Mori Mamiko, Saito Koji, Ohta Yasutaka
Division of Cell Biology, Department of Biosciences, School of Science, Kitasato University, Kanagawa, Japan.
PLoS One. 2014 Jun 16;9(6):e100271. doi: 10.1371/journal.pone.0100271. eCollection 2014.
Rho small GTPases control cell morphology and motility through the rearrangement of actin cytoskeleton. We have previously shown that FilGAP, a Rac-specific GAP, binds to the actin-cross-linking protein Filamin A (FLNa) and suppresses Rac-dependent lamellae formation and cell spreading. ARHGAP22 is a member of FilGAP family, and implicated in the regulation of tumor cell motility. However, little is known concerning the cellular localization and mechanism of regulation at the molecular level. Whereas FilGAP binds to FLNa and localizes to lamellae, we found that ARHGAP22 did not bind to FLNa. Forced expression of ARHGAP22 induced enlarged vesicular structures containing the endocytic markers EEA1, Rab5, and Rab11. Moreover, endogenous ARHGAP22 is co-localized with EEA1- and Rab11-positive endosomes but not with trans-Golgi marker TNG46. When constitutively activated Rac Q61L mutant was expressed, ARHGAP22 is co-localized with Rac Q61L at membrane ruffles, suggesting that ARHGAP22 is translocated from endosomes to membrane ruffles to inactivate Rac. Forced expression of ARHGAP22 suppressed lamellae formation and cell spreading. Conversely, knockdown of endogenous ARHGAP22 stimulated cell spreading. Thus, our findings suggest that ARHGAP22 controls cell morphology by inactivating Rac but its localization is not mediated by its interaction with FLNa.
Rho小GTP酶通过肌动蛋白细胞骨架的重排来控制细胞形态和运动。我们之前已经表明，FilGAP是一种Rac特异性GAP，它与肌动蛋白交联蛋白细丝蛋白A（FLNa）结合，并抑制Rac依赖性片状伪足的形成和细胞铺展。ARHGAP22是FilGAP家族的成员，与肿瘤细胞运动的调节有关。然而，关于其细胞定位和分子水平的调节机制知之甚少。虽然FilGAP与FLNa结合并定位于片状伪足，但我们发现ARHGAP22不与FLNa结合。ARHGAP22的强制表达诱导了包含内吞标记物EEA1、Rab5和Rab11的增大的囊泡结构。此外，内源性ARHGAP22与EEA1和Rab11阳性的内体共定位，但不与反式高尔基体标记物TNG46共定位。当组成性激活的Rac Q61L突变体表达时，ARHGAP22与Rac Q61L在膜皱褶处共定位，表明ARHGAP22从内体转运到膜皱褶处以使Rac失活。ARHGAP22的强制表达抑制了片状伪足的形成和细胞铺展。相反，内源性ARHGAP22的敲低刺激了细胞铺展。因此，我们的研究结果表明，ARHGAP22通过使Rac失活来控制细胞形态，但其定位不是由其与FLNa的相互作用介导的。
