Regulation by Afadin of Cyclical Activation and Inactivation of Rap1, Rac1, and RhoA Small G Proteins at Leading Edges of Moving NIH3T3 Cells
Regulation by Afadin of Cyclical Activation and Inactivation of Rap1, Rac1, and RhoA Small G Proteins at Leading Edges of Moving NIH3T3 Cells
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DOI：
10.1074/jbc.m109.016436
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发表时间：
2009-09-04
影响因子：
4.8
通讯作者：
Takai, Yoshimi
中科院分区：
文献类型：
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作者：
Miyata, Muneaki;Rikitake, Yoshiyuki;Takai, Yoshimi
Rho家族小G蛋白（如Rho、Rac和Cdc 42）的周期性激活和失活是移动细胞以响应化学引诱物形成前缘结构所必需的。然而，其活动的动态调节机制尚未完全了解。我们最近发现，另一个小G蛋白，Rap 1，在血小板源性生长因子（PDGF）诱导的前缘结构的形成和激活Rac 1在NIH 3 T3细胞中起着至关重要的作用。我们发现，在NIH 3 T3细胞中，afadin（一种肌动蛋白结合蛋白）的敲低导致未能形成与Rap 1和Rac 1激活受损和RhoA响应PDGF失活相关的前缘结构。Rap 1的组成型活性突变体（Rap 1-CA）的过表达和SPA-1（一种Rap 1 GTP酶激活蛋白，其通过与其结合而被afadin负调控）的敲低在afadin敲低的NIH 3 T3细胞中恢复了前缘结构的形成和PDGF诱导的Rac 1激活和RhoA失活的减少，提示SPA-1对Rap 1的失活是抑制前缘结构形成的原因。Rap 1-CA对前缘结构形成和RhoA失活的恢复的作用通过另外敲除ARAP 1（Rap-activated Rho GAP，其位于移动的NIH 3 T3细胞的前缘）而减弱。这些结果表明，afadin通过SPA-1和ARAP 1调节Rap 1，Rac 1和RhoA的周期性激活和失活。
Cyclical activation and inactivation of Rho family small G proteins, such as Rho, Rac, and Cdc42, are needed for moving cells to form leading edge structures in response to chemoattractants. However, the mechanisms underlying the dynamic regulation of their activities are not fully understood. We recently showed that another small G protein, Rap1, plays a crucial role in the platelet-derived growth factor (PDGF)-induced formation of leading edge structures and activation of Rac1 in NIH3T3 cells. We showed here that knockdown of afadin, an actin-binding protein, in NIH3T3 cells resulted in a failure to develop leading edge structures in association with an impairment of the activation of Rap1 and Rac1 and inactivation of RhoA in response to PDGF. Overexpression of a constitutively active mutant of Rap1 (Rap1-CA) and knockdown of SPA-1, a Rap1 GTPase-activating protein that was negatively regulated by afadin by virtue of binding to it, in afadin-knockdown NIH3T3 cells restored the formation of leading edge structures and the reduction of the PDGF-induced activation of Rac1 and inactivation of RhoA, suggesting that the inactivation of Rap1 by SPA-1 is responsible for inhibition of the formation of leading edge structures. The effect of Rap1-CA on the restoration of the formation of leading edge structures and RhoA inactivation was diminished by additional knockdown of ARAP1, a Rap-activated Rho GAP, which localized at the leading edges of moving NIH3T3 cells. These results indicate that afadin regulates the cyclical activation and inactivation of Rap1, Rac1, and RhoA through SPA-1 and ARAP1.