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Mechanistic target of rapamycin complex 1 (mTORC1)-mediated phosphorylation is governed by competition between

Michael D Dennis1, Scot R Kimball, Leonard S Jefferson

  • 1Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.

The Journal of Biological Chemistry
|November 28, 2012
PubMed

Insights

Loss of 4E-BP1 and 4E-BP2 proteins enhances mTORC1-mediated phosphorylation of p70S6K1. This occurs because 4E-BP proteins normally compete with p70S6K1 for binding to raptor, a component of mTORC1.

Area of Science:

  • Cellular signaling pathways
  • Molecular biology
  • Biochemistry

Background:

  • The mechanistic target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth and metabolism.
  • 4E-binding proteins (4E-BPs) are known inhibitors of cap-dependent translation by binding to eukaryotic initiation factor 4E (eIF4E).
  • p70S6 kinase 1 (p70S6K1) is a major downstream target of mTORC1, involved in cell growth and proliferation.

Purpose of the Study:

  • To investigate the role of 4E-BP1 and 4E-BP2 in regulating mTORC1 activity and its downstream targets.
  • To elucidate the mechanism by which 4E-BP proteins influence p70S6K1 phosphorylation.
  • To determine the impact of 4E-BP loss on the interaction between mTORC1, p70S6K1, and raptor.

Main Methods:

  • Gene disruption of 4E-BP1 and 4E-BP2 in mouse liver and embryonic fibroblasts.
  • Analysis of p70S6K1 phosphorylation levels using Western blotting.
  • Immunoprecipitation assays to study protein-protein interactions (mTORC1, raptor, p70S6K1, 4E-BP1).
  • Cotransfection experiments in cell culture systems.

Main Results:

  • Combined genetic deletion of 4E-BP1 and 4E-BP2 led to dramatically elevated p70S6K1 phosphorylation in mouse liver following feeding-induced mTORC1 activation.
  • Loss of 4E-BP1 and 4E-BP2 resulted in increased interaction between p70S6K1 and raptor, a component of mTORC1.
  • In cell culture, absence of 4E-BP1/2 enhanced p70S6K1 interaction with IGF1-activated mTORC1 and increased p70S6K1 phosphorylation at Thr-389.
  • Overexpression of 4E-BP1, but not a non-binding mutant, reduced mTORC1 recovery in HA-p70S6K1 immunoprecipitates.

Conclusions:

  • The findings indicate that 4E-BP proteins act as negative regulators of mTORC1-mediated p70S6K1 phosphorylation.
  • The absence of 4E-BP proteins leads to increased p70S6K1 phosphorylation due to reduced competition for binding to raptor.
  • This study reveals a novel mechanism of mTORC1 regulation involving substrate competition for raptor.

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