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

Reeves, E. P., Dekker, L. V., Forbes, L. V., Wientjes, F. B., Grogan, A., Pappin, D. J., Segal, A. W. (December 1999) Direct interaction between p47phox and protein kinase C: evidence for targeting of protein kinase C by p47phox in neutrophils. Biochemical Journal, 344 Pt. pp. 859-66. ISSN 0264-6021 (Print)0264-6021 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/10585874

Abstract

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.

Item Type: Paper
Additional Information: Reeves, E P Dekker, L V Forbes, L V Wientjes, F B Grogan, A Pappin, D J Segal, A W Wellcome Trust/United Kingdom Research Support, Non-U.S. Gov't England The Biochemical journal Biochem J. 1999 Dec 15;344 Pt 3:859-66.
Uncontrolled Keywords: Animals Brain/metabolism Cell Fractionation Cytosol/metabolism Humans Microfilament Proteins/metabolism NADPH Oxidase Neutrophils/enzymology/ metabolism Phosphopeptides/chemistry Phosphoproteins/ metabolism Phosphorylation Protein Binding Protein Isoforms Protein Kinase C/ metabolism Rats
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression > phosphorylation
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > kinase > Protein kinase C
CSHL Authors:
Communities: CSHL labs > Pappin lab
Depositing User: Kathleen Darby
Date: 15 December 1999
Date Deposited: 23 Apr 2014 15:09
Last Modified: 23 Apr 2014 15:09
PMCID: 1220709
Related URLs:
URI: http://repository.cshl.edu/id/eprint/29855

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