Regulation and function of the TSC‐mTOR pathway in cell growth
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The mammalian target of rapamycin (mTOR) plays a central role in the regulation of cell growth. mTOR exists in two distinct complexes, mTORC1 and mTORC2. mTORC1 is known to receive inputs from multiple signaling pathways, including growth factors, nutrients, and cellular energy levels to stimulate protein synthesis by phosphorylating key translation regulators such as ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E binding protein 1 (4EBP1). In contrast, mTORC2 regulates cell morphology and also phosphorylates AKT. Recent studies have established that dysregulated mTOR activity is associated with several hamartoma syndromes and metabolic disorders. The mechanism of mTOR regulation by numerous intracellular signaling pathways and the function of mTOR in physiological regulation will be discussed.Keywords:
mTORC2
RPTOR
Ribosomal protein s6
The atypical Ser/Thr kinase target of rapamycin (TOR) is a central controller of cell growth and proliferation. TOR forms two distinct multiprotein complexes, TORC1 and TORC2, which are structurally and functionally conserved from yeast to humans. Four major inputs control mammalian TOR (mTOR): growth factors, such as insulin; cellular energy levels, such as the AMP:ATP ratio; stress, such as hypoxia; and nutrients, such as amino acids. mTOR controls cell growth by the positive and negative regulation of several anabolic and catabolic processes, respectively, that collectively regulate cell size and proliferation. These cellular processes include autophagy, cytoskeleton rearrangement, glycolysis, lipogenesis, nutrient transport, ribosome biogenesis, and translation. Dysregulation of the mTOR signaling network has been associated with aging, and a multitude of diseases including cancer, cardiovascular disease, diabetes, inflammation, immune dysfunctions, and neurodegeneration. However, relatively few direct substrates of either one of the two mTOR complexes, mTORC1 and mTORC2, are known.
To determine downstream effectors of mammalian TOR (mTOR), we applied a functional, quantitative phosphoproteomics workflow to identify novel mTORC1 or mTORC2 regulated phosphorylations. Raptor and Rictor are essential components of mTORC1 and mTORC2, respectively. To distinguish phosphorylations regulated by mTORC1 or mTORC2, we specifically deleted Raptor or Rictor using an inducible gene knockout system in mouse embryonic fiberblasts (MEFs). We detected 4584 phosphorylation sites on 1398 proteins, and identified 335 novel mTOR effectors. Many of the novel effectors are implicated in cancer and metabolic diseases, but have no known links to mTOR. To distinguish direct mTOR substrates from indirect effectors, we combined peptide array in vitro kinase assays with phosphorylation motif analysis. This revealed that mTORC1 phosphorylates CAD in vivo and in vitro. CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) is the initial and rate limiting enzyme in de novo pyrimidine synthesis. The macrolide rapamycin, which forms a complex with FKBP12, binds and acutely inhibits mTORC1 but not mTORC2. Rapamycin treatment inhibited growth factor stimulated CAD phosphorylation and oligomerization, decreased de novo pyrimidine synthesis, and delayed progression of S-phase where CAD activity is essential. Thus mTORC1 phosphorylates CAD and thereby stimulates de novo pyrimidine synthesis to promote cell proliferation.
Separately, we characterize the autophosphorylation of mTOR on Ser2481. Insulin stimulates the phosphorylation of mTOR at Ser2481 specifically in mTORC2. Knockout of Rictor, but not Raptor, abolished mTOR autophosphorylation at Ser2481. Prolonged treatment with rapamycin, which indirectly inhibits mTORC2 complex formation, inhibited Ser2481 phosphorylation. Surprisingly, mTORC2 autophosphorylation at Ser2481 temporally occurs after the insulin-induced phosphorylation of Akt/PKB and the SGK1 substrate NDGR1. Mutation of Ser2481 to aspartic acid rendered mTOR unable to phosphorylate Akt/PKB in vitro. However the function of mTOR-Ser2481 phosphorylation in vivo remains elusive, as mutation of mTOR-Ser2481 did not alter Akt/PKB phosphorylation in vivo.
In summary, mTORC1 and mTORC2 regulate the phosphorylation of a functionally diverse set of substrates to control several anabolic and catabolic processes that determine cell size and proliferation. As a central controller of cell growth and proliferation, mTOR plays a key role in regulating development, whereas dysregulation of mTOR signaling has been linked to aging and diseases such as cancer and metabolic disorders.
mTORC2
Phosphoproteomics
RHEB
RPTOR
Ribosomal protein s6
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The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr kinase that comprises two complexes, termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 phosphorylates S6K1 at Thr 389, whereas mTORC2 phosphorylates AKT at Ser 473 to promote cell growth. As the mTOR name implies it is the target of natural product called rapamycin, a clinically approved drug used to treat human disease. Short-term rapamycin treatment inhibits the kinase activity of mTORC1 but not mTORC2. However, the ATP-competitive catalytic mTOR inhibitor Torin1 was identified to inhibit the kinase activity of both mTORC1 and mTORC2. Here, we report that H89 (N-(2-(4-bromocinnamylamino) ethyl)-5-isoquinolinesulfonamide), a well-characterized ATP-mimetic kinase inhibitor, renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors across multiple cell lines. Moreover, H89 prevented the dephosphorylation of AKT and S6K1 under nutrient depleted conditions. PKA and other known H89-targeted kinases do not alter the phosphorylation status of S6K1 and AKT. Pharmacological inhibition of some phosphatases also enhanced S6K1 and AKT phosphorylation. These findings suggest a new target for H89 by which it sustains the phosphorylation status of S6K1 and AKT, resulting in mTOR signaling.
