NAD Kinase Is an AKT Substrate

The PI3K–AKT–mTORC1 signaling axis promotes a number of anabolic processes by directly phosphorylating metabolic enzymes and indirectly regulating downstream transcription factors. Several of these processes require significant reducing power in the form of NADPH, which is oxidized to NADP⁺ during biosynthesis. Hoxhaj and colleagues demonstrate that AKT activity regulates the availability of NADPH and NADP⁺ via phosphorylation of NAD kinase (NADK), the sole cytosolic enzyme that produces NADP⁺ from a larger pool of NAD⁺. Stimulation of cells with insulin-like growth factor 1 (IGF1) or insulin resulted in AKT-dependent phosphorylation of NADK and increases in NADP⁺ and NADPH independent of glucose metabolism. Expression of constitutively active AKT increased phosphorylation of NADK on three amino-terminal serine residues: S44, S46, and S48. A phospho-NADK antibody confirmed that growth factor stimulation results in AKT-dependent phosphorylation of NADK, primarily at S44 and S46, and these sites are constitutively phosphorylated in cancer cells with aberrant activation of AKT. Expression of NADK harboring mutated phosphorylation sites impaired NADP⁺ synthesis in response to growth factor stimulation or as a result of constitutively active AKT. Although wild-type and phospho-mutant NADK exhibited similar basal enzymatic activity, in vitro phosphorylation by AKT increased the catalytic activity of wild-type but not mutant NADK. An NADK mutant lacking the N-terminal region where the AKT phosphorylation sites reside exhibited increased basal NADP⁺ synthesis, which was not increased further by AKT. Collectively, these findings indicate that the N-terminal domain exerts an autoinhibitory effect on NADK activity and that phosphorylation by AKT relieves this inhibition to stimulate synthesis of NADP⁺. This signaling axis provides a means to acutely regulate the availability of reducing power in the cell in response to growth factor stimulation, a regulatory pathway predicted to be chronically activated in cells with oncogenic activation of PI3K–AKT signaling.

Hoxhaj G, Ben-Sahra I, Lockwood SE, Timson RC, Byles V, Henning GT, et al. Direct stimulation of NADP⁺ synthesis through Akt-mediated phosphorylation of NAD kinase. Science 2019;363:1088–92.

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