mTORC2 protein complex-mediated Akt (Protein Kinase B) Serine 473 Phosphorylation is not required for Akt1 activity in human platelets [corrected]

J Biol Chem. 2011 Jul 15;286(28):24553-60. doi: 10.1074/jbc.M110.202341. Epub 2011 May 18.

Abstract

Protein kinase B (PKB, Akt) is a Ser/Thr kinase involved in the regulation of cell survival, proliferation, and metabolism and is activated by dual phosphorylation on Thr(308) in the activation loop and Ser(473) in the hydrophobic motif. It plays a contributory role to platelet function, although little is known about its regulation. In this study, we investigated the role of the mammalian target of rapamycin complex (mTORC)-2 in Akt regulation using the recently identified small molecule ATP competitive mTOR inhibitors PP242 and Torin1. Both PP242 and Torin1 blocked thrombin and insulin-like growth factor 1-mediated Akt Ser(473) phosphorylation with an IC(50) between 1 and 5 nm, whereas the mTORC1 inhibitor rapamycin had no effect. Interestingly, PP242 and Torin1 had no effect on Akt Thr(308) phosphorylation, Akt1 activity, and phosphorylation of the Akt substrate glycogen synthase kinase 3β, indicating that Ser(473) phosphorylation is not necessary for Thr(308) phosphorylation and maximal Akt1 activity. In contrast, Akt2 activity was significantly reduced, concurrent with inhibition of PRAS40 phosphorylation, in the presence of PP242 and Torin1. Other signaling pathways, including phospholipase C/PKC and the MAPK pathway, were unaffected by PP242 and Torin1. Together, these results demonstrate that mTORC2 is the kinase that phosphorylates Akt Ser(473) in human platelets but that this phosphorylation is dispensable for Thr(308) phosphorylation and Akt1 activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Antibiotics, Antineoplastic / pharmacology
  • Blood Platelets / cytology
  • Blood Platelets / enzymology*
  • Glycogen Synthase Kinase 3 / metabolism
  • Glycogen Synthase Kinase 3 beta
  • Humans
  • Insulin-Like Growth Factor I / metabolism
  • MAP Kinase Signaling System / drug effects
  • MAP Kinase Signaling System / physiology*
  • Naphthyridines / pharmacology
  • Phosphorylation / drug effects
  • Phosphorylation / physiology
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Serine / metabolism
  • Sirolimus / pharmacology
  • Thrombin / metabolism
  • Transcription Factors / antagonists & inhibitors
  • Transcription Factors / metabolism*
  • Type C Phospholipases / metabolism

Substances

  • 1-(4-(4-propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-(quinolin-3-yl)benzo(h)(1,6)naphthyridin-2(1H)-one
  • AKT1S1 protein, human
  • Adaptor Proteins, Signal Transducing
  • Antibiotics, Antineoplastic
  • CRTC2 protein, human
  • Naphthyridines
  • Transcription Factors
  • Serine
  • Insulin-Like Growth Factor I
  • AKT1 protein, human
  • AKT2 protein, human
  • GSK3B protein, human
  • Glycogen Synthase Kinase 3 beta
  • Proto-Oncogene Proteins c-akt
  • Glycogen Synthase Kinase 3
  • Type C Phospholipases
  • Thrombin
  • Sirolimus