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A Critical Role of Protein Kinase C Activation Loop Phosphorylation in Formyl-Methionyl-Leucyl-Phenylalanine-Induced Phosphorylation of p47^phox and Rapid Activation of Nicotinamide Adenine Dinucleotide Phosphate Oxidase¹

Ni Cheng^*, Rong He^*, Jun Tian^*, Mary C. Dinauer and Richard D. Ye^2,*

^* Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL 60612; and Herman B. Wells Center for Pediatric Research, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, IN 46020

Generation of superoxide by professional phagocytes is an important mechanism of host defense against bacterial infection. Several protein kinase C (PKC) isoforms have been found to phosphorylate p47^phox, resulting in its membrane translocation and activation of the NADPH oxidase. However, the mechanism by which specific PKC isoforms regulate NADPH oxidase activation remains to be elucidated. In this study, we report that PKC phosphorylation in its activation loop is rapidly induced by fMLF and is essential for its ability to catalyze p47^phox phosphorylation. Using transfected COS-7 cells expressing gp91^phox, p22^phox, p67^phox, and p47^phox (COS-phox cells), we found that a functionally active PKC is required for p47^phox phosphorylation and reconstitution of NADPH oxidase. PKCβII cannot replace PKC for this function. Characterization of PKC/PKCβII chimeras has led to the identification of the catalytic domain of PKC as a target of regulation by fMLF, which induces a biphasic (30 and 180 s) phosphorylation of Thr⁵⁰⁵ in the activation loop of mouse PKC. Mutation of Thr⁵⁰⁵ to alanine abolishes the ability of PKC to catalyze p47^phox phosphorylation in vitro and to reconstitute NADPH oxidase in the transfected COS-phox cells. A correlation between fMLF-induced activation loop phosphorylation and superoxide production is also established in the differentiated PLB-985 human myelomonoblastic cells. We conclude that agonist-induced PKC phosphorylation is a novel mechanism for NADPH oxidase activation. The ability to induce PKC phosphorylation may distinguish a full agonist from a partial agonist for superoxide production.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grants AI033503 and HL077806 (to R.D.Y.) and HL045635 and HL069974 (to M.C.D.) from the National Institutes of Health. R.H. is a recipient of a Scientist Development Grant from the American Heart Association, Greater Midwest Affiliate.

² Address correspondence and reprint requests to Dr. Richard Ye, Department of Pharmacology, College of Medicine, MC868, University of Illinois, 835 South Wolcott Avenue, Chicago, IL 60612. E-mail address: yer{at}uic.edu

³ Abbreviations used in this paper: PI(3,4)P₂, phosphatidylinositol 3,4-bisphosphate; PKC, protein kinase C; PDK-1, 3-phosphoinositol-dependent kinase 1; PAF, platelet-activating factor; phox, phagocyte oxidase; FPR, formyl peptide receptor; HA, hemagglutinin.

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