Abstract
The mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a ubiquitously expressed multimeric protein kinase complex that integrates nutrient and growth factor signals for the co-ordinated regulation of cellular metabolism and cell growth. Herein, we demonstrate that suppressing the cellular activity of glycogen synthase kinase-3 (GSK3), by use of pharmacological inhibitors or shRNA-mediated gene silencing, results in substantial reduction in amino acid (AA)-regulated mTORC1-directed signalling, as assessed by phosphorylation of multiple downstream mTORC1 targets. We show that GSK3 regulates mTORC1 activity through its ability to phosphorylate the mTOR-associated scaffold protein raptor (regulatory-associated protein of mTOR) on Ser(859). We further demonstrate that either GSK3 inhibition or expression of a S859A mutated raptor leads to reduced interaction between mTOR and raptor and under these circumstances, irrespective of AA availability, there is a consequential loss in phosphorylation of mTOR substrates, such as p70S6K1 (ribosomal S6 kinase 1) and uncoordinated-51-like kinase (ULK1), which results in increased autophagic flux and reduced cellular proliferation.
Highlights
The mammalian or mechanistic target of rapamycin complex 1 is a multimeric protein assembly composed of mTOR, mLST8, proline-rich Akt substrate of 40 kDa (PRAS40) and raptor that integrates mitogenic and nutrient [amino acid (AA)] signals to regulate diverse cellular responses, including mRNA translation, cell growth/proliferation, metabolism and autophagy [1,2]
Activation of mTORC1 is crucially dependent upon a small G-protein called Rheb (Ras homolog enriched in brain), whose intrinsic GTPase activity is inhibited by the GTPase-activating protein (GAP) activity of the tuberous sclerosis complex (TSC1/2) [3]
glycogen synthase kinase-3 (GSK3) inhibition/silencing has no detectable effect upon lysosomal mTOR localization or upon expression of key protein components of mTORC1, loss of Ser859 phosphorylation is associated with reduced interaction of raptor with mTOR resulting in a consequential reduction in the phosphorylation of downstream targets such as S6K1, ukaryotic initiation factor 4E-binding protein (4E-BP1) and uncoordinated-51-like kinase (ULK1)
Summary
The mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a multimeric protein assembly composed of mTOR, mLST8 (mammalian lethal with SEC13 protein 8), proline-rich Akt substrate of 40 kDa (PRAS40) and raptor (regulatory-associated protein of mTOR) that integrates mitogenic and nutrient [amino acid (AA)] signals to regulate diverse cellular responses, including mRNA translation, cell growth/proliferation, metabolism and autophagy [1,2]. The Rag heterodimer is most active when the RagA–RagB component is GTP-loaded and the RagC–RagD component is in its GDP-bound form. In this state, the Rag heterodimer is able to bind mTORC1 allowing it to make contact with Rheb-GTP at the lysosomal surface where it becomes activated [7]. Very recent work has identified a lysosomal membrane AA transporter belonging to the solute carrier 38 (SLC38) family, SLC38A9 [ known as sodium-coupled neutral amino acid transporter 2 (SNAT9)], as a putative AA sensor whose occupancy by substrate AAs is thought to signal AA sufficiency to mTORC1 via its interaction with the Rag–Ragulator complex [9,10]
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