Participation of cytosolic protein phosphatase in regulation of NADPH oxidase in polymorphonuclear leukocytes.
Calyculin A, a protein phosphatase inhibitor, enhanced phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2-) production and translocation of the cytosolic NADPH oxidase factor, p47phox, to the plasma membrane in guinea pig polymorphonuclear leukocytes (PMNs). When PMNs were treated with t-(5-isoquino-line-sulfonyl)-3-methyl-piperazine (H-7), a protein kinase C (PKC) inhibitor, after exposure to PMA, inhibition of O2- production and of translocation of p47phox to the membrane fraction in PMA-stimulated PMNs were observed. When calyculin A was added to the PMA-stimulated PMNs after the addition of H-7, O2- production was again observed, and translocation of p47phox to the membrane fraction also occurred. The activity of NADPH oxidase, the amount of p47phox and the level of phosphorylation of p47phox in the membrane fraction prepared from PMA-stimulated PMNs, were reduced by the addition of the cytosol fraction from unstimulated PMNs. These reductions were attenuated by calyculin A. These results indicate that the active form of NADPH oxidase in PMNs can be reconstituted after the active complex of the enzyme has disappeared once, and that one of the mechanisms of regulation of this enzyme activity involves the phosphorylation of p47phox in the cyotosol and dephosphorylation of phosphorylated p47phox in the NADPH oxidase complex by protein kinase and protein phosphatase, respectively.
Kawakami
N
N
Department of Environmental Biochemistry, Kyoto Pharmaceutical University, Japan.
Takemasa
H
H
Okamura
N
N
Hayakawa
T
T
Shimohama
S
S
Fujimoto
S
S
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D005260:Female; D006168:Guinea Pigs; D008387:Marine Toxins; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010750:Phosphoproteins / Q000494:pharmacology; D010766:Phosphorylation; D011817:Rabbits; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D008387:Marine Toxins; D010080:Oxazoles; D010750:Phosphoproteins; D013481:Superoxides; C059041:calyculin A; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D010749:Phosphoprotein Phosphatases; D013755:Tetradecanoylphorbol Acetate