Protein kinase C activates NAD kinase in human neutrophils

• Published in: Free radical biology & medicine 2020-12, Vol.161, p.50-59
• Main Authors: Rabani, Razieh, Cossette, Chantal, Graham, François, Powell, William S.
• Format: Article
• Language: English
• Subjects: Host defense; NAD kinase; NADP; NADPH; Neutrophils; Protein kinase C
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2020.09.022

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. [Display omitted] •Phorbol myristate acetate increases NADP and NADPH in neutrophils.•The mechanism for this effect is activation of NAD kinase.•NAD kinase in neutrophils is activated by protein kinase C-induced phosphorylation.•Phosphorylation of NAD kinase alone is insufficient to increase enzyme activity.•Phosphorylation might induce the release of an inhibitory protein from NAD kinase.