Akt mediates insulin-stimulated phosphorylation of Ndrg2-: Evidence for cross-talk with protein kinase C θ

被引:70
作者
Burchfield, JG
Lennard, AJ
Narasimhan, S
Hughes, WE
Wasinger, VC
Corthals, GL
Okuda, T
Kondoh, H
Biden, TJ
Schmitz-Peiffer, C
机构
[1] Garvan Inst Med Res, Cell Signalling Grp, Diabet & Obes Program, Sydney, NSW 2010, Australia
[2] Garvan Inst Med Res, Prot Anal Lab, Sydney, NSW 2010, Australia
[3] Osaka Univ, Grad Sch Frontier Biosci, Dev Biol Lab, Suita, Osaka 5650871, Japan
关键词
D O I
10.1074/jbc.M401504200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 PKCtheta and PKCepsilon 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. PKCtheta 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 [P-32] orthophosphate-labeled C2C12 muscle cells co-overexpressing either PKCtheta 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 PKCtheta phosphorylates Ser-332. PKCtheta 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 PKCtheta may inhibit insulin action in part by reducing its phosphorylation by Akt.
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收藏
页码:18623 / 18632
页数:10
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