Jagnandan Davin, Church Jarrod E, Banfi Botond, Stuehr Dennis J, Marrero Mario B, Fulton David J R
Department of Pharmacology and the Vascular Biology Center, Medical College of Georgia, Augusta, Georgia 30912-2500, USA.
J Biol Chem. 2007 Mar 2;282(9):6494-507. doi: 10.1074/jbc.M608966200. Epub 2006 Dec 12.
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.
与其他Nox亚型不同，Nox5的活性不需要辅助蛋白的存在，完全依赖于细胞内钙的升高。先前的研究表明，Nox5对钙的半数有效浓度（EC50）相对较高，这就提出了一个问题，即在细胞内钙没有极端升高的情况下，Nox5是否能被充分激活。在本研究中，我们发现了一种控制Nox5活性的新机制。将表达Nox5的细胞暴露于佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）中会导致活性氧（ROS）缓慢而持续地增加，这与对离子霉素的快速反应明显不同。PMA极大地增强了Nox5对低浓度离子霉素的反应活性。钙螯合消除了PMA增加Nox5活性的能力，这是对酶活性的直接影响，因为在无细胞测定中PMA增加了Nox5对钙的敏感性。PMA刺激Nox5在苏氨酸（Thr）494和丝氨酸（Ser）498位点的时间依赖性磷酸化。将这些残基突变为丙氨酸消除了PMA依赖性磷酸化和钙敏化。相反，将Thr494和Ser498突变为谷氨酸产生了一个功能获得性突变体，该突变体在低浓度离子霉素下具有增强的活性。在细胞内，在内质网的耐去污剂微区中检测到了Nox5。总之，Nox5关键残基的磷酸化有助于在较低水平的细胞内钙时激活酶，并可能为响应更多种类的细胞外刺激而激活酶提供一条途径。
