Modulation of neutrophil activation by okadaic acid, a protein phosphatase inhibitor.
We determined the effects of okadaic acid (OA), a specific inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), on protein phosphorylation and on the activation of the NADPH oxidase in human neutrophils. In otherwise unstimulated cells, OA induced phosphoprotein accumulation, revealing the presence of constitutively active protein kinases. Pulse-chase experiments in electropermeabilized cells confirmed that this effect was due, at least in part, to inhibition of dephosphorylation. OA potentiated phosphoprotein accumulation induced by phorbol esters and by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). In phorbol ester-stimulated cells, OA prolonged the respiratory response after inhibition of protein kinase C (PKC) with staurosporine, consistent with a reduced rate of dephosphorylation of active phosphorylated components. Similarly, OA delayed the inactivation of the burst after displacement of FMLP from its receptor by a competitive antagonist. This suggests that the substrates of the protein kinases activated by FMLP are dephosphorylated by PP1 and/or PP2A. That phosphatases control the intensity and duration of the respiratory response is suggested by the finding that OA magnified and prolonged the oxidative burst elicited by FMLP. In contrast, pretreatment with OA produced a time-dependent inhibition of the phorbol ester-induced respiratory burst. Under conditions where inhibition of the phorbol ester response was nearly complete, activation by the chemoattractant peptide not only persisted but was in fact accentuated. These findings provide strong evidence that receptor-mediated stimulation of the NADPH oxidase can occur by pathways not involving PKC.
Lu
D J
DJ
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Takai
A
A
Leto
T L
TL
Grinstein
S
S
D002463:Cell Membrane Permeability; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006801:Humans; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D019319:Okadaic Acid; D010101:Oxygen Consumption; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011506:Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D004988:Ethers, Cyclic; D011506:Proteins; D019319:Okadaic Acid; D014443:Tyrosine; D010749:Phosphoprotein Phosphatases