Regulation of proline-rich Akt substrate of 40 kDa (PRAS40) function by mammalian target of rapamycin complex 1 (mTORC1)-mediated phosphorylation.
The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contains mTOR, raptor, mLST8, and PRAS40 (proline-rich Akt substrate of 40 kDa). PRAS40 functions as a negative regulator when bound to mTORC1, and it dissociates from mTORC1 in response to insulin. PRAS40 has been demonstrated to be a substrate of mTORC1, and one phosphorylation site, Ser-183, has been identified. In this study, we used two-dimensional phosphopeptide mapping in conjunction with mutational analysis to show that in addition to Ser-183, mTORC1 also phosphorylates Ser-212 and Ser-221 in PRAS40 when assayed in vitro. Mutation of all three residues to Ala markedly reduces mTORC1-mediated phosphorylation of PRAS40 in vitro. All three sites were confirmed to be phosphorylated in vivo by [(32)P]orthophosphate labeling and peptide mapping. Phosphorylation of Ser-221 and Ser-183 but not Ser-212 is sensitive to rapamycin treatment. Furthermore, we demonstrate that mutation of Ser-221 to Ala reduces the interaction with 14-3-3 to the same extent as mutation of Thr-246, the Akt/protein kinase B-phosphorylated site. We also find that mutation of Ser-221 to Ala increases the inhibitory activity of PRAS40 toward mTORC1. We propose that after mTORC1 kinase activation by upstream regulators, PRAS40 is phosphorylated directly by mTOR, thus contributing to the relief of PRAS40-mediated substrate competition.
Wang
Lifu
L
Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA. lw6j@irginia.edu
Harris
Thurl E
TE
Lawrence
John C
JC
D041721:3T3-L1 Cells; D048868:Adaptor Proteins, Signal Transducing; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D000076223:Regulatory-Associated Protein of mTOR; D012694:Serine / Q000235:genetics / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C484523:AKT1S1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D007328:Insulin; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011506:Proteins; C463659:RPTOR protein, human; D000076223:Regulatory-Associated Protein of mTOR; D014157:Transcription Factors; C508191:proline-rich Akt substrate, 40 kDa protein, mouse; D012694:Serine; D011494:Protein Kinases; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D000409:Alanine