title: A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium.
abstract: Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src's adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24-48 h PO myocardium. Our studies indicate that c-Src's adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium.
authors.affiliation: Division of Cardiology, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, 29425-2221.
authors.forename: Arun P
authors.initials: AP
authors.lastname: Palanisamy
authors.affiliation: Division of Cardiology, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, 29425-2221.
authors.forename: Geetha
authors.initials: G
authors.lastname: Suryakumar
authors.affiliation: Division of Cardiology, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, 29425-2221.
authors.forename: Kavin
authors.initials: K
authors.lastname: Panneerselvam
authors.affiliation: Division of Cardiology, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, 29425-2221.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Willey
authors.affiliation: Division of Cardiology, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, 29425-2221.
authors.forename: Dhandapani
authors.initials: D
authors.lastname: Kuppuswamy
mesh_terms: D000818:Animals; D062186:Arterial Pressure / Q000235:genetics; D016466:CHO Cells; D000081247:CSK Tyrosine-Protein Kinase; D002415:Cats; D006224:Cricetinae; D003412:Cricetulus; D050719:Crk-Associated Substrate Protein / Q000096:biosynthesis* / Q000235:genetics; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D009842:Oligopeptides / Q000008:administration & dosage; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D050719:Crk-Associated Substrate Protein; D016023:Integrins; D009842:Oligopeptides; D012694:Serine; C047981:arginyl-glycyl-aspartic acid; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human
keywords: Cas; FOCAL ADHESIONS; HYPERTROPHY; INTEGRINS; c-Src