title: Immune complex-induced integrin activation and L-plastin phosphorylation require protein kinase A.
abstract: Integrins in resting leukocytes are poorly adhesive, and cell activation is required to induce integrin-mediated adhesion. We recently demonstrated a close correlation between phosphorylation of Ser(5) in L-plastin (LPL), a leukocyte-specific 67-kDa actin bundling protein, and activation of alpha(M)beta(2)-mediated adhesion in polymorphonuclear neutrophils (PMN) (Jones, S. L., Wang, J., Turck, C. W., and Brown, E. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9331-9336). However, the kinase that phosphorylates LPL Ser(5) has not been identified. We found that cAMP-dependent protein kinase (PKA), but not a variety of other serine kinases, can specifically phosphorylate LPL and LPL-derived peptides on Ser(5) in vitro. The cell-permeable cAMP analog 8-bromo-cAMP and the adenylate cyclase activator forskolin both induce LPL phosphorylation in cells. Two PKA inhibitors, H89 and KT5720, inhibited immune complex (IC)-stimulated LPL phosphorylation as well as IC-induced activation of alpha(M)beta(2)-mediated adhesion in PMN. The dose response of H89 inhibition of PMN adhesion correlated with its inhibition of LPL phosphorylation in response to IC. IC stimulation also transiently increased intracellular cAMP concentration in PMN. Thus, PKA functions in an integrin activation pathway initiated by IC binding to Fcgamma receptors in addition to its better known role as a negative regulator of cell activation by G protein-coupled receptors. In contrast, LPL Ser(5) phosphorylation and PMN adhesion induced by formylmethionyl-leucylphenylalanine or phorbol myristate acetate were not affected by PKA inhibitors, suggesting that a different kinase(s) is responsible for LPL phosphorylation in response to these agonists. Phosphoinositidyl 3-kinase also is required for FcgammaR but not formylmethionyl-leucylphenylalanine- or phorbol myristate acetate-induced LPL phosphorylation and activation of alpha(M)beta(2). Two phosphoinositidyl 3-kinase inhibitors blocked FcgammaR-induced cAMP accumulation, demonstrating that this kinase acts upstream of PKA. These data demonstrate a necessary role for PKA in IC-induced integrin activation and LPL phosphorylation.
authors.affiliation: Division of Infectious Diseases and Program in Molecular Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: E J
authors.initials: EJ
authors.lastname: Brown
mesh_terms: D000595:Amino Acid Sequence; D000936:Antigen-Antibody Complex / Q000502:physiology*; D002227:Carbazoles; D002448:Cell Adhesion; D000242:Cyclic AMP / Q000096:biosynthesis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D007211:Indoles / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D016177:Macrophage-1 Antigen / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011758:Pyrroles / Q000494:pharmacology; D017452:Receptors, IgG / Q000502:physiology; D012694:Serine / Q000378:metabolism; D013449:Sulfonamides; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000936:Antigen-Antibody Complex; D002227:Carbazoles; D007211:Indoles; D007546:Isoquinolines; D016177:Macrophage-1 Antigen; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011758:Pyrroles; D017452:Receptors, IgG; D013449:Sulfonamides; C026742:plastin; D012694:Serine; C057416:KT 5720; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D013755:Tetradecanoylphorbol Acetate