方式结合RhoA和Cdc 42的野生型形式。ROCK-I和Kinectin序列的比较揭示了一个短的序列同源性区域，该区域是双杂交测定中相互作用所需的，并且足以在体外进行弱相互作用。与ROCK-I/Kinectin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Skn 7蛋白中。我们表明，β 2和Skn 7可以相互作用与哺乳动物RhoA和Cdc 42和酵母Rho 1，在体内和体外。酵母中的功能测定表明Skn 7 ROCK-I/Kinectin同源区是其体内功能所必需的。
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 beta 2 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.