Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
J Cell Sci. 2010 Jun 1;123(Pt 11):1948-58. doi: 10.1242/jcs.062919. Epub 2010 May 11.
Directional cell migration is crucially dependent on the spatiotemporal control of intracellular signalling events. These events regulate polarized actin dynamics, resulting in protrusion at the front of the cell and contraction at the rear. The actin cytoskeleton is regulated through signalling by Rho-like GTPases, such as RhoA, which stimulates myosin-based contractility, and CDC42 and Rac1, which promote actin polymerization and protrusion. Here, we show that Rac1 binds the adapter protein caveolin-1 (Cav1) and that Rac1 activity promotes Cav1 accumulation at Rac1-positive peripheral adhesions. Using Cav1-deficient mouse fibroblasts and depletion of Cav1 expression in human epithelial and endothelial cells mediated by small interfering RNA and short hairpin RNA, we show that loss of Cav1 induces an increase in Rac1 protein and its activated, GTP-bound form. Cav1 controls Rac1 protein levels by regulating ubiquitylation and degradation of activated Rac1 in an adhesion-dependent fashion. Finally, we show that Rac1 ubiquitylation is not required for effector binding, but regulates the dynamics of Rac1 at the periphery of the cell. These data extend the canonical model of Rac1 inactivation and uncover Cav1-regulated polyubiquitylation as an additional mechanism to control Rac1 signalling.
细胞的定向迁移很大程度上依赖于细胞内信号事件的时空控制。这些事件调节极化的肌动蛋白动态，导致细胞前端的突起和后端的收缩。肌动蛋白细胞骨架通过 Rho 样 GTP 酶（如 RhoA）的信号调节，RhoA 刺激肌球蛋白依赖性收缩，而 CDC42 和 Rac1 则促进肌动蛋白聚合和突起。在这里，我们表明 Rac1 与衔接蛋白 caveolin-1（Cav1）结合，并且 Rac1 活性促进 Rac1 在 Rac1 阳性周边黏附中的积累。使用 Cav1 缺陷型小鼠成纤维细胞和通过小干扰 RNA 和短发夹 RNA 介导的人上皮细胞和内皮细胞中 Cav1 的表达缺失，我们表明 Cav1 的缺失会导致 Rac1 蛋白及其激活的 GTP 结合形式增加。Cav1 通过依赖黏附的方式调节激活的 Rac1 的泛素化和降解来控制 Rac1 蛋白水平。最后，我们表明 Rac1 的泛素化对于效应蛋白结合不是必需的，但调节 Rac1 在细胞周边的动态。这些数据扩展了 Rac1 失活的经典模型，并揭示了 Cav1 调节的多泛素化作为控制 Rac1 信号的另一种机制。
