Protein kinase C activates NAD kinase in human neutrophils.

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.