The 47-kDa protein involved in the NADPH:O2 oxidoreductase activity of human neutrophils is phosphorylated by cyclic AMP-dependent protein kinase without induction of a respiratory burst.
When human neutrophilic granulocytes are stimulated with chemoattractants or phorbol esters, these cells respond with a so-called respiratory burst: such stimuli induce the activation of a NADPH:O2 oxidoreductase, which converts oxygen into superoxide. This activation coincides with the phosphorylation of a number of proteins, amongst which a 47-kDa phosphoprotein. Neutrophils from patients with the autosomal form of chronic granulomatous disease (CGD) fail to mount a respiratory burst and concomitantly lack phosphorylation of the 47-kDa protein. We have shown this protein to be a substrate for protein kinase C. In the present paper we describe the phosphorylation of the 47-kDa phosphoprotein by cyclic AMP-dependent protein kinase. For these studies, we used neutrophil cytoplasts, i.e., neutrophils devoid of nucleus and granules, but with an intact NADPH:O2 oxidoreductase. Addition of dibutyryl cyclic AMP (Bt2cAMP) to intact human neutrophil cytoplasts resulted in an increase in protein phosphorylation. Among the phosphorylated proteins is a 47-kDa phosphoprotein. Increased protein phosphorylation was also observed upon addition of Bt2cAMP to neutrophil cytoplast lysates. In lysates of neutrophil cytoplasts from patients with the autosomal form of CGD, phosphorylation of the 47-kDa protein was absent. This finding (confirmed by analysis on two-dimensional gels) indicates that the 47-kDa phosphoprotein, relevant for the NADPH:O2 oxidoreductase, is a substrate for the cAMP-dependent protein kinase. Unlike phorbol ester-induced phosphorylation, Bt2cAMP-induced phosphorylation is not accompanied by initiation of a respiratory burst. This observation demonstrates that 47-kDa phosphoprotein phosphorylation can be uncoupled from respiratory burst activity and indicates that other modifications of the NADPH:O2 oxidoreductase are required for induction of activity.
Kramer
I M
IM
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
van der Bend
R L
RL
Verhoeven
A J
AJ
Roos
D
D
D003994:Bucladesine / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006105:Granulomatous Disease, Chronic / Q000201:enzymology*; D006801:Humans; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010101:Oxygen Consumption / Q000187:drug effects; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000097:blood*
D010750:Phosphoproteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003994:Bucladesine; D000242:Cyclic AMP; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme; D011494:Protein Kinases