Neutrophils stimulated with a chemotactic peptide or a phorbol ester exhibit different alterations in the activities of a battery of protein kinases.
Protein kinases in neutrophils that undergo changes in activity during cell stimulation have been investigated by two recently described procedures. These methods are based on the ability of renatured kinases to undergo autophosphorylation or to phosphorylate protein substrates fixed in gels. Using these techniques, we report that neutrophils contain a battery of protein kinases with molecular masses of 65-61 kDa (termed "group A kinases") that are rapidly activated upon stimulation of the cells with the chemotactic peptide N-fMet-Leu-Phe (fMLP). Activity was maximal within 30 s with this stimulus and returned to the basal level seen in unstimulated cells within 3 min. In contrast, stimulation of neutrophils with 4 beta-phorbol 12-myristate 13-acetate resulted in a diminution of these kinase activities. Treatment of neutrophils with antagonists of type 1 and 2A protein phosphatases (calyculin A, okadaic acid) inhibited the activation of the group A kinases by fMLP, whereas norokadanone, an analog of okadaic acid that is a poor inhibitor of protein phosphatases, had no effect. Exposure of neutrophils to calyculin A alone resulted in the activation of several additional protein kinases with molecular masses different from the group A kinases. These data indicate that the signal transduction pathways of neutrophils are likely to be far more complicated than previously appreciated and involve a number of uncharacterized protein kinases.
Department of Cell and Molecular Biology, Boston Biomedical Research Institute, Massachusetts 02114.
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Ding
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JA
Badwey
D000818:Animals; D004988:Ethers, Cyclic / Q000494:pharmacology; D006168:Guinea Pigs; D008387:Marine Toxins; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004988:Ethers, Cyclic; D008387:Marine Toxins; D010080:Oxazoles; D047428:Protein Kinase Inhibitors; D019319:Okadaic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; C059041:calyculin A; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D013755:Tetradecanoylphorbol Acetate