Dual NADPH oxidases DUOX1 and DUOX2 synthesize NAADP and are necessary for Ca2+ signaling during T cell activation.

The formation of Ca2+ microdomains during T cell activation is initiated by the production of nicotinic acid adenine dinucleotide phosphate (NAADP) from its reduced form NAADPH. The reverse reaction­NAADP to NAADPH­is catalyzed by glucose 6-phosphate dehydrogenase (G6PD). Here, we identified NADPH oxidases NOX and DUOX as NAADP-forming enzymes that convert NAADPH to NAADP under physiological conditions in vitro. T cells express NOX1, NOX2, and, to a minor extent, DUOX1 and DUOX2. Local and global Ca2+ signaling were decreased in mouse T cells with double knockout of Duoxa1 and Duoxa2 but not with knockout of Nox1 or Nox2. Ca2+ microdomains in the first 15 s upon T cell activation were significantly decreased in Duox2−/− but not in Duox1−/− T cells, whereas both DUOX1 and DUOX2 were required for global Ca2+ signaling between 4 and 12 min after stimulation. Our findings suggest that a DUOX2- and G6PD-catalyzed redox cycle rapidly produces and degrades NAADP through NAADPH as an inactive intermediate.

HN1L/JPT2: A signaling protein that connects NAADP generation to Ca2+ microdomain formation.

NAADP-evoked Ca2+ release through type 1 ryanodine receptors (RYR1) is a major mechanism underlying the earliest signals in T cell activation, which are the formation of Ca2+ microdomains. In our characterization of the molecular machinery underlying NAADP action, we identified an NAADP-binding protein, called hematological and neurological expressed 1-like protein (HN1L) [also known as Jupiter microtubule-associated homolog 2 (JPT2)]. Gene deletion of Hn1l/Jpt2 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca2+ microdomains and delayed the onset and decreased the amplitude of global Ca2+ signaling. Photoaffinity labeling demonstrated direct binding of NAADP to recombinant HN1L/JPT2. T cell receptor/CD3-dependent coprecipitation of HN1L/JPT2 with RYRs and colocalization of these proteins suggest that HN1L/JPT2 connects NAADP formation with the activation of RYR channels within the first seconds of T cell activation. Thus, HN1L/JPT2 enables NAADP to activate Ca2+ release from the endoplasmic reticulum through RYR.