Nuclear transport of nicotinamide phosphoribosyltransferase is cell cycle–dependent in mammalian cells, and its inhibition slows cell growth

Nicotinamide phosphoribosyltransferase (NAMPT) is located in both the nucleus and cytoplasm and has multiple biological functions including catalyzing the rate-limiting step in NAD synthesis. Moreover, up-regulated NAMPT expression has been observed in many cancers. However, the determinants and reg...

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Published in:	The Journal of biological chemistry Vol. 294; no. 22; pp. 8676 - 8689
Main Authors:	Svoboda, Petr, Krizova, Edita, Sestakova, Sarka, Vapenkova, Kamila, Knejzlik, Zdenek, Rimpelova, Silvie, Rayova, Diana, Volfova, Nikol, Krizova, Ivana, Rumlova, Michaela, Sykora, David, Kizek, Rene, Haluzik, Martin, Zidek, Vaclav, Zidkova, Jarmila, Skop, Vojtech
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 31-05-2019 American Society for Biochemistry and Molecular Biology
Subjects:	3T3-L1 Cells Acrylamides - pharmacology Active Transport, Cell Nucleus Animals cancer Cell Biology Cell Cycle Checkpoints Cell Nucleus - metabolism Cell Proliferation Cell Survival - drug effects Cytoplasm - metabolism epigenetics GFP fusion Hep G2 Cells Histones - metabolism Humans Mice Mutagenesis, Site-Directed NAD - metabolism NAMPT nicotinamide adenine dinucleotide (NAD) Nicotinamide Phosphoribosyltransferase - chemistry Nicotinamide Phosphoribosyltransferase - genetics Nicotinamide Phosphoribosyltransferase - metabolism nuclear localization Oxidative Stress Piperidines - pharmacology Poly(ADP-ribose) Polymerases - metabolism pre–B cell colony enhancing factor (PBEF) Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism sirtuin Sirtuins - metabolism visfatin nicotinamide adenine dinucleotide (NAD) sirtuin NAMPT nuclear localization cancer GFP fusion epigenetics pre–B cell colony enhancing factor (PBEF) visfatin
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Summary:	Nicotinamide phosphoribosyltransferase (NAMPT) is located in both the nucleus and cytoplasm and has multiple biological functions including catalyzing the rate-limiting step in NAD synthesis. Moreover, up-regulated NAMPT expression has been observed in many cancers. However, the determinants and regulation of NAMPT’s nuclear transport are not known. Here, we constructed a GFP–NAMPT fusion protein to study NAMPT’s subcellular trafficking. We observed that in unsynchronized 3T3-L1 preadipocytes, 25% of cells had higher GFP–NAMPT fluorescence in the cytoplasm, and 62% had higher GFP–NAMPT fluorescence in the nucleus. In HepG2 hepatocytes, 6% of cells had higher GFP–NAMPT fluorescence in the cytoplasm, and 84% had higher GFP–NAMPT fluorescence in the nucleus. In both 3T3-L1 and HepG2 cells, GFP–NAMPT was excluded from the nucleus immediately after mitosis and migrated back into it as the cell cycle progressed. In HepG2 cells, endogenous, untagged NAMPT displayed similar changes with the cell cycle, and in nonmitotic cells, GFP–NAMPT accumulated in the nucleus. Similarly, genotoxic, oxidative, or dicarbonyl stress also caused nuclear NAMPT localization. These interventions also increased poly(ADP-ribosyl) polymerase and sirtuin activity, suggesting an increased cellular demand for NAD. We identified a nuclear localization signal in NAMPT and amino acid substitution in this sequence (424RSKK to ASGA), which did not affect its enzymatic activity, blocked nuclear NAMPT transport, slowed cell growth, and increased histone H3 acetylation. These results suggest that NAMPT is transported into the nucleus where it presumably increases NAD synthesis required for cell proliferation. We conclude that specific inhibition of NAMPT transport into the nucleus might be a potential avenue for managing cancer.
Bibliography:	Edited by Phyllis I. Hanson
ISSN:	0021-9258 1083-351X
DOI:	10.1074/jbc.RA118.003505