title: Thrombin-stimulated Pyk2 phosphorylation in human endothelium is dependent on intracellular calcium and independent of protein kinase C and Src kinases.
abstract: G-protein-coupled receptor agonists (GPCAs) cause functional responses in endothelial cells including secretion, proliferation, and altering monolayer permeability. These events are mediated in part by activation of the p42/44 mitogen-activated protein kinase (MAPK) cascade. The cytosolic tyrosine kinase Pyk2 is postulated to link GPCA-induced changes in intracellular calcium to activation of the MAP kinase cascade. We have investigated the regulation of Pyk2 in human umbilical vein endothelial cells in response to GPCAs and show that (1) thrombin, a PAR-1 peptide, and histamine cause rapid concentration- and time-dependent phosphorylation on tyrosines 402 (Src kinase binding site), 881 (Grb2 binding site), and 580 (an autophosphorylation site), (2) thrombin-stimulated phosphorylation is dependent on intracellular calcium and independent of PKC and PI-3 kinase, and (3) inhibition of Src kinases has no significant effect on thrombin-stimulated phosphorylation, implying that tyrosine phosphorylation of Pyk2 is independent of Src binding.
authors.affiliation: Royal Veterinary College, Department of Veterinary Basic Sciences, Royal College Street, London NW1 0TU, UK.
authors.forename: Rosemary J
authors.initials: RJ
authors.lastname: Keogh
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Houliston
authors.forename: Caroline P D
authors.initials: CP
authors.lastname: Wheeler-Jones
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology*; D051418:Focal Adhesion Kinase 2; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology; D011494:Protein Kinases / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D019061:src-Family Kinases / Q000502:physiology
chemical_list: D002614:Chelating Agents; D010446:Peptide Fragments; C072032:thrombin receptor peptide (42-55); D019000:Phosphotyrosine; D006632:Histamine; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D019061:src-Family Kinases; D011493:Protein Kinase C; D013917:Thrombin; D002118:Calcium