PerspectiveBiochemistry

New Insights into mTOR Signaling: mTORC2 and Beyond

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Science Signaling  21 Apr 2009:
Vol. 2, Issue 67, pp. pe27
DOI: 10.1126/scisignal.267pe27

Abstract

The mammalian target of rapamycin complex 2 (mTORC2) plays critical roles in regulating cell growth and proliferation. mTORC2 promotes the activation of the serum glucocorticoid–induced protein kinase (SGK). This mTOR complex also promotes the constitutive phosphorylation of proline-directed serine or threonine sites in the turn motif of Akt and protein kinase C isoforms. mTORC2 may control phosphorylation of the turn motif by promoting the activity of a kinase that targets the Ser/Thr-Pro sequence or by inhibiting the activity of a phosphatase.

Two mTOR Complexes

The mammalian target of rapamycin (mTOR) protein kinase operates at the hub of transduction networks that coordinate growth and division with extracellular signals, nutrients, and energy conditions. Many cancer-associated mutations promote expansion and proliferation of tumors through their ability to stimulate the protein kinase activity of two evolutionarily conserved mTOR complexes. The mTOR complex 1 (mTORC1) consists of mTOR, Raptor (regulatory associated protein of mTOR), and mLST8 (mammalian lethal with Sec13 protein 8), and is activated through a pathway involving Akt-mediated phosphorylation of PRAS40 (proline-rich substrate of Akt of 40 kD) and the tuberous sclerosis complex, resulting in activation of the Rheb guanosine triphosphatase (GTPase) (1, 2). mTORC1 is also activated by amino acids through a pathway involving bidirectional transport of amino acids across the plasma membrane (3) and the Rag GTPases (4, 5). When intracellular concentrations of adenosine triphosphate (ATP) are low, mTORC1 activity is suppressed through a pathway involving the kinases LKB1 and AMPK (adenosine monophosphate–activated protein kinase) (6). The second mTOR complex, mTORC2, consists of mTOR, Rictor (rapamycin-insensitive companion of mTOR), Sin1, mLST8, and Protor (protein associated with Rictor) (713). The activity of mTORC2 is controlled by phosphatidylinositol 3-kinase (PI3K) and, in contrast to mTORC1, is largely insensitive to nutrients or energy conditions.

The best-characterized mTOR substrates include a subgroup of related AGC family kinases comprising isoforms of Akt, p70 ribosomal S6 kinase (S6K), and protein kinase C (PKC). mTOR complexes directly phosphorylate these enzymes at a conserved noncatalytic residue within the C-terminal hydrophobic motif, which consists of Phe-X-X-Phe-Ser/Thr-Tyr (where X is any amino acid) and is evolutionarily conserved from yeast to mammals (Table 1). Activation of AGC kinases also requires phosphorylation of an additional site within the T-loop motif in the kinase catalytic domain, which is often mediated by the 3-phosphoinositide–dependent protein kinase 1 (PDK1) (14). mTORC1 phosphorylates the hydrophobic motif of S6K (15, 16), whereas mTORC2 phosphorylates the hydrophobic motifs of specific isoforms of Akt (17) and of PKC (12, 18). In the case of S6K and PKC, hydrophobic motif phosphorylation promotes activation of these enzymes by creating a high-affinity interaction site for PDK1, which then phosphorylates and activates these enzymes (14, 19).

Table 1 Summary of hydrophobic motif sites phosphorylated by mTOR complexes. The phosphorylated sites are bold and underlined. Other conserved sites are bold.
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SGK: An mTORC2-Specific Substrate

A recently identified mTORC2 substrate is the serum glucocorticoid–induced protein kinase 1 (SGK1) (2022). Although less well characterized than other members of the AGC family, SGK isoforms are known to be widely distributed, and their phosphorylation and activity are stimulated by growth factors and other agonists (23, 24). SGK isoforms exhibit overlapping substrate specificity with other AGC kinase family members, such as Akt, but they also phosphorylate a distinct set of substrates (23, 24). For example, SGK1 phosphorylates the Nedd4-2 ubiquitin E3 ligase, which promotes the interaction of Nedd4-2 with 14-3-3 proteins. This interaction prevents Nedd4-2 from binding to the epithelial sodium channel (ENaC) and targeting it for degradation (25, 26), thus enhancing ENaC stability and abundance and stimulating sodium transport (27).

