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Published online before print December 9, 2004

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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.0504284

Received for publication May 11, 2004.
Revised October 27, 2004.
Accepted for publication October 29, 2004.

Article

Protein kinase C regulates p67phox phosphorylation in human monocytes

Xiaoxian Zhao ^*, Bo Xu ^*, Ashish Bhattacharjee ^*, Claudine M. Oldfield ^*, Frans B. Wientjes , Gerald M. Feldman , and Martha K. Cathcart ^*@

*Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio; Department of Medicine, University College London, United Kingdom; and Division of Monoclonal Antibodies, Office of Therapeutics, Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland

^@ To whom correspondence should be addressed. E-mail: cathcam{at}ccf.org.

	Abstract

Phosphorylation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components p67phox and p47phox accompanies the assembly and activation of this enzyme complex. We have previously reported that activation of human monocytes with opsonized zymosan (ZOP), a potent stimulator of NADPH oxidase activity, results in the phosphorylation of p67phox and p47phox. In this study, we investigated the regulation of p67phox phosphorylation. 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 phosphorylation. Our studies demonstrate that pretreatment of monocytes with antisense oligodeoxyribonucleotides specific for PKC or rottlerin, a selective inhibitor for PKC, inhibited the phosphorylation 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 stimulation of monocytes induces PKC and p67phox to form a complex. We also demonstrate that lysates from activated monocytes as well as PKC immunoprecipitates from activated monocytes can phosphorylate p67phox in vitro and that pretreatment of monocytes with rottlerin 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.

Key Words: PKC • NADPH oxidase • superoxide anion • inflammation

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