title: Characterization of human neutrophil ecto-protein kinase activity released by kinase substrates.
abstract: Although most studies of protein phosphorylation have focused on intracellular protein kinases, evidence for protein kinase activity on the surface of several types of cells has been described. Evidence was recently provided for the existence of ecto-protein kinase activity on the surface of human neutrophils. Evidence for three distinct ecto-protein kinase activities was detected, one that phosphorylates endogenous surface proteins, one that phosphorylates exogenous substrates in a cAMP-independent manner and is released in the presence of substrate, and a low level of activity of one that phosphorylates exogenous Kemptide in a cAMP-dependent manner. To begin to elucidate its role in neutrophil function, we have characterized several properties of the releasable ecto-protein kinase activity on human neutrophils. This enzyme activity was inhibited by impermeant stilbene disulfonic acids, which are known to alter neutrophil function, as well as by impermeant sulfhydryl reactive agents. Enzyme activity was detectable at physiologic concentrations of Mg2+, but was higher in the presence of Mn2+. Protein kinase activity was strongly inhibited by heparin, whereas trifluoperazine, cAMP, and cGMP had little effect on kinase activity. Protein kinase activity was selectively removed from the cell surface by incubation with the ecto-kinase substrates casein and phosvitin, but the enzyme was not released by phosphatidylinositol-specific phospholipase C. Repeated exposure of neutrophils to substrate depleted ecto-protein kinase activity from the cell surface, but activity was rapidly restored by incubation in buffer lacking substrate. The released protein kinase had a Km for ATP of approximately 0.5 microM and a pH maximum between 7.0 and 7.5. At least four ecto-protein kinase substrates were detected in serum; vitronectin was identified as one of these substrates by immunoprecipitation studies. Although the exact role of ecto-protein kinase activity in neutrophil function remains undefined, the identification of vitronectin as a serum substrate suggests that it interacts with a physiologically important substrate.
authors.affiliation: Department of Medicine, University of Minnesota Medical School, Minneapolis.
authors.forename: K M
authors.initials: KM
authors.lastname: Skubitz
authors.forename: D D
authors.initials: DD
authors.lastname: Ehresmann
authors.forename: T P
authors.initials: TP
authors.lastname: Ducker
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002463:Cell Membrane Permeability; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology* / Q000648:ultrastructure; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010750:Phosphoproteins / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology; D019096:Vitronectin
chemical_list: D002364:Caseins; D002413:Cations, Divalent; D006023:Glycoproteins; D010750:Phosphoproteins; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; D019096:Vitronectin; D014268:Trifluoperazine; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D006493:Heparin; D000242:Cyclic AMP; D011494:Protein Kinases; C034454:ectoprotein kinase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D006152:Cyclic GMP; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid