Department of Pathology, F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA.
J Cell Commun Signal. 2013 Dec;7(4):279-93. doi: 10.1007/s12079-013-0207-5. Epub 2013 Jun 14.
Cell adhesion and migration are complex processes that require integrin activation, the formation and dissolution of focal adhesion (FAs), and linkage of actin cytoskeleton to the FAs. The IPP (ILK, PINCH, Parvin) complex regulates FA formation via binding of the adaptor protein ILK to β1 integrin, PINCH and parvin. The signaling protein Rsu1 is linked to the complex via binding PINCH1. The role of Rsu1 and PINCH1 in adhesion and migration was examined in non-transformed mammary epithelial cells. Confocal microscopy revealed that the depletion of either Rsu1 or PINCH1 by siRNA in MCF10A cells decreased the number of focal adhesions and altered the distribution and localization of β1 integrin, vinculin, talin and paxillin without affecting the levels of FA protein expression. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. In addition, constitutive phosphorylation of actin regulatory proteins occurred in the absence of PINCH1. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function by regulating levels of PINCH1. However, while both Rsu1- or PINCH1-depleted cells retained the ability to activate adhesion signaling in response to EGF stimulation, only Rsu1 was required for EGF-induced p38 Map Kinase phosphorylation and ATF2 activation, suggesting an Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with an Rsu1 mutant that does not bind to PINCH1 failed to restore FAs or migration but did promote spreading and constitutive p38 activation. These data show that Rsu1-PINCH1 association with ILK and the IPP complex is required for regulation of adhesion and migration but that Rsu1 has a critical role in linking integrin-induced adhesion to activation of p38 Map kinase signaling and cell spreading. Moreover, it suggests that Rsu1 may regulate p38 signaling from the IPP complex affecting other functions including survival.
细胞黏附和迁移是复杂的过程，需要整合素激活、黏附斑（FAs）的形成和溶解，以及肌动蛋白细胞骨架与 FAs 的连接。IPP（ILK、PINCH、Parvin）复合物通过衔接蛋白 ILK 与β1 整合素、PINCH 和 Parvin 的结合来调节 FA 的形成。信号蛋白 Rsu1 通过与 PINCH1 的结合与复合物相连。通过 siRNA 在 MCF10A 细胞中耗尽 Rsu1 或 PINCH1，研究了它们在黏附和迁移中的作用。共焦显微镜显示，这两种蛋白的耗竭都减少了焦点黏附的数量，并改变了β1 整合素、 vinculin、talin 和 paxillin 的分布和定位，而不影响 FA 蛋白的表达水平。这与黏附能力降低、EGF 刺激下无法扩展或迁移以及肌动蛋白应力纤维和 caveolae 的缺失有关。此外，在没有 PINCH1 的情况下，肌动蛋白调节蛋白的组成性磷酸化发生。Rsu1 的耗竭导致 PINCH1 的显著减少，这表明 Rsu1 可能通过调节 PINCH1 的水平来发挥作用。然而，虽然 Rsu1-或 PINCH1-耗尽的细胞在响应 EGF 刺激时仍然能够激活黏附信号，但只有 Rsu1 是 EGF 诱导的 p38 MAP 激酶磷酸化和 ATF2 激活所必需的，这表明 Rsu1 的功能独立于 IPP 复合物。用不与 PINCH1 结合的 Rsu1 突变体重建 Rsu1 耗尽的细胞，未能恢复 FAs 或迁移，但确实促进了扩展和组成性 p38 激活。这些数据表明，Rsu1-PINCH1 与 ILK 和 IPP 复合物的结合对于调节黏附和迁移是必需的，但 Rsu1 在将整合素诱导的黏附与 p38 MAP 激酶信号的激活和细胞扩展联系起来方面起着关键作用。此外，这表明 Rsu1 可能通过影响包括存活在内的其他功能，从 IPP 复合物调节 p38 信号。
