Resveratrol inhibits mTOR signaling by targeting DEPTOR

Abstract

Resveratrol (RSV, trans-3,4,5-Trihydroxystilbene), a type of polyphenol originally found in red wines, shows a great promise for the treatment of cancer, aging, type 2 diabetes and cardiovascular diseases. Recent studies suggest that suppressing the signaling pathway mediated by mTOR, a well-known energy sensor that integrates various hormonal, nutrient and environmental signals to regulate cell growth, metabolism and survival, could play an important role in mediating the beneficial effect of RSV. The underlying mechanisms by which RSV inhibits mTOR signaling remain elusive, but our recent studies show that RSV inhibits amino acid-stimulated mTOR signaling in C2C12 fibroblasts via a Sirt1- and AMPK-independent mechanism. RSV treatment has no effect on the expression levels of mTOR, raptor and DEPTOR, but greatly promotes the interaction between mTOR and its inhibitor DEPTOR. Our results reveal a novel mechanism by which RSV inhibits mTOR signaling and its function.

Resveratrol (RSV) is a natural-polyphenol compound that exerts multiple beneficial effects on cardiovascular, metabolic and immune functions.^1–5 The mechanisms underlying the protective effects of RSV on various cardiovascular and metabolic disorders remain elusive, but there is some evidence suggesting that inhibition of the mammalian target of rapamycin (mTOR) signaling pathway could play a role.^1–3

mTOR is a member of the PI 3-kinase-related protein kinase (PIKK) family that plays a critical role in the regulation of cell homeostasis in response to various upstream stimuli such as growth factors, nutrients and stress.^6,7 mTOR exists in two distinct complexes, TORC1 and TORC2, which differ in subunit compositions and biological functions.⁸ The rapamycin-sensitive mTORC1, which consists of five components including mTOR, the regulatory-associated protein of mTOR (Raptor), mammalian lethal with Sec13 protein 8 (mLST8, also known as GβL), proline-rich Akt substrate 40 kDa (PRAS40), and the DEP-domain-containing and mTOR-interactive protein (DEPTOR),⁹ regulates protein synthesis and cell growth by phosphorylating downstream target proteins such as p70 ribosomal S6 kinase 1 (S6K) and the eukaryotic initiation factor 4E-BP1.^6,10 The rapamycin-insensitive TORC2 contains similar components except that Raptor is replaced with a unique protein named rapamycin-insensitive companion of mTOR (Rictor).

The mechanism by which RSV inhibits mTOR signaling remains elusive. Some studies suggest that activation of the Sirt1 signaling pathway is essential for RSV action.^11–13 However, a number of recent studies indicate that many of the protective effects of RSV could be mediated by Sirt1-independent mechanisms.^14–16 In agreement with this, our recent study showed that RSV inhibits insulin- and leucine-stimulated mTOR signaling in a Sirt1-independent manner.¹⁷

RSV has been shown to directly bind to the ATP-binding site of PI3K and thus function as a class 1A PI3K inhibitor.^16,18 Since activation of the PI3K/PI3K-dependent kinase 1 (PDK1)/Akt signaling pathway is necessary for insulin-stimulated mTOR activation,^19,20 it is possible that RSV inhibits mTOR signaling by inhibiting the PI3K/PDK1/Akt signaling pathway. Consistent with this, we found that treating C2C12 fibroblasts with RSV greatly inhibited insulin-stimulated activation of the PI3K and the mTOR signaling pathways.¹⁷ However, inhibition of Akt or disruption of PDK1 expression had no significant effect on leucine-stimulated mTOR signaling, suggesting that the mechanisms by which insulin and amino acids promote mTOR activation are distinct.¹⁷

The tumor suppressor tuberous sclerosis complex (TSC1/2) plays a crucial role in the negative regulation of Rheb, a small GTPase that activates mTOR.^21,22 While it is possible that RSV may inhibit mTOR signaling by targeting TSC1/2, we found that suppression or disruption of TSC1/2 expression had little effect on leucine-stimulated mTOR signaling in C2C12 cells or MEFs,¹⁷ suggesting that RSV negatively regulates mTOR signaling by targeting at a site downstream of TSC1/2. Consistent with this view, we found that RSV treatment markedly increased the association between mTOR and DEPTOR, a newly identified negative regulator of mTOR.^23,24 Since the binding of DEPTOR to mTOR is critical for the negative regulation of mTOR activity,^23,24 enhancing the interaction between DEPTOR and mTOR could thus provide a mechanism by which RSV inhibits mTORC1 signaling. In agreement with this view, suppressing the expression levels of DEPTOR by RNAi greatly diminished the inhibitory effect of RSV on leucine-stimulated mTORC1 signaling.¹⁷ Our study also showed that RSV treatment negatively regulates Akt activity.¹⁷ Since Akt stimulates mTOR activity by suppressing the negative effect of TSC1/2,²⁵ these findings indicate that RSV is able to negatively regulate mTOR activity via distinct mechanisms in response to different upstream stimuli.

In vitro kinase assay experiments strongly suggest that RSV may suppress PI3K activity through a direct binding to the ATP-binding site of PI3K (Fig. 1).¹⁸ The 4′ hydroxyl group of RSV has been shown to form a hydrogen bond with the catalytic lysine residues in the ATP-binding site of p110α (or p110β), thus leading to the suppression of PI3K activity.^16,18 mTOR is a class IV PI3K protein kinase,^26,27 and its C-terminal catalytic domain is homologous to the p110α catalytic subunit of the class I PI3K.²⁸ The structural similarity between mTOR and PI3K has promoted the identification of highly potent and effective PI3K/mTOR dual inhibitors for the treatment of cancer.^29–31 An interesting question that remains unanswered is whether RSV also targets the ATP-binding pocket of mTOR and if it does, whether the binding provides a mechanism underlying the negative effect of RSV on mTOR activity (Fig. 1).

Figure 1.

Mechanisms by which RSV inhibits insulin and amino acid-induced mTOR activation. (1) RSV binds to the ATP-binding site of the p110 subunit of PI3K and competitively inhibits the binding of ATP to the enzyme, leading to suppression of mTOR activity and downstream signaling. (2) RSV promotes the interaction between mTOR and its inhibitor DEPTOR, resulting in an inhibition of mTOR signaling.

DEPTOR has been shown to interact with the C-terminal portion of mTOR (aa 1,483–2,000) that is upstream of its kinase domain (aa 2,430–2,549) including the ATP-binding site (aa 2,519–2,549).³² It is possible that the interaction between RSV and mTOR leads to a conformational change that promotes the interaction between mTOR and DEPTOR. Another possibility is that RSV may interact with DEPTOR and the formation of the RSV/DEPTOR complex is essential for interaction with mTOR. Further studies will be necessary to address these possibilities. Nevertheless, our finding that RSV promotes the interaction between DEPTOR and mTOR reveals a novel mechanism by which RSV negatively regulates mTOR signaling and function.