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Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The vertebrate-specific DEP domain-containing mTOR interacting protein (DEPTOR), an oncoprotein or tumor suppressor, has important roles in metabolism, immunity, and cancer. It is the only protein that binds and regulates both complexes of mammalian target of rapamycin (mTOR), a central regulator of cell growth. Biochemical analysis and cryo-EM reconstructions of DEPTOR bound to human mTOR complex 1 (mTORC1) and mTORC2 reveal that both structured regions of DEPTOR, the PDZ domain and the DEP domain tandem (DEPt), are involved in mTOR interaction. The PDZ domain binds tightly with mildly activating effect, but then acts as an anchor for DEPt association that allosterically suppresses mTOR activation. The binding interfaces of the PDZ domain and DEPt also support further regulation by other signaling pathways. A separate, substrate-like mode of interaction for DEPTOR phosphorylation by mTOR complexes rationalizes inhibition of non-stimulated mTOR activity at higher DEPTOR concentrations. The multifaceted interplay between DEPTOR and mTOR provides a basis for understanding the divergent roles of DEPTOR in physiology and opens new routes for targeting the mTOR-DEPTOR interaction in disease. Introduction DEP domain-containing mTOR interacting protein (DEPTOR), conserved in vertebrates, modulates the activity of the serine/threonine kinase mammalian target of rapamycin (mTOR), a master regulator of cell growth. mTOR acts in two functionally distinct multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2 (Sabatini et al., 1994; Jacinto et al., 2004; Sarbassov et al., 2004; Liu and Sabatini, 2020; Loewith and Hall, 2011; Loewith et al., 2002), and DEPTOR is the only protein reported to bind and inhibit both mTOR complexes (Peterson et al., 2009). DEPTOR is a 46 kDa protein comprising an N-terminal DEP (Dishevelled, Egl-10, and Pleckstrin) domain tandem, herein referred to as DEPt, and a C-terminal PDZ (postsynaptic density 95, disks large, zonula occludens-1) domain. The PDZ domain has been suggested to interact with mTOR (Peterson et al., 2009), and DEPt mediates phosphatidic acid (PA) binding (Weng et al., 2021). The linker connecting DEPt and the PDZ domain contains a phosphodegron motif. mTOR phosphorylates this motif, leading to subsequent additional phosphorylation, ubiquitylation by the SCFβTrCP E3 ubiquitin ligase, and DEPTOR degradation (Gao et al., 2011; Zhao et al., 2011; Duan et al., 2011). DEPTOR degradation, in turn, leads to activation of mTORC1 and inactivation of mTORC2 via the mTOR negative feedback loop. OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) counteracts this process by deubiquitylating DEPTOR (Zhao et al., 2018). The interplay of mTOR and DEPTOR with the feedback loop from mTORC1 to mTORC2 and other signaling pathways leads to complex response patterns linked to variations in DEPTOR abundance depending on cell type and state (Caron et al., 2018; Catena and Fanciulli, 2017). DEPTOR plays central roles in cancer, obesity, and immunodeficiency (Caron et al., 2018; Laplante et al., 2012; Wedel et al., 2019; Wedel et al., 2016; Caron et al., 2016). It can act as both an oncoprotein and a tumor suppressor (Caron et al., 2018), and its effect in modulating PI3K-AKT signaling is variable depending on cancer type and cellular status. DEPTOR levels are low in most cancers due to active PI3K signaling (Caron et al., 2018). In few cancers, including multiple myeloma (Peterson et al., 2009), DEPTOR is overexpressed and promotes cancer cell survival. DEPTOR expression levels are increased in white adipose tissue in obesity and DEPTOR promotes adipogenesis by tuning down mTORC1 feedback control and thereby activating AKT signaling (Laplante et al., 2012). Despite its relevance to human health, DEPTOR is the only direct protein regulator of mTOR complexes whose molecular mechanism of action is unknown (Yang et al., 2017; Anandapadamanaban et al., 2019; Rogala et al., 2019). Results To investigate the interplay of DEPTOR and mTOR in both human mTOR complexes, we combined cryo-EM analysis of recombinantly expressed and purified DEPTOR-mTORC2 and DEPTOR-mTORC1 complexes at resolutions of 3.2 and 3.7 Å (Figure 1a, b and c; Figure 1—figure supplements 1–3 Video 1 and Video 2), respectively, with crystallographic and in solution structural characterization of the DEPt region of DEPTOR and biochemical analysis. The core architecture of the cryo-EM reconstructions of the two mTOR complexes in association with DEPTOR largely resembles that of their DEPTOR-free states (Figure 1a, b and c; Yang et al., 2017; Scaiola et al., 2020). In the mTORC1 and mTORC2 complexes associated with DEPTOR, the mTOR active site adopts a non-activated conformation (Yang et al., 2017; Scaiola et al., 2020; Figure 1—figure supplement 3g) and is not occupied by substrates. Inositol-hexakis-phosphate (IP6) was recently found to bind to mTORC1 and mTORC2, albeit without clear activating or inhibitory effect (Scaiola et al., 2020; Gat, 2019), and its binding is undisturbed in DEPTOR-bound mTORC1 and mTORC2 complexes. Binding sites for short linear TOS and RAIP motifs in mTORC1 substrates remain empty in the mTORC1-DEPTOR complex (Tee and Proud, 2002; Nojima, 2003; Schalm et al., 2002). Consistent density for DEPTOR is observed in two regions of the FAT domain of mTOR for both mTORC1 and mTORC2, in agreement with a regulatory effect of DEPTOR on both complexes (Peterson et al., 2009). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg The DEP domain-containing mammalian target of rapamycin (mTOR) interacting protein (DEPTOR)-mTOR complex 2 (mTORC2). Overview of cryo-EM reconstruction and model, highlighting functionally relevant sites and interactions discussed in the associated manuscript. