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J. Biol. Chem., Vol. 279, Issue 18, 18623-18632, April 30, 2004

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Akt Mediates Insulin-stimulated Phosphorylation of Ndrg2

EVIDENCE FOR CROSS-TALK WITH PROTEIN KINASE C ^*

James G. Burchfield, Alecia J. Lennard, Sakura Narasimhan, William E. Hughes, Valerie C. Wasinger¶, Garry L. Corthals||, Tomohiko Okuda**, Hisato Kondoh**, Trevor J. Biden, and Carsten Schmitz-Peiffer

From the Cell Signalling Group, Diabetes and Obesity Program and the Protein Analysis Laboratory, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, New South Wales 2010, Australia and the ^**Laboratory of Developmental Biology, Graduate School of Frontier Bioscience, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan

The protein kinase Akt mediates several metabolic and mitogenic effects of insulin, whereas activation of protein kinase C (PKC) isoforms has been implicated in the inhibition of insulin action. We have previously shown that both PKC and PKC are activated in skeletal muscle of insulin-resistant high fat-fed rats, and to identify potential substrates for these kinases, we incubated recombinant PKC isoforms with rat muscle fractions in vitro. PKC specifically phosphorylated a 48-kDa protein that was subsequently identified by mass spectrometry as Ndrg2. Ndrg2 is highly related to N-Myc downstream-regulated protein 1, which has been linked to stress responses, cell proliferation, and differentiation, although Ndrg2 itself is not repressed by N-Myc. Ndrg2 contains several potential phosphorylation sites, including three Akt consensus sequences. Ndrg2 phosphorylation was enhanced in [³²P]orthophosphate-labeled C2C12 muscle cells co-overexpressing either PKC or Akt. Phosphorylation of Ndrg2 was examined further using a phospho (Ser/Thr) Akt substrate antibody. Insulin increased Ndrg2 phosphorylation in C2C12 cells in a wortmannin- and palmitate-inhibitable manner, whereas rapamycin, PD98059, and bisindoylmaleimide I had no effect, supporting a direct role for Akt. Mutation of Ndrg2 indicated that Thr-348 is the major phosphorylation site detected by the antibody and that Akt stimulates phosphorylation of this site, whereas PKC phosphorylates Ser-332. PKC overexpression, however, diminished the effect of insulin on Thr-348 phosphorylation without reducing Akt activation, suggesting that this is mediated through phosphorylation of Ndrg2 at Ser-332. Our data identify Ndrg2 as a novel insulin-dependent phosphoprotein and suggest that PKC may inhibit insulin action in part by reducing its phosphorylation by Akt.

Received for publication, February 11, 2004

^* This work was supported by the National Health and Medical Research Council of Australia. The Garvan Institute Protein Analysis Laboratory was supported by Wellcome Trust Grants 052458 and 053248. 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.

^¶ Current address: Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, Australia.

^|| Current address: Biomedical Proteomics Research Group, Geneva University Hospital, Switzerland.

Current address: National Cardiovascular Center, Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan.

To whom correspondence should be addressed. Tel.: 61-2-9295-8212; Fax: 61-2-9295-8201; E-mail: c.schmitz-peiffer{at}garvan.org.au.

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