Residues of the Rho family GTPases Rho and Cdc42 that specify sensitivity to Dbl-like guanine nucleotide exchange factors
Residues of the Rho family GTPases Rho and Cdc42 that specify sensitivity to Dbl-like guanine nucleotide exchange factors
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DOI：
10.1074/jbc.272.8.4671
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发表时间：
1997-02-21
影响因子：
4.8
通讯作者：
Zheng, Y
中科院分区：
文献类型：
--
作者：
Li, R;Zheng, Y
Dbl样鸟嘌呤核苷酸交换因子（GEF）Lbc癌蛋白特异性激活哺乳动物成纤维细胞中的小GTP结合蛋白Rho以诱导转化和肌动蛋白应力纤维形成，而另一种Dbl相关分子Cdc24刺激Rho家族GTbCdc42的鸟嘌呤核苷酸交换以引起对酿酒酵母中基因诱导和基于肌动蛋白的细胞骨架变化的影响，为了理解这些功能相互作用的机制，我们已经采取了生物化学方法来探测Rho和Cdc42上参与与它们各自的GEF、Lbc和Cdc24蛋白偶联的位点。小G蛋白的开关II区域中的点突变，其中许多点突变在Pas的情况下会影响与GEF的相互作用，或在开关I区中被鉴定为Rab3A和Rab之间的接触位点的突变，GEF在与Lbc或Cdc24相互作用的能力方面对RhoA或Cdc42H几乎没有影响，这表明存在通过它们的GEF调节Rho家族蛋白的独特机制，RhoA和Cdc42H之间产生的嵌合体组的分析，它们都保持了对Db1、它们的共同GEF和GTP酶激活蛋白的应答能力，揭示了每个GTP酶中至少两个不同的位点是由各自的GEF激活所必需的，进一步的定点突变研究表明，两种GTP酶的推定效应区中的保守残基Tyr（32）（编号为Cdc42H）对于GEF的结合是关键的，并且对Lbc或Cdc24的特异性识别至少部分地通过Rho的残基Lys（27）和Cdc42的残基Gln（116）实现。此外，发现RhoA突变（D76Q）的GEF响应性的丧失是由受损的GEF催化引起的，而不是由GEF结合亲和力的变化引起的。总之，这些结果表明Rho GTP酶的多个位点参与GEF的调节，有助于GEF结合或GEF催化，并提出了通过特定GEF激活每个Rho家族G蛋白可能参与不同机制的可能性。
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 Pas, 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 Tyr(32) 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 Lys(27) of Rho and Gln(116) 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.