Biochemical studies of the mechanism of action of the Cdc42-GTPase-activating protein
Biochemical studies of the mechanism of action of the Cdc42-GTPase-activating protein
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DOI：
10.1074/jbc.273.26.16210
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发表时间：
1998-06-26
影响因子：
4.8
通讯作者：
Manor, D
中科院分区：
文献类型：
--
作者：
Leonard, DA;Lin, R;Manor, D
小的gtp结合蛋白Rac， Rho和Cdc42被证明介导多种信号通路，包括细胞骨架重排，细胞周期进展和转化。这些gtp结合蛋白正常发挥作用的关键是确保信号衰减的有效关闭机制。被称为GAPs （GTP酶激活蛋白）的调节蛋白增强GTP结合蛋白的内在水解，从而确保信号终止。我们已经使用位点特异性诱变技术阐明了Cdc42-GAP中GAP活性的极限结构域，并表明除了已知的GAP同源结构域（三个保守框）外，该结构域外的c端区域也是GAP活性所必需的。此外，我们用丙氨酸取代了结构研究认为是关键催化残基的保守精氨酸（Arg(305)），发现R305A Cdc42- gap突变体的催化能力大大降低，但仍然能够高亲和力地结合Cdc42。因此，确认了该残留物的关键催化作用。然而，我们也提供了额外残基参与的证据，因为R305A Cdc42-GAP突变体仍然表现出可测量的活性。由于双突变体R305A/R306A的催化活性进一步下降，一些残留活性可能来自邻近的精氨酸。
The small GTP-binding proteins Rac, Rho, and Cdc42 were shown to mediate a variety of signaling pathways including cytoskeletal rearrangements, cell-cycle progression, and transformation. Key to the proper function of these GTP-binding proteins is an efficient shutoff mechanism that ensures the decay of the signal. Regulatory proteins termed GAPs (GTPase-activating proteins) enhance the intrinsic GTP hydrolysis of the GTP-binding proteins, thereby ensuring signal termination. We have used site specific mutagenesis to elucidate the limit domain for GAP activity in Cdc42-GAP, and show that in addition to the known GAP-homology domain (three conserved boxes), a C-terminal region outside that domain is also essential for GAP activity. In addition, we have replaced the conserved arginine (Arg(305)), which was suggested by structural studies to be a key catalytic residue, with an alanine and found that the R305A Cdc42-GAP mutant has a greatly diminished catalytic capacity but is still able to bind Cdc42 with high affinity. Thus, a key catalytic role for this residue is confirmed. However, we also present evidence for the involvement of an additional residue(s), since the R305A Cdc42-GAP mutant still exhibits measurable activity. Some of this residual activity might result from a neighboring arginine, since a double mutant R305A/R306A shows a further decrease in catalytic activity.