The phosphorylation of the SGK1 hydrophobic motif site and SGK1 activity are ablated in fibroblasts from mice lacking mTORC2-specific subunits but still possessing mTORC1 activity (20). Furthermore, the SGK1 substrate NDRG1 (N-myc downstream regulated 1) is not phosphorylated in mTORC2-deficient fibroblasts, which is consistent with a lack of SGK1 activity (20). Moreover, immunoprecipitated mTORC2, but not mTORC1, phosphorylates the hydrophobic motif site of SGK1 in vitro (20). However, another group has reported that SGK1 is phosphorylated by mTORC1, not mTORC2 (28). A possible explanation for this discrepancy is that the antibody used to detect the phosphorylation of SGK1 at the hydrophobic motif site cross-reacts with S6K1 phosphorylated at its hydrophobic motif site (20). This may explain why the observed phosphorylation was suppressed by the mTORC1 inhibitor rapamycin (28), whereas other studies have shown that SGK1 activity or phosphorylation of the hydrophobic motif in SGK1 are insensitive to rapamycin (20, 29). Genetic studies in Caenorhabditis elegans analyzing the phenotype of Rictor knockout worms have also confirmed that SGK1 is regulated by TORC2 (21, 22).

SGK1 and Akt have similar and potentially overlapping substrate specificities, making it difficult to distinguish whether a substrate is phosphorylated by Akt or SGK. In cells lacking mTORC2 activity, SGK1 is not phosphorylated at its T-loop residue and is therefore inactive, but Akt is still active, because it is still phosphorylated at its T-loop Thr308 residue under these conditions (11, 12). Therefore, in mTORC2-deficient cells, SGK1 substrates are not phosphorylated, whereas Akt substrates are still likely phosphorylated, albeit at a reduced amount. Thus, in mTORC2-deficient cells, phosphorylation of potential SGK substrates, such as Forkhead transcription factors, is suppressed relative to other proteins that are unlikely to be SGK substrates, such as PRAS40 and glycogen synthase kinase–3 (GSK-3) (11, 12). The loss of SGK activity seems more likely to account for the reduced phosphorylation of certain proteins in mTORC2-deficient cells than other explanations that have been proposed, such as Akt possessing distinct substrate specificities depending on whether it is phosphorylated at its hydrophobic motif site (11).

SGK may be a key target of mTORC2, and the importance of SGK is underscored by increasing evidence that it regulates cancer cell growth and survival (30, 31). Furthermore, in C. elegans, the ability of TORC2 to regulate growth, fat metabolism, reproduction, and life span is mediated through Sgk1-1 rather than Akt (21, 22), thus calling into question the widely held view that Akt is the key mediator of signaling downstream of mTORC2. Thus, identifying proteins whose phosphorylation is inhibited in mTORC2-deficient cells may help to elucidate additional substrates and roles for SGK. The phosphorylated form of endogenous NDRG1 is readily detectable in most cells and tissues by commercially available antibodies, and therefore represents a useful marker to assess cellular mTORC2 activity.

Phosphorylation of the Turn Motif

In addition to the sites in the T-loop and hydrophobic motifs, AGC kinases possess a third conserved phosphorylation site in the turn motif. Located 12 to 23 residues before the hydrophobic motif, the turn motif has a Ser/Thr-Pro sequence quite distinct from that of the hydrophobic motif (32). Phosphorylation of the turn motif site stabilizes the active conformation of the kinase and may also protect the hydrophobic motif site from dephosphorylation (32). For some members of the AGC family, the turn motif site is constitutively phosphorylated during or soon after synthesis of the AGC kinases (33), in contrast to the phosphorylation of the hydrophobic motif site, which is induced by extracellular signals (34). Because the amino acid sequences surrounding the turn and hydrophobic motifs are quite different, most protein kinases would not phosphorylate two such different motifs. Unexpectedly, however, mTORC2 also promotes phosphorylation of the turn motif sites of Akt and PKC isoforms (35, 36). In mTORC2-deficient cells, isoforms of Akt and PKC are not phosphorylated at their turn motif site, leading to increased ubiquitination and decreased stability (35, 36). The abundance of specific PKC isoforms is also reduced in cells lacking the upstream kinase, PDK1 (37). Partial dephosphorylation of the turn motif sites of Akt1 and PKCα requires prolonged treatment of cells with mTOR inhibitors (35), which is consistent with phosphorylation of the turn motif site occurring during or immediately after protein translation and lack of accessibility of this site to phosphatases. In contrast to phosphorylation of the turn motif site in isoforms of Akt and PKC, phosphorylation of the turn motif site of S6K1 is not constitutive. Instead, it is induced by insulin, inhibited by rapamycin, and reportedly phosphorylated by mTORC1 in vitro (38).

