Abstract
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.
Highlights
Nisms involve changes in organization of the actin cytoskeleton
Vav2, an exchange factor for Rac1, Cdc42, and RhoA (14), has been shown to be necessary for integrin-dependent activation of Rac leading to lamellipodia formation in fibroblasts (15), suggesting that guanine nucleotide exchange factor (GEF) are involved in integrin-induced activation of Rho GTPases. ␣PIX/Cool-2/ARHGEF6, an exchange factor for Rac1 and Cdc42 (16, 17), mediates PAK activation upon cell adhesion to fibronectin (18)
In this study we describe a novel interaction between the Rac1/Cdc42-specific guanine nucleotide exchange factor ␣PIX and calpain 4, the small subunit of /m-calpain
Summary
We used the yeast two-hybrid system CytoTrap (Stratagene), designated Sos Recruitment System (36), to identify ␣PIX-interacting proteins. For the CytoTrap screening, the pretransformed cdc25H yeast strain (see above) was transformed with 3 g of pMyr-cDNA library plasmids. Protein interactions of putatively positive colonies were confirmed by retransformation of the cdc25H yeast strain with both the cDNA-containing pMyr plasmid and pSos-␣PIX or empty pSos (negative control). Those clones growing on galactose media at the restrictive temperature of 37 °C after 6 days were defined as “true” positives. In subsequent yeast two-hybrid experiments, different domains of ␣PIX were tested for interaction with calpain 4, using various bait constructs (see “Plasmid Constructs”) in combination with plasmid pMyr-calpain 4 as prey. GenBank accession numbers are AF207831 and D25304 for the ␣PIX sequence and NM_001749 for calpain 4
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