Mori Mamiko, Saito Koji, Sekine Ayano, Hasebe Rio, Ohta Yasutaka
Division of Cell Biology, Department of Biosciences, School of Science, Kitasato University, Kanagawa, Japan.
Division of Cell Biology, Department of Biosciences, School of Science, Kitasato University, Kanagawa, Japan
Anticancer Res. 2022 Dec;42(12):5763-5771. doi: 10.21873/anticanres.16083.
BACKGROUND/AIM: Rho small GTPases regulate cancer cell adhesion, migration and invasion through reorganization of the actin cytoskeleton. Rho GTPase Activating Protein 22 (ARHGAP22) is a Rac-specific GAP and suppresses Rac-dependent lamella formation and cell spreading. We have previously shown that ARHGAP22 localizes at endosomes in human melanoma A7 cells. The aim of the present study was to demonstrate the functional significance of its localization at the endosomes in melanoma cells.
The lamella formation and cell spreading were monitored using human melanoma A7 cells. The effect of inhibition of endosome recycling pathway was examined.
We found that dominant negative Rab11 S25N mutant inhibits RacGAP activity of ARHGAP22 and blocks ARHGAP22-dependent suppression of lamella formation and melanoma cell spreading. Furthermore, deletion of 19 amino acid residues at the C-terminal region of ARHGAP22 abolished the localization of ARHGAP22 at enlarged vesicles and stimulated RacGAP activity of ARHGAP22. The deletion mutant accumulated at enlarged vesicles when endosome recycling pathway was blocked either by co-transfection of the Rab11 S25N mutant or treatment of the cells with N-ethylmaleimide, which blocks endosomal vesicular fusion to the plasma membrane. On the other hand, deletion of the pleckstrin homology (PH) domain of ARHGAP22 abolished its RacGAP activity and localization at the plasma membrane.
ARHGAP22 localizes at endosomes and is transported to the plasma membrane to inactivate Rac and suppresses lamella formation and spreading of melanoma cells.
背景/目的：Rho 小 GTP 酶通过肌动蛋白细胞骨架的重组来调节癌细胞的黏附、迁移和侵袭。Rho GTP 酶激活蛋白 22（ARHGAP22）是一种 Rac 特异性 GAP，可抑制 Rac 依赖的片状伪足形成和细胞铺展。我们之前已经表明 ARHGAP22 定位于人黑色素瘤 A7 细胞的内体中。本研究的目的是证明其在内体中的定位在黑色素瘤细胞中的功能意义。
使用人黑色素瘤 A7 细胞监测片状伪足形成和细胞铺展。检测了内体再循环途径抑制的效果。
我们发现显性负性 Rab11 S25N 突变体抑制 ARHGAP22 的 RacGAP 活性，并阻断 ARHGAP22 依赖的片状伪足形成抑制和黑色素瘤细胞铺展。此外，ARHGAP22 C 末端区域缺失 19 个氨基酸残基消除了 ARHGAP22 在扩大囊泡中的定位，并刺激了 ARHGAP22 的 RacGAP 活性。当通过共转染 Rab11 S25N 突变体或用 N - 乙基马来酰亚胺处理细胞来阻断内体再循环途径时，该缺失突变体在扩大的囊泡中积累，N - 乙基马来酰亚胺可阻断内体囊泡与质膜的融合。另一方面，ARHGAP22 的普列克底物蛋白同源（PH）结构域的缺失消除了其 RacGAP 活性和在质膜上的定位。
ARHGAP22 定位于内体，并被转运到质膜以使 Rac 失活，从而抑制黑色素瘤细胞的片状伪足形成和铺展。
