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

Author:

REEVES Emer P.1,DEKKER Lodewijk V.1,FORBES Louisa V.1,WIENTJES Frans B.1,GROGAN Ann1,PAPPIN Darryl J. C.2,SEGAL Anthony W.1

Affiliation:

1. Centre for Molecular Medicine, University College London, 5 University Street, London WC1E 6JJ, U.K.

2. Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, U.K.

Abstract

p47phox is an essential component of the NADPH oxidase, and phosphorylation of p47phox is associated with activation of the enzyme. Here we have used p47phox affinity chromatography to extract a p47phox 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)-βI, -βII and -∆. The C-terminus of p47phox represented the site of interaction with PKC. Co-immunoprecipitation experiments revealed that the interaction between PKC isotypes and p47phox 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 p47phox, we investigated the functional relevance of the interaction between PKC and p47phox. Subcellular fractionation revealed an abnormal recruitment of PKC-βI and -βII, but not PKC-∆, to particulate fractions in p47phox-deficient cells. Phosphorylation of cytosolic proteins was generally increased in stimulated p47phox-deficient neutrophils as compared with normal neutrophils. Furthermore, the cytoskeletal protein coronin was not phosphorylated upon stimulation of p47phox-deficient neutrophils. These findings were confirmed in an in vitro-reconstituted system using rat brain cytosol in which addition of p47phox affected phosphorylation by PKC/PKM (PKM is the catalytic fragment of PKC). These results indicate that p47phox can act as a regulator of PKC in neutrophils.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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