A critical role of protein kinase Cδ activation loop phosphorylation in formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of p47 phox and rapid activation of nicotinamide adenine dinucleotide phosphate oxidase

Ni Cheng, Rong He, Jun Tian, Mary C. Dinauer, Richard D. Ye

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Abstract

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 p47phox, 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 PKCδ phosphorylation in its activation loop is rapidly induced by fMLF and is essential for its ability to catalyze p47phox phosphorylation. Using transfected COS-7 cells expressing gp91phox, p22phox, p67phox, and p47phox (COS-phox cells), we found that a functionally active PKCδ is required for p47phox phosphorylation and reconstitution of NADPH oxidase. PKCβII cannot replace PKCδ for this function. Characterization of PKCδ/PKCβII chimeras has led to the identification of the catalytic domain of PKCδ as a target of regulation by fMLF, which induces a biphasic (30 and 180 s) phosphorylation of Thr505 in the activation loop of mouse PKCδ. Mutation of Thr505 to alanine abolishes the ability of PKCδ to catalyze p47phox 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 PKCδ phosphorylation is a novel mechanism for NADPH oxidase activation. The ability to induce PKCδ phosphorylation may distinguish a full agonist from a partial agonist for superoxide production.

Original languageEnglish
Pages (from-to)7720-7728
Number of pages9
JournalJournal of Immunology
Volume179
Issue number11
DOIs
StatePublished - Dec 1 2007

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