αPIX associates with calpain 4, the small subunit of calpain, and has a dual role in integrin-mediated cell spreading

Abstract

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 αPIX (Cool-2 or ARHGEF6), a Rac1/Cdc42-specific guanine nucleotide exchange factor (GEF), binds to β-parvin/affixin and colocalizes with integrin-linked kinase in actively spreading cells, suggesting that αPIX is involved in integrin-induced signaling leading to activation of Rac1/Cdc42. Here we report calpain 4, the small subunit of the proteases μ-calpain and m-calpain, as a novel binding partner of αPIX. This association was identified by the CytoTrap system and confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays. The αPIX triple domain SH3-DH-PH was found to be required for calpain 4 binding. During integrin-dependent spreading of CHO-K1 cells, αPIX colocalized with μ- and m-calpain, integrin-linked kinase, and β1 integrin in early integrin-containing clusters. Overexpression of αPIX wild type but not the GEF-deficient mutant (L386R/L387S) resulted in enhanced formation of characteristic cellular protrusions during cell spreading, suggesting that αPIX 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 αPIX wild type or the L386R/L387S mutant restored cell spreading. Together, these data suggest that αPIX is a component of early integrin clusters and plays a dual role in integrin-dependent cell spreading. Whereas αPIX 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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.