Knaus U G, Wang Y, Reilly A M, Warnock D, Jackson J H
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
J Biol Chem. 1998 Aug 21;273(34):21512-8. doi: 10.1074/jbc.273.34.21512.
The Rho family GTPases, Rac1 and Rac2, regulate a variety of cellular functions including cytoskeletal reorganization, the generation of reactive oxygen species, G1 cell cycle progression and, in concert with Ras, oncogenic transformation. Among the many putative protein targets identified for Rac (and/or Cdc42), the Ser/Thr kinase p21-activated kinase (PAK) is a prime candidate for mediating some of Rac's cellular effects. This report shows that Rac1 binds to and stimulates the kinase activity of PAK1 approximately 2- and 4-5-fold, respectively, better than Rac2. Mutational analysis was employed to determine the structural elements on Rac and PAK that are important for optimal binding and activation. The most notable difference between the highly homologous Rac isomers is the composition of their C-terminal polybasic domains. Mutation of these six basic residues in Rac1 to neutral amino acids dramatically decreased the ability of Rac1 to bind PAK1 and almost completely abolished its ability to stimulate PAK activity. Moreover, replacing the highly charged polybasic domain of Rac1 with the less charged domain of Rac2 (and vice versa) completely reversed the PAK binding/activation properties of the two Rac isomers. Thus, polybasic domain differences account for the disparate abilities of Rac1 and Rac2 to activate PAK. PAK proteins also contain a basic region, consisting of three contiguous lysine residues (Lys66-Lys67-Lys68), which lies outside of the previously identified Cdc42/Rac-binding domain. Mutation of these Lys residues to neutral residues decreased PAK binding to activated Rac1 and Rac2 (but not Cdc42) and greatly reduced PAK1 activation by Rac1, Rac2, and Cdc42 proteins in vivo. In contrast, mutation of lysines 66-68 to basic Arg residues did not decrease (and in some cases enhanced) the ability of Rac1, Rac2, and Cdc42 to bind and activate PAK1. Our studies suggest that the polybasic domain of Rac is a novel effector domain that may allow the two Rac isomers to activate different effector proteins. In addition, our results indicate that a basic region in PAK is required for PAK activation and that binding of Rac/Cdc42 to PAK is not sufficient for kinase activation.
Rho家族GTP酶Rac1和Rac2可调节多种细胞功能，包括细胞骨架重组、活性氧的产生、G1期细胞周期进程，并且与Ras协同作用促进致癌转化。在已确定的众多Rac（和/或Cdc42）假定蛋白靶点中，丝氨酸/苏氨酸激酶p21活化激酶（PAK）是介导Rac某些细胞效应的主要候选者。本报告表明，Rac1与PAK1结合并刺激其激酶活性，分别比Rac2高约2倍和4 - 5倍。采用突变分析来确定Rac和PAK上对最佳结合和激活至关重要的结构元件。高度同源的Rac异构体之间最显著的差异在于其C末端多碱性结构域的组成。将Rac1中的这六个碱性残基突变为中性氨基酸，显著降低了Rac1与PAK1结合的能力，并几乎完全消除了其刺激PAK活性的能力。此外，用Rac2电荷较少的结构域替换Rac1电荷较多的多碱性结构域（反之亦然），完全逆转了两种Rac异构体的PAK结合/激活特性。因此，多碱性结构域的差异解释了Rac1和Rac2激活PAK能力的不同。PAK蛋白还包含一个碱性区域，由三个连续的赖氨酸残基（Lys66 - Lys67 - Lys68）组成，位于先前确定 的Cdc42/Rac结合结构域之外。将这些赖氨酸残基突变为中性残基会降低PAK与活化的Rac1和Rac2（但不包括Cdc42）的结合，并在体内极大地降低Rac1、Rac2和Cdc42蛋白对PAK1的激活作用。相反，将赖氨酸66 - 68突变为碱性的精氨酸残基不会降低（在某些情况下还会增强）Rac1、Rac2和Cdc42结合并激活PAK1的能力。我们的研究表明，Rac的多碱性结构域是一个新的效应结构域，可能使两种Rac异构体激活不同的效应蛋白。此外，我们的结果表明，PAK中的一个碱性区域是PAK激活所必需的，并且Rac/Cdc42与PAK的结合不足以激活激酶。
