Rabani Razieh, Cossette Chantal, Graham François, Powell William S
Meakins-Christie Laboratories, Centre for Translational Biology, Research Institute of the McGill University Health Centre, 1001 Decarie Blvd, Montreal, QC H4A 3J1, Canada.
Meakins-Christie Laboratories, Centre for Translational Biology, Research Institute of the McGill University Health Centre, 1001 Decarie Blvd, Montreal, QC H4A 3J1, Canada.
Free Radic Biol Med. 2020 Dec;161:50-59. doi: 10.1016/j.freeradbiomed.2020.09.022. Epub 2020 Oct 2.
NAD kinase (NADK) is required for the de novo synthesis of NADP from NAD. In neutrophils, NADK plays an essential role by providing sufficient levels of NADPH to support a robust oxidative burst. Activation of NADPH oxidase-2 (NOX-2) in neutrophils by stimulators of protein kinase C (PKC), such as phorbol myristate acetate (PMA), results in the rapid generation of superoxide at the expense of oxidation of NADPH to NADP. In this study, we measured the levels of pyridine nucleotides following the addition of PMA to neutrophils. PMA elicited a rapid increase in NADP in neutrophils, which was not due to oxidation of NADPH, the levels of which also rose. This was mirrored by a rapid reduction in NAD levels, suggesting that NADK had been activated. PMA-induced depletion of NAD in neutrophils was blocked by PKC inhibitors, but was not dependent on NOX-2, as it was not blocked by the NOX inhibitor, diphenyleneiodonium. PMA also increased NADK activity in neutrophil lysates as well as NADK phosphorylation, as revealed by a monoclonal antibody selective for phospho-NADK. Human recombinant NADK was phosphorylated by PKCδ, resulting in increased immunoreactivity, but unchanged enzyme activity, suggesting that PKC-induced phosphorylation alone is insufficient to increase NADK activity in neutrophils. This leads us to speculate that phosphorylation of NADK promotes the dissociation of an inhibitory molecule from a complex, thereby increasing enzyme activity. Activation of NADK by PKC in phagocytic cells could be critical for the rapid provision of sufficient levels of superoxide for host defence against invading microorganisms.
NAD激酶（NADK）是从NAD从头合成NADP所必需的。在中性粒细胞中，NADK通过提供足够水平的NADPH来支持强大的氧化爆发发挥着至关重要的作用。蛋白激酶C（PKC）的刺激物，如佛波酯肉豆蔻酸酯乙酸酯（PMA），激活中性粒细胞中的NADPH氧化酶-2（NOX-2），导致以NADPH氧化为NADP为代价快速产生超氧化物。在本研究中，我们测量了向中性粒细胞中添加PMA后吡啶核苷酸的水平。PMA引起中性粒细胞中NADP的快速增加，这不是由于NADPH的氧化，NADPH的水平也升高了。这反映在NAD水平的快速降低上，表明NADK已被激活。PKC抑制剂可阻断PMA诱导的中性粒细胞中NAD的消耗，但不依赖于NOX-2，因为它不受NOX抑制剂二苯基碘鎓的阻断。PMA还增加了中性粒细胞裂解物中的NADK活性以及NADK磷酸化，如对磷酸化NADK具有选择性的单克隆抗体所揭示的。人重组NADK被PKCδ磷酸化，导致免疫反应性增加，但酶活性不变，这表明单独的PKC诱导的磷酸化不足以增加中性粒细胞中的NADK活性。这使我们推测NADK的磷酸化促进了抑制分子从复合物中的解离，从而增加了酶活性。PKC在吞噬细胞中激活NADK对于快速提供足够水平的超氧化物以抵御入侵微生物的宿主防御可能至关重要。