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg The DEP domain-containing mammalian target of rapamycin (mTOR) interacting protein (DEPTOR)-mTOR complex 1 (mTORC1). Overview of cryo-EM reconstruction and model, highlighting functionally relevant sites and interactions discussed in the associated manuscript. Figure 1 with 3 supplements see all Download asset Open asset Cryo-EM reconstruction of DEP domain-containing mammalian target of rapamycin (mTOR) interacting protein (DEPTOR)-bound mTOR complexes 1 and 2 (mTORC1 and mTORC2). (a) Composite map of overall and local focused cryo-EM reconstructions of DEPTOR-bound mTORC2. (b) Schematic representation of the domain architecture of mTORC1, mTORC2, and DEPTOR. (c) Composite map of overall and local focused cryo-EM reconstructions of DEPTOR-mTORC1. In (a) and (c) proteins are colored according to the schemes in (b). DEPTOR binds to mTORC1 and mTORC2 in virtually identical manner via its extended PDZ-linker and DEP domain tandem (DEPt) regions associating with the FAT domain of mTOR. The DEPTOR-mTOR interaction occurs in two steps. In one step, the DEPTOR PDZ domain binds the mTOR FAT domain. The PDZ domain core (aaDEPTOR326–409) adopts a canonical PDZ fold and binds the TRD2 subdomain (Yang et al., 2013) of the mTOR FAT domain (Figure 2a and b; Figure 2—figure supplement 1a). The interaction interface is formed by a conserved surface of the PDZ domain and three mTOR helices (aamTOR1525–1578) (Figures 1, 2a and b; Figure 2—figure supplement 1a,b,c). The canonical PDZ domain peptide binding groove (Lee and Zheng, 2010) is present, but remains unoccupied in the interaction of the DEPTOR PDZ domain with mTOR (Figure 2—figure supplement 1d). This opens the possibility that binding of other, yet unknown protein partners via a canonical PDZ-peptide interaction to the DEPTOR PDZ domain could further strengthen or inhibit the mTOR-PDZ interaction. To the best of our knowledge, the mode of interaction of the DEPTOR PDZ domain with mTOR has not been observed for other PDZ domains. The binding interface between mTOR and the DEPTOR PDZ domain is considerably enlarged by contributions from regions which are known or predicted to be disordered in isolated mTOR complexes or DEPTOR. A loop in the Horn (also known as N-HEAT) (Yang et al., 2017; Aylett et al., 2016) region of mTOR (aamTOR290–350, DEPTOR-binding loop) (Figure 2c) was disordered in previous reconstructions of mTOR complexes in the absence of DEPTOR, and its function remained elusive (Yang et al., 2017; Scaiola et al., 2020). In complex with DEPTOR, residues aamTOR304–317 are ordered and the backbone of residues aamTOR290–303 connecting to the Horn is visible at lower resolution (Figure 2—figure supplement 1e). Residues aamTOR304–306 interact with the DEPTOR PDZ domain and residue FmTOR306 is inserted between the DEPTOR PDZ and mTOR FAT domains as an integral part of the interface (Figure 2—figure supplement 1f). The DEPTOR PDZ domain together with the DEPTOR-binding loop forms a structural link between the Horn and FAT domain of mTOR, positioned to mediate conformational crosstalk between different subregions of the mTOR complexes. The DEPTOR linker connecting DEPt and the PDZ domain remains largely unresolved (aaDEPTOR231–303), and only the C-terminal region of the linker (aaDEPTOR 304–325) is ordered when DEPTOR is bound to mTOR complexes. Residues aaDEPTOR309–325 provide an N-terminal extension to the PDZ core domain, while aaDEPTOR304–308 bind a groove formed by alpha-helices 14–16 of the mTOR FAT domain (Figure 2d). The linker-mTOR interaction enlarges the interface formed by the PDZ core domain suggesting functional relevance of linker residues aaDEPTOR304–308 for DEPTOR-mediated regulation of mTOR. Figure 2 with 1 supplement see all Download asset Open asset Architecture of the DEP domain-containing mammalian target of rapamycin (mTOR) interacting protein (DEPTOR) PDZ domain and its interaction with mTOR. (a, b) Front (a) and back (b) view of DEPTOR PDZ bound to the mTOR FAT domain. The PDZ domain (shown as transparent surface with red cartoon) binds to a hinge in the FAT domain of mTOR. (c) The PDZ domain N-terminal extension stretches toward the FAT domain. The adjacent N-terminal linker inserts into a groove on the FAT domain and substantially contributes the PDZ-mTOR interface. (d) Loop region (aamTOR290–350) in the mTOR Horn-region (transparent with cartoon) is disordered in free mTOR complexes and contributes to the mTOR-PDZ interface and thereby creates a link between the Horn-region and the FAT domain of mTOR and the DEPTOR PDZ domain. The other step of the DEPTOR-mTOR interaction is mediated by the DEPTOR DEPt region and the mTOR FAT domain (Figure 1a and c; Figure 3a). The DEPt region bound to mTOR is less well resolved in overall high-resolution reconstructions as a consequence of local flexibility and partial occupancy. 3D variability analysis, focused classification, and local refinement (Figure 1—figure supplements 1–S.1a and 2a) led to clear visualization of the overall fold and individual secondary structure elements (Figure 3—figure supplement 1a) at a local resolution of approximately 4–6 Å (Figure 1—figure supplement 3e,f). To obtain a pseudo-atomic model of the second DEPTOR-mTOR binding interface, we determined an X-ray crystal structure of a recombinant DEPt region (aaDEPTOR 1–230) at 1.93 Å resolution in a domain-swapped conformation as revealed by small-angle X-ray scattering (SAXS) in solution (Figure 3—figure supplement 1b,c). Each of the two domains in DEPt adopts a characteristic DEP domain fold comprising an alpha-helical core and a protruding beta-hairpin arm. In the DEPt, the two DEP domains are interacting via their N-terminal alpha-helices and a C-terminal extension of the second DEP domain that folds back onto the first DEP domain (Figure 3—figure supplement 1b). Binding of DEPt to mTOR preserves the overall fold of DEPt, but is linked to a 3.6 Å translation and 39° rotation between the two DEP domains (Figure 3—figure supplement 1d). Figure 3 with 2 supplements see all Download asset Open asset Interactions of the DEP domain-containing mammalian target of rapamycin (mTOR) interacting protein (DEPTOR) DEP domain tandem (DEPt) region with mTOR. (a) Surface representation of DEPTOR (transparent with cartoon in red) bound to mTOR complex 2 (mTORC2). The DEPt region binds centrally on top of the helical repeats of the FAT domain. (b) The protruding hairpin of the first DEP domain of DEPt inserts into a crevice between the