Akt mediates insulin-stimulated phosphorylation of Ndrg2: evidence for cross-talk with protein kinase C theta.
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 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. 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.
Cell Signalling Group, Diabetes and Obesity Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.
James G
JG
Burchfield
Alecia J
AJ
Lennard
Sakura
S
Narasimhan
William E
WE
Hughes
Valerie C
VC
Wasinger
Garry L
GL
Corthals
Tomohiko
T
Okuda
Hisato
H
Kondoh
Trevor J
TJ
Biden
Carsten
C
Schmitz-Peiffer
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007527:Isoenzymes / Q000502:physiology; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000166:cytology; D018482:Muscle, Skeletal / Q000166:cytology; D055456:Nodal Signaling Ligands; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000502:physiology; D000074121:Protein Kinase C-theta; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017208:Rats, Wistar; D020239:Receptor Cross-Talk; D016415:Sequence Alignment; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D014162:Transfection
D007328:Insulin; D007527:Isoenzymes; C113775:Ndr2 protein, vertebrate; D055456:Nodal Signaling Ligands; D011506:Proteins; D011518:Proto-Oncogene Proteins; C494918:AKT1 protein, human; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C076233:PRKCQ protein, human; C489593:Prkcq protein, mouse; C489591:Prkcq protein, rat; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta