¹Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands

Correspondence: Dr FJT Zwartkruis, Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. E-mail: g.j.t.zwartkruis@med.uu.nl

Received 19 April 2005; Revised 18 July 2005; Accepted 29 July 2005; Published online 19 September 2005.

Abstract

The mTOR/S6K/4E-BP1 pathway integrates extracellular signals derived from growth factors, and intracellular signals, determined by the availability of nutrients like amino acids and glucose. Activation of this pathway requires inhibition of the tumor suppressor complex TSC1/2. TSC2 is a GTPase-activating protein for the small GTPase Ras homologue enriched in brain (Rheb), GTP loading of which activates mTOR by a yet unidentified mechanism. The level at which this pathway senses the availability of amino acids is unknown but is suggested to be at the level of TSC2. Here, we show that amino-acid depletion completely blocks insulin- and TPA-induced Rheb activation. This indicates that amino-acid sensing occurs upstream of Rheb. Despite this, amino-acid depletion can still inhibit mTOR/S6 kinase signaling in TSC2-/- fibroblasts. Since under these conditions Rheb-GTP levels remain high, a second level of amino-acid sensing exists, affecting mTOR activity in a Rheb-independent fashion.