Direct interaction between p47phox and protein kinase C: evidence for targeting of protein kinase C by p47phox in neutrophils

p47^phox is an essential component of the NADPH oxidase, and phosphorylation of p47^phox is associated with activation of the enzyme. Here we have used p47^phox affinity chromatography to extract a p47^phox kinase from neutrophil cytosol. The kinase activity was purified by gel filtration and Mini Q chromatography and shown to be indistinguishable from the catalytic fragments of protein kinase C (PKC)-β_I, -β_II and -∆. The C-terminus of p47^phox represented the site of interaction with PKC. Co-immunoprecipitation experiments revealed that the interaction between PKC isotypes and p47^phox takes place in intact cells. However PKC-β and -∆ showed different time courses of co-immunoprecipitation, suggesting that the interactions may serve different functions for the various PKC isotypes. Using cells lacking p47^phox, we investigated the functional relevance of the interaction between PKC and p47^phox. Subcellular fractionation revealed an abnormal recruitment of PKC-β_I and -β_II, but not PKC-∆, to particulate fractions in p47^phox-deficient cells. Phosphorylation of cytosolic proteins was generally increased in stimulated p47^phox-deficient neutrophils as compared with normal neutrophils. Furthermore, the cytoskeletal protein coronin was not phosphorylated upon stimulation of p47^phox-deficient neutrophils. These findings were confirmed in an in vitro-reconstituted system using rat brain cytosol in which addition of p47^phox affected phosphorylation by PKC/PKM (PKM is the catalytic fragment of PKC). These results indicate that p47^phox can act as a regulator of PKC in neutrophils.