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Characterisation of a kinase involved in the phosphorylation of the p47phox component of the neutrophil NADPH oxidase

Lal, Aroon Steven; (1999) Characterisation of a kinase involved in the phosphorylation of the p47phox component of the neutrophil NADPH oxidase. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

One of the crucial stages in bacterial killing by neutrophil phagocytes is the generation of superoxide (O2-) by the enzyme complex known as the NADPH oxidase. The phosphorylation of one of the cytosolic components, p47phox, is viewed as a crucial step in oxidase activation but, to date, the exact nature of the kinase(s) responsible remains to be elucidated. Studies have shown that activation of neutrophils by formyl-peptides (fMLP) and phorbol esters (PMA) leads to the activation of the extracellular regulated/mitogen activated protein kinase (ERX/MAPK) system. More recently the related p38 MAP kinase pathway has been shown to be activated by both these substances in other cell systems. The role of these pathways in the activation of the NADPH oxidase of human neutrophils, however, has not been elucidated. In this study, a functional p38 MAPK pathway was identified in human neutrophils and its rapid and potent activation upon stimulation of the neutrophil NADPH oxidase by either fMLP or PMA demonstrated. Treatment with SB 203580 (a specific inhibitor of the p38 MAPK pathway) produced complete inhibition of this activation and a partial suppression of the production of O2-. The concentration of SB 203580 that suppressed both activation and O2- production was similar to that which inhibited p38 MAPK in vitro. The effect on O2- production was not due to an inhibition of neutrophil priming, phosphorylation of p47phox, or the translocation of the oxidase components p47phox, p67phox or p21rac to the plasma membrane. Inhibition of the ERK pathway (with the inhibitor PD 098059) did not influence the production of superoxide. Since neither p38 MAPKnor ERK were responsible for the phosphorylation of p47phox, recombinant p47phox was used as substrate to detect other potential kinases. Activity was detected on the membranes of neutrophils activated by either PMA or fMLP with a time course similar to O2- production. The responsible kinase was not intrinsic to the neutrophil membrane and could be eluted with 0.5 M NaCl. The activity was partially purified and was found to be distinct from previously suggested kinases, including PKC isotypes, MAPK and PKB. Gel filtration, renaturation in substrate gels and labelling with FSBA suggested a molecular weight of between 45 and 55 kDa. The kinase activity was independent of calcium and lipids but potently inhibited by staurosporine. Treatment with protein phosphatase 2Ac suggested that the kinase was activated by serine/threonine phosphorylation. Finally, phospho-amino acid analysis and phospho-peptide mapping indicated that the kinase phosphorylated p47phox on similar residues and sites to those found in vivo. These data indicate that although neutrophils possess a functional p38 MAPK pathway it is not directly involved in the phosphorylation of p47phox. It may modulate the NADPH oxidase indirectly, perhaps via effects on the cytoskeleton. It has been shown that activation of neutrophils by PMA results in the activation of a novel membrane associated kinase that is likely to play a direct role in the regulation of the neutrophil NADPH oxidase through its ability to phosphorylate p47phox on biologically relevant sites.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Characterisation of a kinase involved in the phosphorylation of the p47phox component of the neutrophil NADPH oxidase
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Phosphorylation
URI: https://discovery.ucl.ac.uk/id/eprint/10102556
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