Perrin Benjamin J, Amann Kurt J, Huttenlocher Anna
Cellular and Molecular Biology Program, University of Wisconsin-Madison, Madison, WI 53706, USA.
Mol Biol Cell. 2006 Jan;17(1):239-50. doi: 10.1091/mbc.e05-06-0488. Epub 2005 Nov 9.
Calpain 2 regulates membrane protrusion during cell migration. However, relevant substrates that mediate the effects of calpain on protrusion have not been identified. One potential candidate substrate is the actin binding protein cortactin. Cortactin is a Src substrate that drives actin polymerization by activating the Arp2/3 complex and also stabilizes the cortical actin network. We now provide evidence that proteolysis of cortactin by calpain 2 regulates membrane protrusion dynamics during cell migration. We show that cortactin is a calpain 2 substrate in fibroblasts and that the preferred cleavage site occurs in a region between the actin binding repeats and the alpha-helical domain. We have generated a mutant cortactin that is resistant to calpain proteolysis but retains other biochemical properties of cortactin. Expression of the calpain-resistant cortactin, but not wild-type cortactin, impairs cell migration and increases transient membrane protrusion, suggesting that calpain proteolysis of cortactin limits membrane protrusions and regulates migration in fibroblasts. Furthermore, the enhanced protrusion observed with the calpain-resistant cortactin requires both the Arp2/3 binding site and the Src homology 3 domain of cortactin. Together, these findings suggest a novel role for calpain-mediated proteolysis of cortactin in regulating membrane protrusion dynamics during cell migration.
钙蛋白酶2在细胞迁移过程中调节膜突出。然而，介导钙蛋白酶对突出作用的相关底物尚未确定。一种潜在的候选底物是肌动蛋白结合蛋白皮层肌动蛋白。皮层肌动蛋白是一种Src底物，通过激活Arp2/3复合体驱动肌动蛋白聚合，还能稳定皮层肌动蛋白网络。我们现在提供证据表明，钙蛋白酶2对皮层肌动蛋白的蛋白水解作用在细胞迁移过程中调节膜突出动力学。我们发现皮层肌动蛋白是成纤维细胞中的钙蛋白酶2底物，且优先切割位点位于肌动蛋白结合重复序列和α螺旋结构域之间的区域。我们构建了一种对钙蛋白酶蛋白水解具有抗性但保留皮层肌动蛋白其他生化特性的突变型皮层肌动蛋白。表达抗钙蛋白酶的皮层肌动蛋白而非野生型皮层肌动蛋白会损害细胞迁移并增加瞬时膜突出，这表明钙蛋白酶对皮层肌动蛋白的蛋白水解作用限制了膜突出并调节成纤维细胞的迁移。此外，抗钙蛋白酶的皮层肌动蛋白所观察到的增强突出需要皮层肌动蛋白的Arp2/3结合位点和Src同源3结构域。总之，这些发现表明钙蛋白酶介导的皮层肌动蛋白蛋白水解在调节细胞迁移过程中的膜突出动力学方面具有新作用。
