Inhibition of neutrophil NADPH oxidase assembly by a myristoylated pseudosubstrate of protein kinase C.
To further define the role played by protein kinase C (PKC) in the activation of the neutrophil NADPH oxidase, we have utilized a pseudosubstrate of PKC which was myristoylated at the N terminus. In electropermeabilized neutrophils, the myristoylated pseudosubstrate Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (myr-psi PKC) inhibited PMA-induced protein phosphorylations and activation of the NADPH oxidase, induced either by PMA or by the receptor agonist formyl-methionyl-leucyl-phenylalanine. Both the pseudosubstrate lacking the N-terminal myristate (psi PKC) and a myristoylated control peptide (Phe-Ala-Glu-Asp-Gly-Ala-Leu-Glu-Gln, myr-CP) were without effect on these responses. The myristoylated pseudosubstrate was also tested in a cell-free system, in which NADPH oxidase activation can be achieved by addition of SDS and guanosine 5'-3-O-(thio)triphosphate in a staurosporine-insensitive manner. Myr-psi PKC, but not psi PKC or myr-CP, proved to be a potent inhibitor of NADPH oxidase activity in the cell-free system, indicating that the inhibition observed in permeabilized neutrophils may have been caused by an effect other than PKC inhibition. In the presence of myr-psi PKC, translocation in the cell-free system of the cytosolic oxidase components p47-phox and p67-phox to the plasma membrane was inhibited. From these results we conclude that myristoylation profoundly increases the ability of pseudosubstrates of PKC to inhibit not only PKC-mediated phosphorylations, but also NADPH oxidase activation. The latter effect, however, is most probably not related to PKC inhibition but may indicate a critical role of the membrane surface charge in the translocation of the cytosolic oxidase components p47-phox and p67-phox.
Verhoeven
A J
AJ
Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Leusen
J H
JH
Kessels
G C
GC
Hilarius
P M
PM
de Bont
D B
DB
Liskamp
R M
RM
D000595:Amino Acid Sequence; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D004789:Enzyme Activation / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009226:Myristates / Q000737:chemistry / Q000494:pharmacology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors*; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D009226:Myristates; D009842:Oligopeptides; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D012967:Sodium Dodecyl Sulfate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate