Abstract
The removal of extracellular amino acids or leucine alone inhibits the ability of the mammalian target of rapamycin (mTOR) to signal to the raptor-dependent substrates, p70 S6 kinase and 4E-BP. This inhibition can be overcome by overexpression of the Rheb GTPase. Rheb binds directly to the amino-terminal lobe of the mTOR catalytic domain, and activates mTOR kinase in a GTP-dependent manner. Herein we show that the binding of Rheb to endogenous and recombinant mTOR is reversibly inhibited by withdrawal of all extracellular amino acids or just leucine. The effect of amino acid withdrawal is not attributable to changes in Rheb-GTP charging; amino acid withdrawal does not alter the GTP charging of recombinant Rheb. Moreover, the binding of mTOR to Rheb mutants that are unable to bind guanyl nucleotide in vivo is also inhibited by amino withdrawal. The inhibitory effect of amino acid withdrawal is exerted through an action on mTOR, at a site largely distinct from that responsible for the binding of Rheb; deletion of the larger, carboxyl-terminal lobe of the mTOR catalytic domain eliminates the inhibitory effect of amino acid withdrawal on Rheb binding, without altering Rheb binding per se. The lesser ability of the mTOR catalytic domain to bind Rheb after amino acid withdrawal does not persist after extraction and purification of the mTOR polypeptide. Amino acid withdrawal may generate an inhibitor of the Rheb-mTOR interaction that interferes with the signaling function of TOR complex 1.
Highlights
From the Diabetes Unit and Medical Services and the Department of Molecular Biology, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114
We recently showed that recombinant Rheb can bind directly to mammalian target of rapamycin (mTOR) and that the protein kinase activity of mTOR is positively regulated by Rheb-GTP [8]
Replacement of the usual medium, DMEM plus 10% serum, with D-PBS for 1.5 to 2 h greatly reduces the recovery of endogenous (Fig. 2A) or recombinant (Fig. 2B) mTOR with Rheb; removal of serum alone has no effect on the recovery of HA-mTOR with GST-Rheb (Fig. 2B, compare lane 1 with lane 3), whereas the addition of an amino acid mixture alone to D-PBS is sufficient to fully restore the Rheb-bound mTOR to the levels recovered from cells in DMEM plus 10% serum (Fig. 2B, compare lane 1 with lane 4)
Summary
The removal of extracellular amino acids or leucine alone inhibits the ability of the mammalian target of rapamycin (mTOR) to signal to the raptor-dependent substrates, p70 S6 kinase and 4E-BP. As regards the mechanisms by which Rheb acts as a positive regulator of TOR signaling, we recently demonstrated [8] that Rheb binds directly to the smaller, amino-terminal lobe of the mTOR catalytic domain, and the kinase activity of the TOR polypeptides bound to Rheb is determined by the state of Rheb nucleotide charging.
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