Li R, Zheng Y
Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163, USA.
J Biol Chem. 1997 Feb 21;272(8):4671-9. doi: 10.1074/jbc.272.8.4671.
The Dbl-like guanine nucleotide exchange factor (GEF) Lbc oncoprotein specifically activates the small GTP-binding protein Rho in mammalian fibroblasts to induce transformation and actin stress fiber formation, whereas another Dbl-related molecule, Cdc24, stimulates guanine nucleotide exchange of the Rho family GTPase Cdc42 to elicit effects on both gene induction and actin-based cytoskeleton change in Saccharomyces cerevisiae. To understand the mechanism of these functional interactions, we have taken a biochemical approach to probe the sites on Rho and Cdc42 that are involved in coupling to their respective GEFs, the Lbc and Cdc24 proteins. Point mutations in the switch II region of the small G-proteins, many of which would affect the interaction with GEF in the case of Ras, or a mutation in the switch I region that was identified as a contact site between Rab3A and Rab GEF had little effect on RhoA or Cdc42Hs with regard to the ability to interact with Lbc or Cdc24, suggesting that there exists a unique mechanism of regulation of the Rho family proteins by their GEFs. Analysis of a panel of chimeras made between RhoA and Cdc42Hs, which all maintained the ability to respond to Dbl, their mutual GEF, and to GTPase-activating protein, revealed that at least two distinct sites in each of the GTPases are required for activation by the respective GEFs. Further site-directed mutagenesis studies showed that the conserved residue Tyr32 in the putative effector region of both GTPases (numbered by Cdc42Hs) is critical for binding of the GEFs and that specific recognition for Lbc or Cdc24 is achieved at least in part through residues Lys27 of Rho and Gln116 of Cdc42. Moreover, the loss of GEF responsiveness of a RhoA mutation (D76Q) was found to be caused by the impaired GEF catalysis, not by a change in the GEF binding affinity. Together, these results indicate that multiple sites of the Rho GTPases are involved in the regulation by GEFs, contributing to GEF binding or GEF catalysis, and raise the possibility that activation of each Rho family G-protein by a specific GEF may engage in a distinct mechanism.
Dbl样鸟嘌呤核苷酸交换因子（GEF）Lbc癌蛋白在哺乳动物成纤维细胞中特异性激活小GTP结合蛋白Rho，以诱导细胞转化和肌动蛋白应力纤维形成，而另一种与Dbl相关的分子Cdc24则刺激Rho家族GTP酶Cdc42的鸟嘌呤核苷酸交换，从而对酿酒酵母中的基因诱导和基于肌动蛋白的细胞骨架变化产生影响。为了理解这些功能相互作用的机制，我们采用了一种生化方法来探究Rho和Cdc42上与它们各自的GEF（即Lbc和Cdc24蛋白）偶联相关的位点。小G蛋白开关II区域的点突变，其中许多在Ras的情况下会影响与GEF的相互作用，或者开关I区域中被确定为Rab3A与Rab GEF之间接触位点的突变，对RhoA或Cdc42Hs与Lbc或Cdc24相互作用的能力几乎没有影响，这表明Rho家族蛋白由其GEF调控存在独特的机制。对一组在RhoA和Cdc42Hs之间构建的嵌合体进行分析，这些嵌合体都保持了对它们共同的GEF Dbl以及GTP酶激活蛋白作出反应的能力，结果显示每个GTP酶中至少有两个不同的位点是各自GEF激活所必需的。进一步的定点诱变研究表明，两种GTP酶（以Cdc42Hs编号）假定效应器区域中的保守残基Tyr32对于GEF的结合至关重要，并且对Lbc或Cdc24的特异性识别至少部分是通过Rho的Lys27残基和Cdc42的Gln116残基实现的。此外，发现RhoA突变体（D76Q）丧失GEF反应性是由GEF催化受损导致的，而非GEF结合亲和力的改变。总之，这些结果表明Rho GTP酶的多个位点参与了GEF的调控，对GEF结合或GEF催化有贡献，并增加了一种可能性，即特定GEF对每个Rho家族G蛋白的激活可能涉及不同的机制。
