Effects of antagonists of protein phosphatases on superoxide release by neutrophils.
Neutrophils stimulated with 4 beta-phorbol 12-myristate 13-acetate (PMA) release large quantities of superoxide (O2-) and exhibit phosphorylation of two proteins with molecular masses of 47(p47) and 49 kDa (p49). Addition of inhibitors of protein kinases (e.g. 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) to these cells after stimulation with PMA results in the loss of 32P from these proteins and a rapid cessation of O2- release (e.g. Heyworth, P. G., and Badwey, J. A. (1990) Biochim. Biophys. Acta 1052, 299-305). In this paper we report that antagonists of type 1 and 2A protein phosphatases (okadaic acid, calyculin A) prevented both the loss of 32P from p47 and the termination of O2- release in stimulated neutrophils treated with H-7. Calyculin A also caused a remarkable hyperphosphorylation of a number of proteins in neutrophils and increased O2- release from these cells in response to a suboptimal amount of PMA. Enzymes present in both the soluble and particulate fractions of neutrophils catalyzed the near complete dephosphorylation of 32P-labeled p47 and p49 bound to Immobilon-P membranes. Dephosphorylation of these blotted phosphoproteins occurred at physiological rates and was inhibited by okadaic acid and calyculin A. These data strongly suggest that p47 undergoes a continual cycle of phosphorylation and dephosphorylation throughout the period of O2- release when PMA is the stimulus. Moreover, we show that antagonists of type 1 and 2A protein phosphatases block dephosphorylation of p47 both in vivo and in vitro, indicating that these enzymes may modulate O2- release under certain circumstances.
Ding
J
J
Department of Cell and Molecular Biology, Boston Biomedical Research Institute, Massachusetts 02114.
Badwey
J A
JA
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006168:Guinea Pigs; D007546:Isoquinolines / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008387:Marine Toxins; D008954:Models, Biological; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology*; D010710:Phosphates / Q000097:blood; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010761:Phosphorus Radioisotopes; D010879:Piperazines / Q000494:pharmacology; D013481:Superoxides / Q000097:blood*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D004988:Ethers, Cyclic; D007546:Isoquinolines; D008387:Marine Toxins; D010080:Oxazoles; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D010879:Piperazines; D013481:Superoxides; D019319:Okadaic Acid; C059041:calyculin A; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D010749:Phosphoprotein Phosphatases; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium