Forbes, Louisa Vivien; (1998) Phosphorylation of the p67phox component of the NADPH oxidase in human phagocytes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

p67phox is a component of the NADPH oxidase, the multicomponent enzyme that causes the production of superoxide in phagocytes. The phosphorylation of p67phox upon cellular activation was studied in neutrophils and B lymphocytes. p67phox showed low basal phosphorylation which increased up to three-fold upon stimulation. Phosphopeptide mapping revealed one major tryptic peptide, for p67phox from the cytosol of resting and stimulated cells. The same phosphopeptide was produced by different agonists. In vitro phosphorylation provided insight to the phosphorylation of p67phox in the intact cell. Neutrophil cytosol or mitogen-activated protein (MAP) kinase produced the same phosphopeptide. Identification of the phosphorylation site threonine 233, involved high pressure liquid chromatography-mass spectrometry and site mutagenesis. In vitro phosphorylation of recombinant p67phox was lost by the mutation of threonine 233 to alanine. The structural significance of this site was pursued by attempts at crystallisation, but these were unsuccessful. A series of studies into the mechanism of p67phox phosphorylation were performed. Although a MAP kinase pathway inhibitor (PD098059) reduced the in vitro phosphorylation of p67phox by cytosol, intact cell experiments did not clearly provide evidence for MAP kinase in native p67phox phosphorylation. The inhibitors staurosporine and okadaic acid gave results similar to those for p47phox in that phosphorylation of p67phox correlates with activity, and that dephosphorylation may be involved in deactivation of the NADPH oxidase. Experiments using p47phox-deficient cells showed that p67phox phosphorylation is independent of p47phox and is unlikely to require membrane interaction. Tyrosine phosphorylation was found to occur very weakly but was not associated with the formation of active NADPH oxidase. These results form a foundation of research into the phosphorylation of p67phox The functional importance of this event in the regulation of the NADPH oxidase is the subject of further study. Models are proposed for a structure-function relationship of p67phox phosphorylation within the NADPH oxidase.

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