kinase and FAT domain of mTOR. The DEPTOR-displacing mutant R2505P (Grabiner et al., 2014) is located in close proximity. (c) Analysis of the impact of wild-type and mutant forms of DEPTOR on Rheb-stimulated mTORC1 activity. Mutants are described in Figure 3—figure supplement 2. mTORC1 was incubated with 4E-BP1 and Rheb for stimulation, in the presence of DEPTOR wild-type and mutants. Reactions were separated by SDS-PAGE and analyzed by western blot. 4E-BP1 phosphorylation was detected with an antibody specific to phosphorylation of residues T37/46. Quantification (mean ± SD) of western blots in 4E-BP1-pT37/46 signals were normalized to total 4E-BP1 signals and the statistical significance of changes between control (0 µM DEPTOR) and DEPTOR variants determined by one-way ANOVA. ****p < 0.0001, ***p < 0.001, *p < 0.05, nsp >0.05, n = 4. Figure 3—source data 1 Source data of kinase assay. Uncropped blots of all four replicates (bands shown in Figure 3c indicated) and statistical analysis of western blot quantification. https://cdn.elifesciences.org/articles/70871/elife-70871-fig3-data1-v1.pdf Download elife-70871-fig3-data1-v1.pdf The DEPt region binds on top of the helical repeats of the mTOR FAT domain in the region aamTOR1680–1814 (Figure 3a). The binding interface of DEPt with mTOR is smaller than that of the PDZ-and-linker interaction site (~950 Å2 compared to ~1100 Å2). The N-terminal DEP domain, including the linker to the second DEP domain, forms the major part of the interface (~700 Å2 compared to ~250 Å2) and is better ordered than the C-terminal DEP domain (Figure 1—figure supplement 3e,f; Figure 3—figure supplement 1a). Notably, the N-terminal DEP domain is absent in one of the two known isoforms of human DEPTOR (Ota et al., 2004), likely abolishing DEPt-mTOR association. The protruding beta-hairpin of the N-terminal DEP domain inserts into a crevice between the FAT and kinase domains of mTOR (Figure 3b), where residue RmTOR2505 is located. This residue is altered to proline in a cancer-associated mutation that weakens DEPTOR binding to mTOR (Grabiner et al., 2014; Sato et al., 2010) and cannot be compensated by DEPTOR overexpression, underlining the functional relevance of this interaction (Figure 3b; Xu et al., 2016). mTOR interacting residues of DEPt are highly conserved (Figure 3—figure supplement 1e) and the surface electrostatic potentials around the interface are complementary (Figure 3—figure supplement 1f). Notably, two positively charged patches in DEPt, which are involved in mTOR interaction, were found to bind PA (Weng et al., 2021). PA has been reported to displace DEPTOR from mTOR complexes (Yoon et al., 2015). Previously described mechanisms of mTOR inhibition include ATP-competitive binding to the kinase active site in mTORC1 and mTORC2 (e.g. Torin1) (Thoreen et al., 2009), steric hindrance of access to the active site by the FKBP12-Rapamycin complex (Yang et al., 2017; Choi et al., 1996), and competition with substrate-guiding interactions specific to mTORC1 by the FRB domain binding protein inhibitor PRAS-40. Competitive binding at other substrate recognition elements, such as the TOS and RAIP motif binding sites in mTORC1 (Yang et al., 2017; Tee and Proud, 2002; Nojima, 2003; Schalm et al., 2002; Böhm, 2021; Wang et al., 2007) or C-terminal parts of SIN1 (Tatebe et al., 2017) in mTORC2, provides alternative target sites for mTOR inhibition. Recently developed small molecule mTOR inhibitors either utilize the above inhibitory mechanisms or their detailed mode of action is still unknown (Benavides-Serrato et al., 2017; Benjamin et al., 2011; Wang et al., 2020; Guenzle et al., 2020). Notably, DEPTOR is not only a modulator of mTORC1 and mTORC2 activity, but also a substrate of mTOR in mTORC1 and mTORC2 (Peterson et al., 2009; Gao et al., 2011; Zhao et al., 2011; Duan et al., 2011). Indeed, we observe weak residual density in the DEPTOR-mTORC1 complex at a binding site for helical peptide segments of substrates and the inhibitor PRAS40 on the FRB domain, which might represent a dynamically interacting segment of DEPTOR or copurified interacting proteins (Figure 4—figure supplement 1a). Based on distance constraints, binding of the linker of DEPTOR with an extended helix as in PRAS40 to the FRB site is incompatible with the DEPTOR association to mTOR via its PDZ and DEPt regions; a smaller association patch cannot be ruled out, but would require fully extended surrounding linker regions. Still, it is sterically impossible that all sites for mTOR-mediated phosphorylation in the DEPTOR linker (aaDEPTOR 244, 265, 286, 293, 295, 296, 299; Peterson et al., 2009; Gao et al., 2011; Zhao et al., 2011; Duan et al., 2011) could reach the mTOR active site when DEPTOR is associated with mTORC1 via its PDZ and DEPt regions. Thus, a secondary linker-mediated, low-affinity binding mode of DEPTOR (or in trans-phosphorylation without recruiting signal) is required and provides a plausible explanation for the residual signal at the FRB domain. To test the relevance of DEPTOR interactions in the regulation of mTOR activity, we analyzed phosphorylation of 4E-BP1 by Rheb-activated mTORC1. An equivalent in vitro activity assay with activated mTORC2 has not been described. Insect cell and Escherichia coli expressed DEPTOR, which are partially phosphorylated or unphosphorylated, respectively, show a 60–70% inhibition of 4E-BP1 phosphorylation at T4E-BP137/46 (Figure 3c, Figure 3—source data 1). This inhibition is partially abolished by a single mutation and fully reverted by a triple mutation of the core PDZ interface (Figure 3c, Figure 3—figure supplement 2). Notably, mutations in the interface between DEPt and mTOR lead to stimulation of 4E-BP1 phosphorylation (Figure 3c, Figure 3—figure supplement 2), suggesting that DEPt mediates the inhibitory effect of DEPTOR on mTOR complexes, while the binding of DEPt interface mutants only via the PDZ domain even mildly activates Rheb-bound mTORC1 (Figure 3c). Discussion Our structural and mutational analyses suggest a model for DEPTOR action on mTOR complexes, in which DEPTOR provides an additional layer of control with the ability to stimulate or inhibit the mTOR complexes (Figure 4a). In this model DEPTOR associates with high affinity via its PDZ domain anchor, possibly modulated by other PDZ-binding proteins, followed by lower-affinity association of DEPt based on avidity. DEPTOR partially inhibits mTOR activity by a dominant negative effect of DEPt association or moderately stimulates mTOR activity via the influence of the PDZ domain, if DEPt is prevented from mTOR association by PA binding (Figure 4b). A suppression of non-stimulated basal mTORC1 or mTORC2 would only be observed at high concentration of DEPTOR (Figure 4b) that result in additional substrate-like binding of DEPTOR to mTORCs. Figure 4 with 1 supplement see all Download asset Open asset Model for the DEP domain-containing mammalian target of rapamycin (mTOR) interacting protein (DEPTOR)-mediated regulation of mTOR activity. (a) Structure-based representation of (1) the basal state of non-activated mTOR complex 1 (mTORC1) (based on PDB: 6BCX), (2) the allosteric activation of mTORC1 by Rheb binding (based on PDB: 6BCU), and (3) the impact of DEPTOR association via the PDZ domain and DEP domain tandem (DEPt) on the conformational state and activity of mTORC1. Possible transitions in subpopulations of conformational states are indicated by shadowing. (b) Schematic diagram of the suggested regulatory interactions between DEPTOR and mTOR complexes. Structurally characterized states shown in (a) are indicated by numbers. DEPTOR binding via the PDZ domain and DEPt prevents allosteric activation. At high concentrations, DEPTOR binds to mTORC1 in a secondary binding mode as a substrate and sterically influences access of other substrates to the active site. Phosphatidic acid (PA) may interfere with the DEPt-mTOR association, relieving the allosteric inhibition of mTORCs. The remaining bound PDZ domain mildly stimulates kinase activity in activated and non-activated mTOR complexes. The isolated DEPt region of DEPTOR has been reported to lack significant binding to mTORC1 and to have no effect on mTORC1 activity, resulting in the hypothesis that DEPt is not involved in controlling mTOR activity in the context of full-length DEPTOR (Peterson et al., 2009; Heimhalt et al., 2021). However, our data show that DEPt, when anchored via the PDZ domain, binds to a region of the mTOR FAT domain and suppresses allosteric activation of mTOR. Avidity of combined strong PDZ and weak DEPt interactions is supported by the earlier observation that full-length DEPTOR inhibits mTORC1 activation already at a lower concentration than required for binding of the isolated PDZ domain: The isolated PDZ domain binds with a Kd of 0.6 μM to the mTORC1 variant AmTOR1459P that mimics activation by Rheb, but the IC50 value for DEPTOR in the same system is 30–50 nM (Heimhalt et al., 2021). The functional relevance of a similar interplay of strong and extremely weak association has recently been demonstrated for two mTORC1-binding motifs in 4E-BP1, the high affinity TOS motif and the very low-affinity RAIP motif (Böhm, 2021). We observed an unexpected, weak activation of Rheb-stimulated mTORC1 activity in mutants of the DEPt interface which we attributed to an effect of PDZ domain binding. This effect is consistent with the observation of increased 4E-BP1 phosphorylation by mTORC1 in the presence of equimolar isolated PDZ domain (at overall nanomolar concentrations, Figure 2—figure supplement 1/Figure 6—figure supplement 1 in Heimhalt et al., 2021). It has also been reported that the PDZ domain has an approximately 10-fold higher affinity (Kd 0.6 μM vs. 7 μM) for binding to activated vs. non-activated mTORC1 (Heimhalt et al., 2021), despite a lack of differences in the interface in static structures of activated and non-activated mTOR complexes (Yang et al., 2017). Together, these data suggest that association of the PDZ domain is linked to changes in the dynamics of its binding site on mTOR, which are allosterically coupled to mTOR activation. Binding of other proteins to DEPTOR based on a canonical peptide-PDZ domain interaction via the empty PDZ-peptide binding groove may modulate the affinity of the PDZ to mTOR or even its effect on activity when bound to mTOR. Why are low concentrations of DEPTOR inhibiting Rheb-stimulated mTORC1 but much higher concentrations of DEPTOR are required (Heimhalt et al., 2021) for the reported inhibition of non-stimulated mTORCs (Peterson et al., 2009; Gao et al., 2011; Laplante et al., 2012; Yoon et al., 2015)? Facilitated by PDZ binding to mTOR, DEPt associates with non-activated mTOR complexes without inducing structural changes in the FAT region, as visualized here for mTORC1 and mTORC2. We suggest that DEPt association with a region of the FAT domain, that transduces allosteric activation by Rheb, specifically suppresses the conformational coupling between the Rheb binding site and the kinase site and consequently reduces only the stimulation of mTORC1 activity. This may occur by increasing the population of a state of the FAT region that is less competent for transmitting allosteric activation, either directly the state observed in non-activated mTOR or other intermediate states. Such a mode of action suggests the absence of inhibition of non-stimulated basal mTORC activity at low concentrations of DEPTOR. An alternative explanation for the lack of inhibition at low DEPTOR concentrations (Heimhalt et al., 2021) could be the failure to associate with mTORCs as a result of the differential interaction of the PDZ domain with stimulated vs. non-stimulated mTORC1, Kd of 0.6 μM vs. 7 μM (Heimhalt et al., 2021), respectively. However, based on our demonstration of an additional interface for DEPt binding, the avidity of combined DEPt and PDZ association suggests that the association of full-length DEPTOR still occurs at a concentration lower than the approximately 15–50 μM required for effective inhibition of non-stimulated mTORC1 (Heimhalt et al., 2021). A plausible explanation for inhibition of mTORC1 and mTORC2 at higher concentrations could be a secondary, lower-affinity binding mode at excess concentrations of DEPTOR over mTOR that does not involve interactions of PDZ and DEPt with mTOR. Indeed, DEPTOR is a substrate for mTORC1 