The mechanism by which mTORC2 controls turn motif phosphorylation is unclear. One study reported that in vitro, immunoprecipitated mTORC2 phosphorylated Akt1 at its turn motif site (36). However, other groups, including ours (L.R.P. and D.R.A., unpublished observations), have been unable to demonstrate direct phosphorylation of the turn motif site of isoforms of Akt or PKC in vitro (35). One possible reason for this discrepancy may be the presence of a contaminating kinase in the assay used by Facchinetti et al., which is suggested by their finding that overexpressed catalytically inactive mTOR phosphorylated the turn motif site of Akt1 to only a slightly lower extent than did wild-type mTOR (36).

mTORC2 may regulate turn motif site phosphorylation through several potential mechanisms. One possibility is that only newly translated unfolded Akt or PKC is a substrate for mTORC2 (Fig. 1A). Fully folded Akt or PKC treated with a protein phosphatase to dephosphorylate the turn motif site may be in a conformation such that the turn motif site is not recognized and phosphorylated by mTORC2. Another possibility is that mTORC2 may need to interact with other adaptors or regulatory components in order to phosphorylate the turn motif site (Fig. 1B). A third option is that mTORC2 regulates turn motif site phosphorylation through an intermediate protein kinase (Fig. 1C). This role could be filled by a kinase from the CMGC family, such as cyclin-dependent kinases, which tend to phosphorylate Ser or Thr residues followed by Pro (39). It remains to be determined whether any CMGC kinases phosphorylate the turn motif sites of isoforms of Akt or PKC. Finally, it cannot be ruled out that mTORC2 regulates the phosphorylation status of the turn motif site by inhibiting the activity of a turn motif protein phosphatase (Fig. 1D). Indeed, elegant work carried out in yeast shows that TOR regulates the activity of protein phosphatases (40).

Fig. 1

Alternative mechanisms by which mTORC2 may regulate phosphorylation of turn motif sites. (A) mTORC2 phosphorylates newly synthesized Akt. (B) mTORC2 requires an adaptor to phosphorylate Akt. (C) mTORC2 directs a kinase that phosphorylates Ser or Thr residues that are followed by Pro. (D) mTORC2 inhibits the activity of a phosphatase. PH, pleckstrin homology domain.

Phosphorylation of 4E-BP1 by mTOR

mTOR also controls the phosphorylation of other substrates, such as 4E-BP1, an inhibitor of the eukaryotic translation initiation factor 4E (eIF4E). Phosphorylation of 4E-BP1 by mTOR induces dissociation from eIF4E to promote initiation of protein translation required during cell growth and proliferation (15, 16, 41). Ser and Thr residues in 4E-BP1 that are phosphorylated by mTOR are followed by Pro residues, which is similar to the turn motifs of Akt and PKC isoforms (Table 2). If the phosphorylation state of the turn motif sites of Akt and PKC isoforms is regulated by mTOR through an intermediate proline-directed kinase or phosphatase, it would be necessary to evaluate whether this mechanism is also involved in 4E-BP1 phosphorylation. Alternatively, there may be other rapamycin-resistant forms of mTOR distinct from mTORC2 that control phosphorylation of 4E-BP1, as suggested by work using small-molecule inhibitors that target both complexes of mTOR (42, 43). A distinct mTOR complex may exist that is capable of directly phosphorylating 4E-BP1 at its Ser/Thr-Pro sequences, as well as phosphorylating the turn motif sites of AGC kinases. The combined use of chemical and genetic approaches to inhibit mTOR complexes will undoubtedly lead to the discovery of additional roles for this master regulator of cell growth.

Table 2 Summary of confirmed and potential proline-directed sites phosphorylated by mTOR complexes. The phosphorylated sites are bold and underlined. The conserved proline residues are bold.
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Acknowledgments

48.Research in the Alessi laboratory is supported by the Medical Research Council and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit.

References

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