Mammalian Target of Rapamycin Complex I (mTORC1) Activity in Ras Homologue Enriched in Brain (Rheb)-Deficient Mouse Embryonic Fibroblasts

Marlous J Groenewoud,Jorien Kassies,Wendy Pellis-Van Berkel,Richard F Lamb,Ype Elgersma,Susan M I Goorden,Fried J T Zwartkruis

doi:10.1371/journal.pone.0081649

Abstract

The Ras-like GTPase Rheb has been identified as a crucial activator of mTORC1. Activation most likely requires a direct interaction between Rheb and mTOR, but the exact mechanism remains unclear. Using a panel of Rheb-deficient mouse embryonic fibroblasts (MEFs), we show that Rheb is indeed essential for the rapid increase of mTORC1 activity following stimulation with insulin or amino acids. However, mTORC1 activity is less severely reduced in Rheb-deficient MEFs in the continuous presence of serum or upon stimulation with serum. This remaining mTORC1 activity is blocked by depleting the cells for amino acids or imposing energy stress. In addition, MEK inhibitors and the RSK-inhibitor BI-D1870 interfere in mTORC1 activity, suggesting that RSK acts as a bypass for Rheb in activating mTORC1. Finally, we show that this rapamycin-sensitive, Rheb-independent mTORC1 activity is important for cell cycle progression. In conclusion, whereas rapid adaptation in mTORC1 activity requires Rheb, a second Rheb-independent activation mechanism exists that contributes to cell cycle progression.

Highlights

The mTORC1 complex plays a vital role in adapting cellular metabolism of mammalian organisms to changing conditions like progress through development, food intake, prolonged starvation or acute stress
The results demonstrate that while Ras Homologue Enriched in Brain (Rheb) is required for the strong insulin and amino acid-induced mTORC1 activity, residual mTORC1 activity in Rheb-deficient cells is present, which is still subject to negative regulation by energy stress and amino acid withdrawal
Since this phenotype is much milder than that of mTOR or Raptor deletion, this result suggested that mTORC1 is active in the absence of Rheb or alternatively, that other signaling pathways can substitute for mTORC1

Summary

Introduction

The mTORC1 complex plays a vital role in adapting cellular metabolism of mammalian organisms to changing conditions like progress through development, food intake, prolonged starvation or acute stress (reviewed in 1). Information about the availability of nutrients and growth factors is integrated by various proteins present in mTORC1 and transmitted via the kinase activity of mTOR (reviewed in 2). Two functionally distinct protein complexes, mTORC1 and mTORC2, exist that share mTOR and LST8 as subunits. S6K is activated upon phosphorylation by mTORC1 and its activity is crucial for cell growth. 4E-BPs inhibit translation (reviewed in 3) and proliferation by binding to the eukaryotic initiation factor eIF4E. MTORC1-mediated phosphorylation leads to a release of 4E-BP from eIF4E, overcoming this inhibition [3,4] S6K is activated upon phosphorylation by mTORC1 and its activity is crucial for cell growth. 4E-BPs inhibit translation (reviewed in 3) and proliferation by binding to the eukaryotic initiation factor eIF4E. mTORC1-mediated phosphorylation leads to a release of 4E-BP from eIF4E, overcoming this inhibition [3,4]

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Conclusion