title: Agonist-dependent phosphorylation of the G protein-coupled receptor kinase 2 (GRK2) by Src tyrosine kinase.
abstract: GRK2 is a member of the G protein-coupled receptor kinase (GRK) family, which phosphorylates the activated form of a variety of G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. It has been recently reported that stimulation of the mitogen-activated protein kinase cascade by GPCRs involves tyrosine phosphorylation of docking proteins mediated by members of the Src tyrosine kinase family. In this report, we have investigated the possible role of c-Src in modulating GRK2 function. We demonstrate that c-Src can directly phosphorylate GRK2 on tyrosine residues, as shown by in vitro experiments with purified proteins. The phosphorylation reaction exhibits an apparent K(m) for GRK2 of 12 nM, thus suggesting a physiological relevance in living cells. Consistently, overexpression of the constitutively active c-Src Y527F mutant in COS-7 cells leads to tyrosine phosphorylation of co-expressed GRK2. In addition, GRK2 can be detected in phosphotyrosine immunoprecipitates from HEK-293 cells transiently transfected with this Src mutant. Interestingly, phosphotyrosine immunoblots reveal a rapid and transient increase in GRK2 phosphorylation upon agonist stimulation of beta(2)-adrenergic receptors co-transfected with GRK2 and wild type c-Src in COS-7 cells. This tyrosine phosphorylation is maximal within 5 min of isoproterenol stimulation and reaches values of approximately 5-fold over basal conditions. Furthermore, GRK2 phosphorylation on tyrosine residues promotes an increased kinase activity toward its substrates. Our results suggest that GRK2 phosphorylation by c-Src is inherent to GPCR activation and put forward a new mechanism for the regulation of GPCR signaling.
authors.affiliation: Departamento de Biología Molecular, Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid) Universidad Autónoma, E-28049 Madrid, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Sarnago
authors.forename: A
authors.initials: A
authors.lastname: Elorza
authors.forename: F
authors.initials: F
authors.lastname: Mayor
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology*; D019169:Jurkat Cells; D007700:Kinetics; D010766:Phosphorylation / Q000187:drug effects; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000318:Adrenergic beta-Agonists; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D019061:src-Family Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D007545:Isoproterenol