and mTORC2 (Peterson et al., 2009; Gao et al., 2011; Zhao et al., 2011; Duan et al., 2011). Substrate recruitment by TOR complexes involves specific substrates recruitment via medium- and low-affinity interactions outside the kinase domain for many substrates (Yang et al., 2017; Tee and Proud, 2002; Nojima, 2003; Schalm et al., 2002; Böhm, 2021; Tatebe et al., 2017), but the primary DEPTOR binding via PDZ domain and DEPt is not suitable for recruitment, suggesting lower-affinity secondary binding modes to mTORC1 and mTORC2. Indeed, such an alternative, substrate-like weak interaction is indicated by residual density observed here in a known substrate recruitment site for mTORC1. We have previously shown that the core substrate recognition regions in mTORC2 are flexibly disposed, explaining why substrate interactions are not visualized in the current type of cryo-EM analysis (Scaiola et al., 2020). Substrate-like association of DEPTOR, via the FRB domain or other regions in mTORC2, and simultaneous recruitment of other substrates with their respective binding motifs, for example, 4E-BP1 with its TOS and RAIP motifs, would result in a mutual restriction of access to the mTOR active site that may partially be uncoupled from solution concentrations and dominated by local protein dynamics. At the same time, we consider that the core mechanism for the high-affinity mode of inhibition of activated mTOR complexes by DEPTOR is unlikely to be based on a PRAS40-like FRB interaction because (1) the binding site on the FRB domain is not accessible in mTORC2 (Figure 4—figure supplement 1b), (2) this would leave the conserved characteristic DEPt and its mTOR interaction involving RmTOR2505 without assigned function, and (3) it provides no additional explanation for differential effects on stimulated and non-stimulated mTORC1, as the binding site on the FRB domain is not coupled to allosteric activation. Notably, the interface of DEPt and mTOR suggested here to mediate inhibition of mTOR inhibition involves regions of DEPt that have been recently implicated in PA interaction (Weng et al., 2021). We hypothesize that DEPt interaction with PA may control DEPt association with mTOR, resulting in either PDZ-based activation or DEPt-based down-regulation of activated mTOR complexes. This would create a mechanistic link between PA signaling and mTOR activation on membranes (Figure 4b; Takahara et al., 2020). DEPTOR has been characterized as a modulator of mTOR activity with a profound impact on metabolism and cancer (Liu and Sabatini, 2020). However, its divergent and orthogonal effects on cell physiology, including its apparently antagonistic roles as an oncoprotein and tumor suppressor, have remained enigmatic. Here, we provide a structure-guided model of the complex interplay of DEPTOR with mTORC1 and mTORC2 that identifies orthogonal contributions by different interacting regions of DEPTOR, and further potential for modulation by crosstalk from other signaling pathways. The molecular insights provided here will be a crucial component for targeted dissection of DEPTOR effects on mTOR signaling to further understand the divergent effects of DEPTOR in physiology and disease. Materials and methods Protein expression and purification Request a detailed protocol Sf21 insect cells (Expression Systems) were grown in HyClone insect cell media (GE Life Sciences) and baculovirus was generated according to Fitzgerald et al., 2006. mTORC2 was expressed and purified as previously described with an internal FLAG-tag inserted after D258 (Scaiola et al., 2020). Purified mTORC2 was concentrated in gel filtration buffer, supplemented with 5% w/v glycerol and stored at –80°C until further use. For expression of human mTORC1, Sf21 insect cells were infected with baculovirus as described previously (Aylett et al., 2016). Cells were harvested 72 hr after infection by centrifugation at 800×g for 15 min and stored at −80°C until further use. Cell pellet was lysed in 50 mM bicine (pH 8), 250 mM NaCl, and 5 mM MgCl2 using a dounce homogenizer and the lysate was cleared by ultracentrifugation. Soluble protein was incubated with 7 ml of anti-DYKDDDDK agarose beads (Genscript, Piscataway, NJ) for 1 hr at 4°C. The beads were transferred to a 50 ml gravity flow column (Bio-Rad) and washed four times with 200 ml of wash buffer containing 50 mM bicine (pH 8), 150 mM NaCl, and 5 mM MgCl2. Protein was elut
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ABSTRACT We treated the thymoma cell line (EL4) with two model immunosuppressants, rapamycin and tributyltin oxide (TBTO), and compared their effects on the expression levels of proteins that are downstream targets of mTOR kinase 1 (mammalian target of rapamycin, known also as mechanistic target of rapamycin): p70 ribosomal S6 kinase1 and 4E‐binding protein 1, a repressor of the cap‐binding protein eIF4E. In addition, we evaluated the levels of ribosomal protein S6, p‐eIF4B, substrates of p70S6 kinase1, matrin 3 and ribonucleotide reductase, subunit RRM2. The levels of these proteins were evaluated in cell lysates by immunoblot. We found that both compounds inhibited the phosphorylation state of p70S6 kinase 1 and its substrates; however, TBTO, in contrast to rapamycin, reduced the level of the total p70S6k1. Besides, we detected a band with a molecular weight of c . 32 kDa only in the TBTO‐treated lysates. This band was detected with a monoclonal antibody specific for S6k1, suggesting that this band might be a degradation product of the kinase. Further, TBTO and rapamycin differentially affected 4E‐binding protein 1; the former compound stimulated its phosphorylation state whereas the latter inhibited it. The two immunosuppressants did not affect the level of ribonucleotide reductase, but TBTO downregulated matrin3, in agreement with a previous report, whereas rapamycin had no effect on the expression level of this latter protein. We conclude that TBTO inhibits, like rapamycin, the p70 S6 kinase 1 pathway, but with a different mechanism. However, in contrast to rapamycin, which inhibits the cap‐dependent translation, TBTO increases the phosphorylation of 4E‐binding protein1. Copyright © 2013 John Wiley & Sons, Ltd.
