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.
Burchfield
James G
JG
Cell Signalling Group, Diabetes and Obesity Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.
Lennard
Alecia J
AJ
Narasimhan
Sakura
S
Hughes
William E
WE
Wasinger
Valerie C
VC
Corthals
Garry L
GL
Okuda
Tomohiko
T
Kondoh
Hisato
H
Biden
Trevor J
TJ
Schmitz-Peiffer
Carsten
C
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