From the Departments of Pathology, Pittsburgh, Pennsylvania 15261.
Bioengineering, Pittsburgh, Pennsylvania 15261.
J Biol Chem. 2010 Oct 22;285(43):33549-33566. doi: 10.1074/jbc.M110.123604. Epub 2010 Aug 20.
m-Calpain plays a critical role in cell migration enabling rear de-adhesion of adherent cells by cleaving structural components of the adhesion plaques. Growth factors and chemokines regulate keratinocyte, fibroblast, and endothelial cell migration by modulating m-calpain activity. Growth factor receptors activate m-calpain secondary to phosphorylation on serine 50 by ERK. Concurrently, activated m-calpain is localized to its inner membrane milieu by binding to phosphatidylinositol 4,5-bisphosphate (PIP(2)). Opposing this, CXCR3 ligands inhibit cell migration by blocking m-calpain activity secondary to a PKA-mediated phosphorylation in the C2-like domain. The failure of m-calpain activation in the absence of PIP(2) points to a key regulatory role, although whether this PIP(2)-mediated membrane localization is regulatory for m-calpain activity or merely serves as a docking site for ERK phosphorylation is uncertain. Herein, we report the effects of two CXCR3 ligands, CXCL11/IP-9/I-TAC and CXCL10/IP-10, on the EGF- and VEGF-induced redistribution of m-calpain in human fibroblasts and endothelial cells. The two chemokines block the tail retraction and, thus, the migration within minutes, preventing and reverting growth factor-induced relocalization of m-calpain to the plasma membrane of the cells. PKA phosphorylation of m-calpain blocks the binding of the protease to PIP(2). Unexpectedly, we found that this was due to membrane anchorage itself and not merely serine 50 phosphorylation, as the farnesylation-induced anchorage of m-calpain triggers a strong activation of this protease, leading notably to an increased cell death. Moreover, the ERK and PKA phosphorylations have no effect on this membrane-anchored m-calpain. However, the presence of PIP(2) is still required for the activation of the anchored m-calpain. In conclusion, we describe a novel mechanism of m-calpain activation by interaction with the plasma membrane and PIP(2) specifically, this phosphoinositide acting as a cofactor for the enzyme. The phosphorylation of m-calpain by ERK and PKA by growth factors and chemokines, respectively, act in cells to regulate the enzyme only indirectly by controlling its redistribution.
钙蛋白酶 m 在细胞迁移中起着关键作用，通过切割黏附斑的结构成分，使黏附细胞的后脱离。生长因子和趋化因子通过调节钙蛋白酶 m 的活性来调节角质形成细胞、成纤维细胞和内皮细胞的迁移。生长因子受体通过 ERK 对丝氨酸 50 的磷酸化作用激活钙蛋白酶 m。同时，激活的钙蛋白酶 m 通过与磷脂酰肌醇 4,5-二磷酸（PIP（2））结合而定位于其内膜环境中。相反，CXCR3 配体通过 PKA 介导的 C2 样结构域中的磷酸化作用抑制细胞迁移，从而阻断钙蛋白酶 m 的活性。在没有 PIP（2）的情况下，钙蛋白酶 m 的激活失败表明其发挥着关键的调节作用，尽管这种 PIP（2）介导的膜定位是调节钙蛋白酶 m 活性的关键，还是仅仅作为 ERK 磷酸化的对接位点还不确定。在此，我们报告了两种 CXCR3 配体，CXCL11/IP-9/I-TAC 和 CXCL10/IP-10，对 EGF 和 VEGF 诱导的人成纤维细胞和内皮细胞中钙蛋白酶 m 再分布的影响。这两种趋化因子在数分钟内阻止了尾部回缩，从而阻止并逆转了生长因子诱导的钙蛋白酶 m 向细胞质膜的再定位。钙蛋白酶 m 的 PKA 磷酸化阻断了该蛋白酶与 PIP（2）的结合。出乎意料的是，我们发现这是由于膜锚定本身，而不仅仅是丝氨酸 50 磷酸化，因为钙蛋白酶 m 的法尼酰化锚定触发了该蛋白酶的强烈激活，导致细胞死亡显著增加。此外，ERK 和 PKA 的磷酸化对这种膜锚定的钙蛋白酶 m 没有影响。然而，对于锚定的钙蛋白酶 m 的激活，仍然需要 PIP（2）的存在。总之，我们描述了一种钙蛋白酶 m 通过与质膜相互作用和 PIP（2）特异性激活的新机制，这种磷酸肌醇作为该酶的辅助因子。生长因子和趋化因子分别通过 ERK 和 PKA 对钙蛋白酶 m 的磷酸化作用，在细胞中仅通过控制其再分布来间接调节酶的活性。
