Rosenberger Georg, Gal Andreas, Kutsche Kerstin
Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, D-22529 Hamburg, Germany.
J Biol Chem. 2005 Feb 25;280(8):6879-89. doi: 10.1074/jbc.M412119200. Epub 2004 Dec 20.
Binding of integrins to the extracellular matrix results in actin cytoskeletal rearrangements, e.g. during cell spreading, by regulating the activity of Rho GTP-ases. We have shown previously that alphaPIX (Cool-2 or ARHGEF6), a Rac1/Cdc42-specific guanine nucleotide exchange factor (GEF), binds to beta-parvin/affixin and colocalizes with integrin-linked kinase in actively spreading cells, suggesting that alphaPIX is involved in integrin-induced signaling leading to activation of Rac1/Cdc42. Here we report calpain 4, the small subunit of the proteases mu-calpain and m-calpain, as a novel binding partner of alphaPIX. This association was identified by the CytoTrap system and confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays. The alphaPIX triple domain SH3-DH-PH was found to be required for calpain 4 binding. During integrin-dependent spreading of CHO-K1 cells, alphaPIX colocalized with mu- and m-calpain, integrin-linked kinase, and beta1 integrin in early integrin-containing clusters. Overexpression of alphaPIX wild type but not the GEF-deficient mutant (L386R/L387S) resulted in enhanced formation of characteristic cellular protrusions during cell spreading, suggesting that alphaPIX GEF activity is necessary for this specific actin cytoskeletal reorganization. The calpain inhibitors calpeptin and calpain inhibitor IV significantly inhibited integrin-dependent cell spreading. However, concomitant overexpression of alphaPIX wild type or the L386R/L387S mutant restored cell spreading. Together, these data suggest that alphaPIX is a component of early integrin clusters and plays a dual role in integrin-dependent cell spreading. Whereas alphaPIX GEF activity contributes to enhanced formation of cellular protrusions, the GEF-independent association with calpain 4 leads to induction of a yet unknown signaling cascade resulting in cell spreading.
整合素与细胞外基质的结合通过调节Rho GTP酶的活性导致肌动蛋白细胞骨架重排，例如在细胞铺展过程中。我们之前已经表明，αPIX（Cool-2或ARHGEF6），一种Rac1/Cdc42特异性鸟嘌呤核苷酸交换因子（GEF），与β-帕文/亲和素结合，并在活跃铺展的细胞中与整合素连接激酶共定位，这表明αPIX参与整合素诱导的信号传导，导致Rac1/Cdc42的激活。在这里，我们报告钙蛋白酶4，即蛋白酶μ-钙蛋白酶和m-钙蛋白酶的小亚基，作为αPIX的一种新型结合伴侣。这种关联通过CytoTrap系统鉴定，并通过免疫共沉淀和谷胱甘肽S-转移酶下拉实验得到证实。发现αPIX的三联结构域SH3-DH-PH是钙蛋白酶4结合所必需的。在CHO-K1细胞的整合素依赖性铺展过程中，αPIX在早期含整合素的簇中与μ-钙蛋白酶和m-钙蛋白酶、整合素连接激酶以及β1整合素共定位。αPIX野生型而非GEF缺陷型突变体（L386R/L387S）的过表达导致细胞铺展过程中特征性细胞突起的形成增强，这表明αPIX的GEF活性对于这种特定的肌动蛋白细胞骨架重组是必需的。钙蛋白酶抑制剂钙肽素和钙蛋白酶抑制剂IV显著抑制整合素依赖性细胞铺展。然而，αPIX野生型或L386R/L387S突变体的共过表达恢复了细胞铺展。总之，这些数据表明αPIX是早期整合素簇的一个组成部分，并且在整合素依赖性细胞铺展中起双重作用。虽然αPIX的GEF活性有助于增强细胞突起的形成，但与钙蛋白酶4的非GEF依赖性关联导致诱导一种未知的信号级联反应，从而导致细胞铺展。
