El-Sibai Mirvat, Nalbant Peri, Pang Huan, Flinn Rory J, Sarmiento Corina, Macaluso Frank, Cammer Michael, Condeelis John S, Hahn Klaus M, Backer Jonathan M
Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
J Cell Sci. 2007 Oct 1;120(Pt 19):3465-74. doi: 10.1242/jcs.005942. Epub 2007 Sep 12.
Cdc42 plays a central role in regulating the actin cytoskeleton and maintaining cell polarity. Here, we show that Cdc42 is crucial for epidermal growth factor (EGF)-stimulated protrusion in MTLn3 carcinoma cells. When stimulated with EGF, carcinoma cells showed a rapid increase in activated Cdc42 that is primarily localized to the protruding edge of the cells. siRNA-mediated knockdown of Cdc42 expression caused a decrease in EGF-stimulated protrusion and reduced cell motility in time-lapse studies. These changes were correlated with a decrease in barbed-end formation and Arp2/3 localization at the cell edge, and a marked defect in actin filament branching, as revealed by rotary-shadowing scanning electron microscopy. Upstream of Arp2/3, Cdc42 knockdown inhibited EGF-stimulated activation of PI 3-kinase at early (within 1 minute) but not late (within 3 minutes) time points. Membrane targeting of N-WASP, WAVE2 and IRSp53 were also inhibited. Effects on WAVE2 were not owing to Rac1 inhibition, because WAVE2 recruitment is unaffected by Rac1 knockdown. Our data suggest that Cdc42 activation is crucial for the regulation of actin polymerization in carcinoma cells, and required for both EGF-stimulated protrusion and cell motility independently of effects on Rac.
Cdc42在调节肌动蛋白细胞骨架和维持细胞极性方面发挥着核心作用。在此，我们表明Cdc42对于MTLn3癌细胞中表皮生长因子（EGF）刺激的突起形成至关重要。在用EGF刺激时，癌细胞中活化的Cdc42迅速增加，且主要定位于细胞的突出边缘。在延时研究中，siRNA介导的Cdc42表达敲低导致EGF刺激的突起减少以及细胞运动性降低。这些变化与细胞边缘处带刺末端形成和Arp2/3定位的减少相关，并且如旋转阴影扫描电子显微镜所显示的，肌动蛋白丝分支存在明显缺陷。在Arp2/3的上游，Cdc42敲低在早期（1分钟内）而非晚期（3分钟内）时间点抑制了EGF刺激的PI 3激酶活化。N-WASP、WAVE2和IRSp53的膜靶向也受到抑制。对WAVE2的影响并非由于Rac1抑制，因为Rac1敲低不影响WAVE2的募集。我们的数据表明，Cdc42活化对于癌细胞中肌动蛋白聚合的调节至关重要，并且是EGF刺激的突起形成和细胞运动性所必需的，且独立于对Rac的影响。
