AMPK-dependent S314 phosphorylation activates NAMPT by facilitating NAMPT/PRPP association. (a) Bacterially purified unheated WT His/Flag-NAMPT or S314 mutant protein was incubated with purified active AMPK proteins for an in vitro kinase assay. His/Flag-NAMPT proteins were precipitated using anti-Flag antibody, and the NAMPT enzymatic activity in the precipitates was measured. ***p < 0.001. (b) HOK and HUVEC cells with expression of WT Flag-NAMPT or Flag-NAMPT S314A were treated with 10 Gy ionizing radiation. Flag-NAMPT proteins were precipitated from cell lysates 30 min after irradiation, washed twice with PBS, and the NAMPT enzymatic activity in the precipitates was measured. IR, ionizing radiation; WCL, whole cell lysate; **p < 0.01. (c) The NAMPT catalytic domain in human NAMPT protein (shown in cyan) structure was boxed by dotted line, and enlarged to show the spatial location of S314 (side chain shown in red), PRPP (oxygen atom shown in purple, PDB code: 2E5C), and nicotinamide (oxygen atom shown in purple, PDB code: 2E5D). (d) Human NAMPT protein structure (PDB code: 2E5C) shows the spatial location of S314 (side chain shown in red), PRPP (oxygen atom shown in purple), and H247 (side chain shown in orange). (e) Bacterially purified unheated WT His/Flag-NAMPT or S314 mutant protein was incubated with purified active AMPK proteins for an in vitro kinase assay. His/Flag-NAMPT proteins were precipitated using anti-Flag antibody, and the binding affinity between NAMPT protein and PRPP was measured. ***p < 0.001. (f) HOK cells with expression of WT Flag-NAMPT or Flag-NAMPT S314A were treated with 10 Gy ionizing radiation. Flag-NAMPT proteins were precipitated from cell lysates 30 min after irradiation, washed twice with PBS, and the binding affinity between NAMPT protein and PRPP was measured. **p < 0.01. (g) HOK cells with expression of WT Flag-NAMPT were pre-treated with 5 µM Compound C for 2 h, and cells were then treated with 10 Gy ionizing radiation. Flag-NAMPT proteins were precipitated from cell lysates 30 min after irradiation, washed twice with PBS and the binding affinity between NAMPT protein and PRPP was measured. **p < 0.01.

A schematic of AMPK-mediated NAMPT activation under ionizing radiation. Ionizing radiation causes rapid activation of AMPK, which in turn phosphorylates NAMPT S314 and enhances NAMPT enzymatic activity. NAMPT activation promotes NAD⁺ synthesis, thereby facilitating DNA repair and cell viability.

Nicotinamide adenine dinucleotide (NAD⁺) is widely established as an essential cofactor for electron transfer functioning in diverse metabolic pathways [1]. Particularly, NAD⁺ plays critical roles in glycolysis in cytosol and the tricarboxylic acid cycle in mitochondria where it generates reducing force in the form of NADH, which then transfers electrons from various sources to the mitochondrial complex I and downstream components of the electron transport chain, ultimately leading to the production of ATP [2].

NAD⁺ can be synthesized through the de novo pathway from tryptophan, the Preiss-handler pathway from nicotinic acid, or the salvage pathway by recycling of nicotinamide, and the latter is the major route for NAD⁺ biosynthesis for mammalians [3]. The rate-limiting step of salvage pathway, by which nicotinamide and PRPP were condensated to generate nicotinamide mononucleotide (NMN), is catalysed by nicotinamide phosphoribosyltransferase (NAMPT) [4]. NAD⁺ is eventualy produced from NMN in mammalian cells by three nicotinamide mononucleotide adenylyltransferases (NMNATs), with NMNAT1 located in the nucleus, NMNAT2 located in the Golgi apparatus, and NMNAT3 located in mitochondria [5].

In addition to being an electron carrier, NAD⁺ also function as the substrate for protein poly(ADP-ribosyl)ation, by which polymers of ADP-ribose are covalently linked to proteins through poly (ADP-ribose) polymerases (PARPs) [6]. Poly(ADP-ribosyl)ation has significant impacts on the cellular responses to DNA strand breaks under ionizing radiation [7]. As early DNA damage sensors, PARPs are rapidly activated by binding with the ionizing radiation-elicited DNA breaks, and catalyse poly(ADP-ribosyl)ation on itself as well as adjacent histones and other proteins, thereby marking the DNA lesion sites along the chromatin. The poly(ADP-ribose) chain of the modified proteins can then recruit other effector proteins to the lesion site and locally assemble the DNA damage-responsive complexes, thereby promoting chromatin relaxation and initiating DNA repair process [8].

Radiation-induced oral mucositis (RIOM) is the most common complication for patients who receive head and neck radiotherapy, which may cause multiple temporary or irreversible damages in oral mucosa [9]. RIOM-mediated inflammation, which may lead to ulceration, is highly toxic to epithelial and endothelial cells in oral mucosa [10]. During the repair of ionizing radiation-induced DNA breaks, the length of poly(ADP-ribose) chains may attain a size of 200–300 residues, and their synthesis increases up to 500-fold owing to the activation of PARPs, which can result in rapid and significant consumption of cellular NAD⁺ [11,12]. Given the essential role of poly(ADP-ribosyl)ation in DNA repair, NAD⁺ availability becomes a critical factor that may modulate DNA repair capacity [13]. Addition of NAD⁺ largely promoted DNA repair capacity of soluble cell extracts on ionizing radiation or radiomimetic agents-treated DNA [14]. However, whether NAMPT, as the rate-limiting enzyme in the salvage pathway for NAD⁺ biosynthesis, is co-opted under ionizing radiation to timely fine-tune NAD⁺ homeostasis remains elusive. In this study, we demonstrate that AMPK rapidly phosphorylates NAMPT at Serine (S)314 in oral keratinocyte and endothelial cells under ionizing radiation, which reinforces the enzymatic activity of NAMPT by increasing its binding with PRPP. AMPK-mediated NAMPT S314 phosphorylation substantially restores NAD⁺ level in the irradiated cells and facilitates cell viability.