Novel mechanism of activation of NADPH oxidase 5. calcium sensitization via phosphorylation
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Published on: October 22, 2012
Davin Jagnandan1, Jarrod E Church, Botond Banfi
1Department of Pharmacology and the Vascular Biology Center, Medical College of Georgia, Augusta, Georgia 30912-2500, USA.
Abstract:
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.
Insights
Phorbol 12-myristate 13-acetate (PMA) enhances the activity of NADPH oxidase 5 (Nox5) by increasing its calcium sensitivity through phosphorylation. This novel mechanism allows Nox5 activation at lower intracellular calcium levels.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- NADPH oxidase 5 (Nox5) activity is calcium-dependent and typically requires high intracellular calcium levels for activation.
- Previous research questioned Nox5's activation in cells with moderate calcium fluctuations.
- Accessory proteins are not required for Nox5 activity, unlike other Nox isoforms.
Purpose of the Study:
- To investigate a novel mechanism regulating Nox5 activity.
- To determine if Nox5 can be activated by stimuli other than extreme calcium elevations.
- To elucidate the role of phorbol 12-myristate 13-acetate (PMA) in Nox5 regulation.
Main Methods:
- Cellular assays measuring reactive oxygen species (ROS) production in response to ionomycin and PMA.
- Cell-free assays to assess the direct effect of PMA on Nox5 calcium sensitivity.
- Site-directed mutagenesis to investigate the role of specific phosphorylation sites (Thr494 and Ser498).
- Cellular localization studies using detergent-resistant microdomains.
Main Results:
- PMA induced a slow, sustained ROS increase, distinct from ionomycin's rapid response.
- PMA potentiated Nox5 activity at low ionomycin concentrations, indicating enhanced calcium sensitivity.
- PMA's effect was dependent on calcium and directly modulated enzyme activity.
- PMA stimulated phosphorylation of Nox5 at Thr494 and Ser498, which was crucial for calcium sensitization.
- Mutating these sites abolished PMA-induced sensitization, while mimicking phosphorylation (glutamic acid substitution) created a gain-of-function mutant.
- Nox5 was localized to detergent-resistant microdomains of the endoplasmic reticulum.
Conclusions:
- Phosphorylation of Nox5 at Thr494 and Ser498 by PMA facilitates enzyme activation at lower intracellular calcium concentrations.
- This phosphorylation-dependent mechanism broadens the range of extracellular stimuli that can activate Nox5.
- Nox5 activation may occur in response to diverse cellular signals beyond just acute calcium spikes.
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