Phosphorylation of PRAS40 on Thr246 by PKB/AKT facilitates efficient phosphorylation of Ser183 by mTORC1.
Type 2 diabetes is associated with alterations in protein kinase B (PKB/Akt) and mammalian target of rapamycin complex 1 (mTORC1) signalling. The proline-rich Akt substrate of 40-kDa (PRAS40) is a component of mTORC1, which has a regulatory function at the intersection of the PKB/Akt and mTORC1 signalling pathway. Phosphorylation of PRAS40-Thr246 by PKB/Akt, and PRAS40-Ser183 and PRAS40-Ser221 by mTORC1 results in dissociation from mTORC1, and its binding to 14-3-3 proteins. Although all phosphorylation sites within PRAS40 have been implicated in 14-3-3 binding, substitution of Thr246 by Ala alone is sufficient to abolish 14-3-3 binding under conditions of intact mTORC1 signalling. This suggests that phosphorylation of PRAS40-Thr246 may facilitate efficient phosphorylation of PRAS40 on its mTORC1-dependent sites. In the present study, we investigated the mechanism of PRAS40-Ser183 phosphorylation in response to insulin. Insulin promoted PRAS40-Ser183 phosphorylation after a euglycaemic-hyperinsulinaemic clamp in human skeletal muscle. The insulin-induced PRAS40-Ser183 phosphorylation was further evidenced in vivo in rat skeletal and cardiac muscle, and in vitro in A14 fibroblasts, 3T3L1 adipocytes and L6 myotubes. Inhibition of mTORC1 by rapamycin or amino acid deprivation partially abrogated insulin-mediated PRAS40-Ser183 phosphorylation in cultured cell lines. However, lowering insulin-induced PRAS40-Thr246 phosphorylation using wortmannin or palmitate in cell lines, or by feeding rats a high-fat diet, completely abolished insulin-mediated PRAS40-Ser183 phosphorylation. In addition, replacement of Thr246 by Ala reduced insulin-mediated PRAS40-Ser183 phosphorylation. We conclude that PRAS40-Ser183 is a component of insulin action, and that efficient phosphorylation of PRAS40-Ser183 by mTORC1 requires the phosphorylation of PRAS40-Thr246 by PKB/Akt.
Department of Molecular Cell Biology, Leiden University Medical Centre, Leiden, The Netherlands.
Emmani B M
EB
Nascimento
Marieke
M
Snel
Bruno
B
Guigas
Gerard C M
GC
van der Zon
Jan
J
Kriek
J Antonie
JA
Maassen
Ingrid M
IM
Jazet
Michaela
M
Diamant
D Margriet
DM
Ouwens
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007333:Insulin Resistance; D051379:Mice; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology; D041681:NIH 3T3 Cells; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D000077191:Wortmannin
C484523:AKT1S1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; C519510:Crtc1 protein, rat; D004791:Enzyme Inhibitors; D007328:Insulin; D010750:Phosphoproteins; D014157:Transcription Factors; D013912:Threonine; D012694:Serine; D051057:Proto-Oncogene Proteins c-akt; D020123:Sirolimus; D000077191:Wortmannin