αPIX associates with calpain 4, the small subunit of calpain, and has a dual role in integrin-mediated cell spreading
αPIX associates with calpain 4, the small subunit of calpain, and has a dual role in integrin-mediated cell spreading
复制标题
DOI：
10.1074/jbc.m412119200
复制
发表时间：
2005-02-25
影响因子：
4.8
通讯作者：
Kutsche, K
中科院分区：
文献类型：
--
作者：
Rosenberger, G;Gal, A;Kutsche, K
整合素与细胞外基质的结合导致肌动蛋白细胞骨架重排，例如在细胞扩散过程中，通过调节 Rho GTPases 的活性。我们之前已经证明，αPIX（Cool-2或ARHGEF6）是一种Rac1/Cdc42特异性鸟嘌呤核苷酸交换因子（GEF），与β-parvin/affixin结合，并与活跃扩散的细胞中的整合素连接激酶共定位，表明aPIX参与整合素诱导的信号传导，从而导致Rac1/Cdc42的激活。在这里，我们报告了钙蛋白酶 4（蛋白酶 mu-钙蛋白酶和 m-钙蛋白酶的小亚基）作为 alphaPIX 的新型结合伴侣。这种关联通过 CytoTrap 系统进行了鉴定，并通过免疫共沉淀和谷胱甘肽 S-转移酶下拉分析进行了证实。发现 alphaPIX 三重结构域 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扩散过程中，aPIX 与 mu-和 m-钙蛋白酶、整合素连接激酶和早期含有整合素的簇中的 β1 整合素共定位。 aPIX 野生型而非 GEF 缺陷突变体 (L386R/L387S) 的过表达导致细胞扩散过程中特征性细胞突起的形成增强，表明 alphaPIX 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显着抑制整合素依赖性细胞扩散。然而，αPIX野生型或L386RAL387S突变体的同时过度表达恢复了细胞扩散。总之，这些数据表明 alphaPIX 是早期整合素簇的组成部分，并在整合素依赖性细胞扩散中发挥双重作用。虽然 alphaPIX GEF 活性有助于增强细胞突起的形成，但 GEF 与钙蛋白酶 4 的独立关联会导致诱导未知的信号级联，从而导致细胞扩散。
Binding of integrins to the extracellular matrix results in actin cytoskeletal rearrangements, e.g. during cell spreading, by regulating the activity of Rho GTPases. 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 aPIX 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, aPIX colocalized with mu-and m-calpain, integrin-linked kinase, and beta1 integrin in early integrin-containing clusters. Overexpression of aPIX 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 L386RAL387S 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.