title: AlphaPIX 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 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.
authors.affiliation: Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, D-22529 Hamburg, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Rosenberger
authors.forename: Andreas
authors.initials: A
authors.lastname: Gal
authors.forename: Kerstin
authors.initials: K
authors.lastname: Kutsche
mesh_terms: D019943:Amino Acid Substitution; D002154:Calpain / Q000378:metabolism* / Q000502:physiology; D002448:Cell Adhesion; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D048430:Cell Shape; D022081:Cell Surface Extensions; D003599:Cytoskeleton / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D016023:Integrins / Q000502:physiology*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D021122:Protein Subunits; D064067:Rho Guanine Nucleotide Exchange Factors
chemical_list: C576603:ARHGEF6 protein, human; D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D016023:Integrins; D021122:Protein Subunits; D064067:Rho Guanine Nucleotide Exchange Factors; D002154:Calpain