Trp56 of Rac1 specifies interaction with a subset of guanine nucleotide exchange factors
Trp56 of Rac1 specifies interaction with a subset of guanine nucleotide exchange factors
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DOI：
10.1074/jbc.m108865200
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发表时间：
2001-12-14
影响因子：
4.8
通讯作者：
Zheng, Y
中科院分区：
文献类型：
--
作者：
Gao, Y;Xing, J;Zheng, Y
Rho GTPase通路的信号特异性部分是通过DBL家族鸟嘌呤核苷酸交换因子(GEF)成员与其Rho GTPase底物之间的选择性相互作用实现的。例如，Trio、gef-h1和tiam1是专门激活rac1而不是密切相关的cdc42的GEF的子集。这些GEF的rac1特异性似乎是由rac1与全球环境基金的结合作用决定的。为了了解全球环境基金特异性问题背后的详细机制，我们分析了rac1和cdc42之间的一组嵌合体，并检测了在Switch I、Switch II和beta(2)/beta(3)区域产生的一系列racl点突变，以了解它们与GEF相互作用和被GEF激活的能力。结果表明，开关I和开关II区域的rac1残基都参与了全球环境基金对接和全球环境基金介导的核苷酸破坏，因为天冬氨酸(38)、天冬氨酸(39)、谷氨酰胺(61)、酪氨酸()或精氨酸(66)/亮氨酸(67)突变导致全球环境基金失去结合，而酪氨酸(32)、天冬氨酸(65)或亮氨酸(70)/丝氨酸(71)突变导致全球环境基金催化丧失，但结合能力保持不变。对于GEF的特异性识别和激活来说，rac1的53-72个氨基酸之间的区域是必需的，而β(3)中的Trp(56)似乎是关键的决定因素。将色氨酸(56)引入到Cdc42中，使其在体外和细胞内对RAC特定的环境基金具有完全的反应能力。此外，从rac1的β(3)区衍生的多肽，包括Trp，56残基，作为rac1与GEF相互作用的特异性抑制物。综上所述，这些结果表明，Trp-56是通过rac1特异性GEF的子集来区分rac1的必要和充分的决定因素，并表明可以探索一种模拟Trp 56作用的化合物作为一种专门针对rac1激活的干扰试剂。
Signaling specificity of Rho GTPase pathways is achieved in part by selective interaction between members of the Dbl family guanine nucleotide exchange factors (GEFs) and their Rho GTPase substrates. For example, Trio, GEF-H1, and Tiam1 are a subset of GEFs that specifically activate Rac1 but not the closely related Cdc42. The Rac1 specificity of these GEFs appears to be governed by Rac1-GEF binding interaction. To understand the detailed mechanism underlying the GEF specificity issue, we have analyzed a panel of chimeras made between Rac1 and Cdc42 and examined a series of point mutants of Racl made at the switch I, switch II, and beta (2)/beta (3) regions for their ability to interact with and to be activated by the GEFs. The results reveal that Rac1 residues of both the switch I and switch II regions are involved in GEF docking and GEF-mediated nucleotide disruption, because mutation of Asp(38), Asn(39), Gln(61), Tyr(64), or Arg(66)/Leu(67) into Ala results in the loss of GEF binding, whereas mutation at Tyr(32), Asp(65), or Leu(70)/ Ser(71) leads to the loss of GEF catalysis while retaining the binding capability. The region between amino acids 53-72 of Rac1 is required for specific recognition and activation by the GEFs, and Trp(56) in beta (3), appears to be the critical determinant. Introduction of Trp(56) to Cdc42 renders it fully responsive to the Rac-specific GEF in vitro and in cells. Further, a polypeptide derived from the beta (3) region of Rac1 including the Trp,56 residue serves as a specific inhibitor for Rac1 interaction with the GEFs. Taken together, these results indicate that Trp-56 is the necessary and sufficient determinant of Rac1 for discrimination by the subset of Rac1-specific GEFs and suggest that a compound mimicking Trp 56 action could be explored as an interfering reagent specifically targeting Rac1 activation.