Protein kinase C regulates p67phox phosphorylation in human monocytes

Abstract: Phosphorylation of the reduced nicotin-amide adenine dinucleotide phosphate (NADPH) oxidase components p67phox and p47phox ac-companies the assembly and activation of this enzyme complex. We have previously reported that activation of human monocytes with opso-nized zymosan (ZOP), a potent stimulator of NADPH oxidase activity, results in the phosphor-ylation of p67phox and p47phox. In this study, we investigated the regulation of p67phox phos-phorylation. Although protein kinase C (PKC) has previously been shown to regulate NADPH oxidase activity, we found that inhibition of PKC had no effect on p67phox phosphoryla-tion. Our studies demonstrate that pretreatment of monocytes with antisense oligodeoxyribo-nucleotides specific for PKC or rottlerin, a se-lective inhibitor for PKC, inhibited the phos-phorylation of p67phox in monocytes, and Go6976, a specific inhibitor for conventional PKCs, PKC and PKC, had no such inhibitory effect. Additional studies indicate that ZOP stim-ulation of monocytes induces PKC and p67phox to form a complex. We also demon-strate that lysates from activated monocytes as well as PKC immunoprecipitates from activated monocytes can phosphorylate p67phox in vitro and that pretreatment of monocytes with rot-tlerin blocked the phosphorylation in each case. We further show that recombinant PKC can phosphorylate p67phox in vitro. Finally, we show that PKC-deficient monocytes produce significantly less superoxide anion in response to ZOP stimulation, thus emphasizing the functional significance of the PKC regulation of p67phox phosphorylation. Taken together, this is the first report to describe the requirement of PKC in regulating the phosphorylation of p67phox and the related NADPH oxidase activity in primary human monocytes. J. Leukoc. Biol. 77