Direct interaction between p47(phox) and protein kinase C: evidence for targeting of protein kinase C by p47(phox) in neutrophils

p47(phox) is 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 neutr ophil cytosol. The kinase activity was purified by gelfiltration and Mini Q chromatography and shown to be indistinguishable from the catalytic fragme nts of protein kinase C (PKC)-beta(I), -beta(II), and -delta. The C-terminu s of p47(phox) represented the site of interaction with PKC. Coimmunoprecip itation experiments revealed that the interaction between PKC isotypes and p47(phox) takes place in intact cells. However PKC-beta and -delta showed d ifferent time courses of coimmunoprecipitation, suggesting that the interac tions may serve different functions for the various PKC isotypes. Using cel ls lacking p47(phox), we investigated the functional relevance of the inter action between PKC and p47(phox). Subcellular fractionation revealed an abn ormal recruitment of PKC-beta(I) and -beta(II), but not PKC-delta, to parti culate fractions in p47(phox)-deficient cells. Phosphorylation of cytosolic proteins was generally increased in stimulated p47(phox)-deficient neutrop hils as compared with normal neutrophils. Furthermore, the cytoskeletal pro tein coronin was not phosphorylated upon stimulation of p47(phox)-deficient neutrophils. These findings were confirmed in an in vitro-reconstituted sy stem using rat brain cytosol in which addition of p47(phox) affected phosph orylation 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.