The Journal of Immunology, Vol 147, Issue 2 638-650, Copyright © 1991 by American Association of Immunologists

ARTICLES

Characterization of human neutrophil ecto-protein kinase activity released by kinase substrates

KM Skubitz, DD Ehresmann and TP Ducker
Department of Medicine, University of Minnesota Medical School, Minneapolis.

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.

This article has been cited by other articles:

				A.-C. Schlaeppi, T. Malherbe, and P. Butikofer Coordinate Expression of GPEET Procyclin and Its Membrane-associated Kinase in Trypanosoma brucei Procyclic Forms J. Biol. Chem., December 12, 2003; 278(50): 49980 - 49987. [Abstract] [Full Text] [PDF]

				A. Babinska, M. V. Hogan, T. Sobocki, M. B. Sobocka, Y. H. Ehrlich, and E. Kornecki Identification of ecto-PKC on surface of human platelets: role in maintenance of latent fibrinogen receptors Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H2008 - H2019. [Abstract] [Full Text] [PDF]

				D. Seger, Z. Gechtman, and S. Shaltiel Phosphorylation of Vitronectin by Casein Kinase II. IDENTIFICATION OF THE SITES AND THEIR PROMOTION OF CELL ADHESION AND SPREADING J. Biol. Chem., September 18, 1998; 273(38): 24805 - 24813. [Abstract] [Full Text] [PDF]

				V. Nickel, S. Prehm, M. Lansing, A. Mausolf, A. Podbielski, J. Deutscher, and P. Prehm An Ectoprotein Kinase of Group C Streptococci Binds Hyaluronan and Regulates Capsule Formation J. Biol. Chem., September 11, 1998; 273(37): 23668 - 23673. [Abstract] [Full Text] [PDF]

				N. Sacerdoti-Sierra and C. L. Jaffe Release of Ecto-protein Kinases by the Protozoan Parasite Leishmania major J. Biol. Chem., December 5, 1997; 272(49): 30760 - 30765. [Abstract] [Full Text] [PDF]

				J. Walter, M. Schnölzer, W. Pyerin, V. Kinzel, and D. Kübler Induced Release of Cell Surface Protein Kinase Yields CK1- and CK2-like Enzymes in Tandem J. Biol. Chem., January 5, 1996; 271(1): 111 - 119. [Abstract] [Full Text] [PDF]

				J. Walter, A. Schindzielorz, B. Hartung, and C. Haass Phosphorylation of the beta -Amyloid Precursor Protein at the Cell Surface by Ectocasein Kinases 1 and 2 J. Biol. Chem., July 28, 2000; 275(31): 23523 - 23529. [Abstract] [Full Text] [PDF]

				D. Seger, R. Seger, and S. Shaltiel The CK2 Phosphorylation of Vitronectin. PROMOTION OF CELL ADHESION VIA THE alpha vbeta 3-PHOSPHATIDYLINOSITOL 3-KINASE PATHWAY J. Biol. Chem., May 11, 2001; 276(20): 16998 - 17006. [Abstract] [Full Text] [PDF]