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The mammalian target of rapamycin(mTOR) is a serine /threonine kinase in cytoplasm and a central regulator of mRNA translation that plays a critical role in sensing the signals of nutrients and regulating cell growth and proliferation. mTOR complex 1(mTORC1) is more sensitive to rapamycin,which mediates the postprandial increase in muscle protein synthesis. Besides,it regulates the accumulation of protein by enhancing the activity of eukaryotic initiation factor 4E binding protein-1(4E-BP1) and ribosomal protein S6 kinase1(S6K1) to promote translation initiation. This paper reviewed the regulation mechanisms of protein deposition by mTOR and its influencing factors,and provided certain references for further investigation on the regulation of mTOR in protein deposition of piglets.
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The "nutrient sensor" mammalian target of rapamycin (mTOR) performs important functions of cell growth and proliferation. mTOR is known to exist in two distinct complexes, the rapamycin-sensitive mTOR complex 1 (mTORC1), which is bound to Raptor; and the rapamycin-insensitive mTOR complex 2 (mTORC2), which is bound to Rictor. mTORC1 is a known activator of the ribosomal protein S6 kinase (S6K1), while mTORC2 has recently been shown to phosphorylate Akt on Serine (S)473. PURPOSE: To determine the effects of a high-fat diet and exercise training on mTORC 1/2 formation and the activation of its downstream substrates. METHODS: We examined the effects of an 8 wk high fat diet (HF) and 4 wktreadmill running (EX) intervention on the mTOR signalling pathway in skeletal muscle. Co-immunoprecipitation of mTOR and its binding partners, Raptor and Rictor, followed by Western blot analysis were used to determine mTORC 1/2 formation. The activation and total protein content of mTORC 1/2 and its downstream substrates were also determined. RESULTS: The association of mTOR and its binding partners Raptor (p=0.02) and Rictor (p=0.03) were increased in HF by ~70% and ~60%, respectively. The increase in mTORC 1/2 formation was attenuated by EX. Compared to HF, EX (p=0.01) increased S2448 phosphorylation of mTOR by ~30%. Both Threonine (T)389 and S421/T424 phosphorylation of S6K1 were increased by ~20% in HF (P=0.004 and p=0.04, respectively). The activation of S6K1 was associated with similar increases in the phosphorylation of its direct sites (S636/S639) on insulin receptor substrate 1 (IRS1) in HF (p=0.01). EX prevented the activation of S6K1 and the serine phosphorylation of IRS1. Insulin stimulated S473 phosphorylation of Akt1 was increased by ~25% in HF (P<0.001). EX prevented the increase of Akt1 S473 phosphorylation induced by HF (P=0.02). No changes were observed in the S473 phosphorylation of the Akt2 isoform. CONCLUSIONS: Our results suggest that high fat feeding increases mTORC 1/2 complex formation, resulting in the activation of Akt1 and S6K1, and inhibitory serine phosphorylation of IRS1. Exercise training reverses the effects of a high-fat diet on mTOR complex formation and downstream signalling events. Supported by an RMIT SMS Grant (SJL), ARC Grant DP0663862 (JAH) and NIH Grant GM-48680, GM-08395 and DK-57625 (BBY)
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Abstract The prevalence of mTOR activation in cancer has led to the development of two classes of inhibitors of the protein as therapeutics: the natural product rapamycin and its analogs as well as direct inhibitors of mTOR kinase. mTOR exists in at least two multi-protein complexes. The mTORC1 complex phosphorylates S6K and 4EBP and stimulates protein translation, metabolism as well as other processes. The mTORC2 complex phosphorylates and activates AKT. Rapamycin binds to the immunophillin FKBP12. Drug-bound FKBP12 complex binds to mTOR FRB domain and selectively inhibits the activity of mTORC1. However, rapalogs preferentially inhibit S6K phosphorylation compared to 4EBP phosphorylation. Rapalogs have undergone extensive clinical testing and have significant antitumor activity in renal cell and pancreatic neuroendocrine tumors and, in combination with aromatase inhibitors, in resistant, ER positive breast cancers. In contrast, mTOR kinase inhibitors suppress both mTORC1 and mTORC2 functions and potently inhibit S6K, 4EBP and AKT S473 phosphorylation, these drugs are in early clinical testing. In order to better understand the mechanism of action of these drugs and potential mechanisms of tumor escape from mTOR inhibition, we selected breast tumor cells for resistance to growth inhibition in cell culture by treatment with either rapamycin or an mTOR kinase inhibitor. In rapamycin resistant cells, phosphorylation of S6K and S6 were insensitive to the drug, but remained sensitive to mTOR kinase inhibitors. Conversely, in clones resistant to mTOR kinase inhibitors, mTORC1 and mTORC2 substrates were insensitive to the drugs, but S6K and S6 phosphorylation remained sensitive to rapamycin. Deep sequencing results explained these findings: rapamycin resistant clones harbored mutations in the FRB domain of mTOR, in the sites shown to interact with the FKBP12-rapamycin complex; mTOR kinase resistant clones harbored mutations in the mTOR catalytic domain. These mutations were not observed in the parental cells. It is likely that the