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Novel mechanism of activation of NADPH oxidase 5. calcium sensitization via phosphorylation.
J Biol Chem 2007; 282(9):6494-507JB

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.

Authors+Show Affiliations

Department of Pharmacology and the Vascular Biology Center, Medical College of Georgia, Augusta, Georgia 30912-2500, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

17164239

Citation

Jagnandan, Davin, et al. "Novel Mechanism of Activation of NADPH Oxidase 5. Calcium Sensitization Via Phosphorylation." The Journal of Biological Chemistry, vol. 282, no. 9, 2007, pp. 6494-507.
Jagnandan D, Church JE, Banfi B, et al. Novel mechanism of activation of NADPH oxidase 5. calcium sensitization via phosphorylation. J Biol Chem. 2007;282(9):6494-507.
Jagnandan, D., Church, J. E., Banfi, B., Stuehr, D. J., Marrero, M. B., & Fulton, D. J. (2007). Novel mechanism of activation of NADPH oxidase 5. calcium sensitization via phosphorylation. The Journal of Biological Chemistry, 282(9), pp. 6494-507.
Jagnandan D, et al. Novel Mechanism of Activation of NADPH Oxidase 5. Calcium Sensitization Via Phosphorylation. J Biol Chem. 2007 Mar 2;282(9):6494-507. PubMed PMID: 17164239.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Novel mechanism of activation of NADPH oxidase 5. calcium sensitization via phosphorylation. AU - Jagnandan,Davin, AU - Church,Jarrod E, AU - Banfi,Botond, AU - Stuehr,Dennis J, AU - Marrero,Mario B, AU - Fulton,David J R, Y1 - 2006/12/12/ PY - 2006/12/14/pubmed PY - 2007/6/19/medline PY - 2006/12/14/entrez SP - 6494 EP - 507 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 282 IS - 9 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/17164239/Novel_mechanism_of_activation_of_NADPH_oxidase_5__calcium_sensitization_via_phosphorylation_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=17164239 DB - PRIME DP - Unbound Medicine ER -