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)-beta(I), -beta(II) and -delta. 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-beta and -delta 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-beta(I) and -beta(II), but not PKC-delta, 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.
Reeves
E P
EP
Centre for Molecular Medicine, University College London, 5 University Street, London WC1E 6JJ, U.K.
Dekker
L V
LV
Forbes
L V
LV
Wientjes
F B
FB
Grogan
A
A
Pappin
D J
DJ
Segal
A W
AW
D000818:Animals; D001921:Brain / Q000378:metabolism; D002458:Cell Fractionation; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats
D008840:Microfilament Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D020033:Protein Isoforms; C072061:coronin proteins; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D011493:Protein Kinase C