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J. Biol. Chem., Vol. 282, Issue 46, 33562-33571, November 16, 2007

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NAD Kinase Levels Control the NADPH Concentration in Human Cells^*

From the Department of Molecular Biology, University of Bergen, Thormøhlensgate 55, N-5008 Bergen, Norway

NAD kinases (NADKs) are vital, as they generate the cellular NADP pool. As opposed to three compartment-specific isoforms in plants and yeast, only a single NADK has been identified in mammals whose cytoplasmic localization we established by immunocytochemistry. To understand the physiological roles of the human enzyme, we generated and analyzed cell lines stably deficient in or overexpressing NADK. Short hairpin RNA-mediated down-regulation led to similar (about 70%) decrease of both NADK expression, activity, and the NADPH concentration and was accompanied by increased sensitivity toward H₂O₂. Overexpression of NADK resulted in a 4–5-fold increase in the NADPH, but not NADP⁺, concentration, although the recombinant enzyme phosphorylated preferentially NAD⁺. Surprisingly, NADK overexpression and the ensuing increase of the NADPH level only moderately enhanced protection against oxidant treatment. Apparently, to maintain the NADPH level for the regeneration of oxidative defense systems human cells depend primarily on NADP-dependent dehydrogenases (which re-reduce NADP⁺), rather than on a net increase of NADP. The stable shifts of the NADPH level in the generated cell lines were also accompanied by alterations in the expression of peroxiredoxin 5 and Nrf2. Because the basal oxygen radical level in the cell lines was only slightly changed, the redox state of NADP may be a major transmitter of oxidative stress.

Received for publication, May 30, 2007 , and in revised form, September 13, 2007.

^* This study was supported in part by Deutsche Forschungsgemeinschaft, INTAS Genomics (Grant 05-1000004-7753) and the University of Bergen. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 47-55584591; Fax: 47-55589683; E-mail: Mathias.Ziegler{at}mbi.uib.no.

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