mutations identified in each domain prevent binding of the drug. Consistent with these data, growth of rapamycin resistant cells retain sensitivity to mTOR kinase inhibitors and mTOR kinase resistant cells retain sensitivity to rapalogs, both in tissue culture and in vivo. The results suggest that tumor cells with acquired resistance to mTOR inhibitors retain a requirement for mTOR signaling for proliferation. Furthermore, tumors resistant to either class of drug may not be cross-resistant to the other and combined therapy with both might delay the onset of resistance. Further studies on the genetics of human tumors with acquired resistance to these agents will determine the clinical importance of these findings. Citation Format: Vanessa S. Rodrik-Outmezguine, Zhan Yao, Radha Mukherjee, Liqun Cai, Derek Barratt, Richard Ward, Teresa Klinowska, Elisa De Stanchina, Michael Berger, Jose Baselga, Neal Rosen. Acquired resistance to rapamycin and mTOR kinase inhibitors is mediated by non-overlapping mutations in distinct sites in the mTOR protein. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1726. doi:10.1158/1538-7445.AM2014-1726
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The mammalian target of rapamycin (mTOR) kinase occurs in mTOR complex 1 (mTORC1) and complex 2 (mTORC2), primarily differing by the substrate specificity factors raptor (in mTORC1) and rictor (in mTORC2). Both complexes are activated during human cytomegalovirus (HCMV) infection. mTORC1 phosphorylates eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) and p70S6 kinase (S6K) in uninfected cells, and this activity is lost upon raptor depletion. In infected cells, 4E-BP1 and S6K phosphorylation is maintained when raptor or rictor is depleted, suggesting that either mTOR complex can phosphorylate 4E-BP1 and S6K. Studies using the mTOR inhibitor Torin1 show that phosphorylation of 4E-BP1 and S6K in infected cells depends on mTOR kinase. The total levels of 4E-BP1 and viral proteins representative of all temporal classes were lowered by Torin1 treatment and by raptor, but not rictor, depletion, suggesting that mTORC1 is involved in the production of all classes of HCMV proteins. We also show that Torin1 inhibition of mTOR kinase is rapid and most deleterious at early times of infection. While Torin1 treatment from the beginning of infection significantly inhibited translation of viral proteins, its addition at later time points had far less effect. Thus, with respect to mTOR's role in translational control, HCMV depends on it early in infection but can bypass it at later times of infection. Depletion of 4E-BP1 by use of short hairpin RNAs (shRNAs) did not rescue HCMV growth in Torin1-treated human fibroblasts as it has been shown to in murine cytomegalovirus (MCMV)-infected 4E-BP1(-/-) mouse embryo fibroblasts (MEFs), suggesting that during HCMV infection mTOR kinase has additional roles other than phosphorylating and inactivating 4E-BP1. Overall, our data suggest a dynamic relationship between HCMV and mTOR kinase which changes during the course of infection.
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Abstract Purpose: Rapamycin inhibits the serine-threonine kinase mammalian target of rapamycin (mTOR), blocking phosphorylation of p70 S6 kinase (S6K1) and 4E-binding protein 1 (4E-BP1) and inhibiting protein translation and cell cycle progression. Rapamycin and its analogues are currently being tested in clinical trials as novel-targeted anticancer agents. Although rapamycin analogues show activity in clinical trials, only some of the treated patients respond. The purpose of this study is to identify determinants of rapamycin sensitivity that may assist the selection of appropriate patients for therapy. Experimental Design: Breast cancer cell lines representing a spectrum of aberrations in the mTOR signaling pathway were tested for rapamycin sensitivity. The expression and phosphorylation state of multiple components of the pathway were tested by Western blot analysis, in the presence and absence of rapamycin. Results: Cell proliferation was significantly inhibited in response to rapamycin in 12 of 15 breast cancer cell lines. The ratio of total protein levels of 4E-BP1 to its binding partner eukaryotic initiation factor 4E did not predict rapamycin sensitivity. In contrast, overexpression of S6K1, and phosphorylated Akt independent of phosphatase and tensin homologue deleted from chromosome 10 status, were associated with rapamycin sensitivity. Targeting S6K1 and Akt with small interfering RNA and dominant-negative constructs, respectively, decreased rapamycin sensitivity. Rapamycin inhibited the phosphorylation of S6K1, ribosomal S6 protein, and 4E-BP1 in rapamycin-resistant as well as -sensitive cells, indicating that its ability to inhibit the mTOR pathway is not sufficient to confer sensitivity to rapamycin. In contrast, rapamycin treatment was associated with decreased cyclin D1 levels in the rapamycin-sensitive cells but not in rapamycin-resistant cells. Conclusions: Overexpression of S6K1 and expression of phosphorylated Akt should be evaluated as predictors of rapamycin sensitivity in breast cancer patients. Furthermore, changes in cyclin D1 levels provide a potential pharmacodynamic marker of response to rapamycin.
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