Novel mechanism of activation of NADPH oxidase 5. calcium sensitization via phosphorylation.
In contrast to other Nox isoforms, the activity of Nox5 does not require the presence of accessory proteins and is entirely dependent on the elevation of intracellular calcium. Previous studies have shown that the EC(50) of Nox5 for calcium is relatively high and raises the question of whether Nox5 can be sufficiently activated in cells that do not experience extreme elevations of intracellular calcium. In the current study, we have identified a novel mechanism governing the activity of Nox5. Exposure of cells expressing Nox5 to phorbol 12-myristate 13-acetate (PMA) resulted in a slow and sustained increase in ROS, which was markedly different from the rapid response to ionomycin. PMA greatly potentiated the activity of Nox5 in response to low concentrations of ionomycin. The ability of PMA to increase Nox5 activity was abolished by calcium chelation and was a direct effect on enzyme activity, since PMA increased the calcium sensitivity of Nox5 in a cell-free assay. PMA stimulated the time-dependent phosphorylation of Nox5 on Thr(494) and Ser(498). Mutation of these residues to alanine abolished both PMA-dependent phosphorylation and calcium sensitization. Conversely, mutation of Thr(494) and Ser(498) to glutamic acid produced a gain of function mutant that had increased activity at low concentrations of ionomycin. Within the cell, Nox5 was detected in detergent-resistant microdomains of the endoplasmic reticulum. In summary, the phosphorylation of Nox5 at key residues facilitates enzyme activation at lower levels of intracellular calcium and may provide an avenue for enzyme activation in response to a greater variety of extracellular stimuli.
Jagnandan
Davin
D
Department of Pharmacology and the Vascular Biology Center, Medical College of Georgia, Augusta, Georgia 30912-2500, USA.
Church
Jarrod E
JE
Banfi
Botond
B
Stuehr
Dennis J
DJ
Marrero
Mario B
MB
Fulton
David J R
DJ
D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000032:analysis*; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D008565:Membrane Proteins; D017382:Reactive Oxygen Species; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases; C431571:NOX5 protein, human; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium