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The activities of both mTORC1 and mTORC2 are negatively regulated by their endogenous inhibitor, DEPTOR. As such, the abundance of DEPTOR is a critical determinant in the activity status of the mTOR network. DEPTOR stability is governed by the 26S-proteasome through a largely unknown mechanism. Here we describe an mTOR-dependent phosphorylation-driven pathway for DEPTOR destruction via SCFβ-TRCP. DEPTOR phosphorylation by mTOR in response to growth signals, and in collaboration with casein kinase I (CKI), generates a phosphodegron that binds β-TRCP. Failure to degrade DEPTOR through either degron mutation or β-TRCP depletion leads to reduced mTOR activity, reduced S6 kinase activity, and activation of autophagy to reduce cell growth. This work expands the current understanding of mTOR regulation by revealing a positive feedback loop involving mTOR and CKI-dependent turnover of its inhibitor, DEPTOR, suggesting that misregulation of the DEPTOR destruction pathway might contribute to aberrant activation of mTOR in disease.
The Mammalian Target of Rapamycin (mTOR) protein is an evolutionarily conserved Serine/Threonine kinase belonging to the phosphoinositide-3-kinase (PI 3K)-related family (PIKKs) of kinases (Sengupta et al., 2010). mTOR plays a central role in regulating a variety of cellular processes, including cell growth, cell metabolism, autophagy and cell cycle progression (Sabatini, 2006; Zoncu et al., 2010). This is achieved primarily by stress-induced modulation of mTOR kinase activity, thereby promoting downstream phosphorylation cascades (Efeyan and Sabatini, 2010; Zoncu et al., 2010). Similar to other members of the PIKK family of kinases such as ATR and ATM that respond to genotoxic stresses (Harper and Elledge, 2007), mTOR behaves as a sensor of metabolic or nutrient stress, thereby allowing cells to survive under non-optimal conditions. mTOR exists in two distinct multi-component complexes referred to as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (Alessi et al., 2009; Guertin and Sabatini, 2007; Reiling and Sabatini, 2006), which function to control largely independent arms of the mTOR network. Both complexes include the mLST8/GβL subunit but mTORC1 also contains the RAPTOR and PRAS40 (Sengupta et al., 2010) while mTORC2 contains RICTOR, SIN1 and PROTOR (Alessi et al., 2009). Subunits unique to each mTORC complex are thought to control their unique target specificity and/or regulatory properties, with mTORC1 functioning primarily to control protein synthesis via S6 kinase (S6K) phosphorylation (Yang and Guan, 2007), and mTORC2 primarily controlling cell survival in response to growth factors via phosphorylation and activation of AKT (Ser473) (Sarbassov et al., 2005) and SGK1 (Ser422) (Garcia-Martinez and Alessi, 2008). mTORC1 also suppresses autophagy via phosphorylation and inactivation of the ULK1/RB1CC1/ATG13/ATG101 complex (Jung et al., 2009).
Given the pivotal role of mTOR kinase in sensing the environmental conditions to control a wide range of cellular processes, its kinase activity is tightly controlled. As such, deregulated mTOR activity has been recently documented in numerous disease states including cancer, diabetes, and aging (Zoncu et al., 2010). Therefore, it is critical to understand the molecular mechanisms that govern mTOR kinase activity. mTORC1 kinase activity is negatively controlled by TSC2 (Inoki et al., 2002) and PRAS40 (Sancak et al., 2007; Vander Haar et al., 2007). Following growth factor stimulation, the PI3K kinase pathway is activated, leading to subsequent phosphorylation of both TSC2 and PRAS40 by AKT, which triggers activation of the mTORC1 kinase (Manning and Cantley, 2007). Additionally, in low energy states, activation of AMP-activated protein kinase (AMPK) leads to repression of mTORC1 activity mediated by AMPK-dependent phosphorylation of RAPTOR (Gwinn et al., 2008). In contrast with mTORC1, mTORC2 kinase activity is not sensitive to low dose rapamycin treatment (Sarbassov et al., 2006) and is not controlled by TSC2 and PRAS40. Aberrant mTORC2 activity has been implicated in cancer via activation of the growth promoting kinases AKT and SGK (Guertin and Sabatini, 2007; Manning and Cantley, 2007; Sahoo et al., 2005). While the cancer linkage with mTORC2 is strong, relatively little is known about the mechanisms that control mTORC2 activity status, and the extent to whether there is crosstalk between the mTORC1 and mTORC2 networks.
Further insight into the regulation of mTORC1 and mTORC2 has come with the discovery of an endogenous inhibitor of both mTORC1 and mTORC2 called DEPTOR (Peterson et al., 2009). DEPTOR, also known as DEPDC6, directly suppresses mTOR by interacting with the FAT domain of mTOR via a PDZ (postsynaptic density 95, discs large, zonula occludens-1) domain located at its C-terminus (Peterson et al., 2009). DEPTOR also contains two DEP (disheveled, egl-10, pleckstrin) domains of largely unknown function at its N-terminus. Importantly, DEPTOR is an unstable protein and is rapidly degraded following growth factor stimulation (Peterson et al., 2009). Rapid DEPTOR destruction may underlie activation of mTOR kinase activity when cells are shifted from unfavorable to favorable environments. However, the mechanism and cellular signals controlling DEPTOR stability remain unknown. In particular, while proteasomal inhibition blocks growth factor-dependent DEPTOR turnover and decreased S6K phosphorylation (Ghosh et al., 2008; Peterson et al., 2009), it has recently been shown that mTOR activation is linked to the lysosomal compartment (Sancak et al., 2010), raising the question of whether the effects of proteasome inhibition on mTOR activity reflects DEPTOR stabilization directly or effects on upstream signaling pathways. Here we report that the stability of DEPTOR is controlled by the SCFβ-TRCP E3 ubiquitin ligase. Our studies thus provide important insight into how the ubiquitin-proteasomal pathway controls mTOR activity and suggest that inappropriate activation of DEPTOR destruction may contribute to the deregulated mTOR activity observed in many disease states.
Consistent with a previous report (Peterson et al., 2009), we found that DEPTOR accumulates upon serum starvation and is rapidly degraded upon serum re-addition (Figure 1A). This behavior was unique to DEPTOR, as no significant fluctuation was observed in the expression of other mTORC components we examined (Figure 1A). Reduced DEPTOR expression upon serum stimulation reflects reduced DEPTOR mRNA abundance (Peterson et al., 2009) (Figures S1A–B), and 26S proteasome-mediated degradation, as the application of the proteasomal inhibitor, MG132, efficiently blocked DEPTOR degradation following serum re-addition (Figure 1B). Furthermore, an inability to degrade DEPTOR in the presence of proteasome inhibitor correlated with reduced phosphorylation of T389 in S6K (Figure 1B).
In an attempt to identify candidate E3 ubiquitin ligases that might be responsible for ubiquitin-dependent DEPTOR turnover, we expressed HA-tagged DEPTOR in 293T cells and analyzed associated proteins by mass spectrometry. In addition to mTOR and mLST8, we also identified β-TRCP2, a substrate specific adaptor of the SCF complex (Figure 1C). β-TRCP1 and β-TRCP2 are closely related in sequence, associate with the same phosphodegron sequences in substrates, and are generally thought to function redundantly. Reciprocal proteomic analysis of HA-β-TRCP2 purified from 293T cells cultured in the presence or absence of bortezomib revealed several known SCFβ-TRCP substrates as well as DEPTOR (Figure 1D). Consistent with these data, endogenous β-TRCP1 co-precipitates with endogenous DEPTOR (Figure 1E) or ectopically expressed DEPTOR (Figure S1C–D). Furthermore, β-TRCP1/DEPTOR interaction was abolished by phosphatase treatment (Figure S1E) and a point mutation within the substrate interaction site of β-TRCP1 (Wu et al., 2003) also resulted in a reduction in the interaction between β-TRCP1 and DEPTOR (Figures 1F and S1F–G). As expected, MYC-CUL1 associated with FLAG-DEPTOR (Figure S2B) in transfected cells. Thus, SCFβ-TRCP emerged as a candidate E3 ubiquitin ligase for DEPTOR.
In order to examine the role of β-TRCP in the control of DEPTOR stability, we first examined the steady-state abundance of DEPTOR in proliferating cells. Depletion of β-TRCP, but not SKP2, FBW7, or CDH1 in either HeLa (Figure 2A) or T98G (Figure 2B) cells led to a significant increase in DEPTOR abundance. Importantly, depletion of β-TRCP did not affect the expression of other key mTOR components, including mTOR, RICTOR, RAPTOR and SIN-1 (Figure 2A), suggesting that β-TRCP specifically targets DEPTOR for destruction. Furthermore, depletion of either β-TRCP1 or β-TRCP2 by multiple independent shRNA lentiviral vectors led to a sharp increase in DEPTOR expression, excluding a possible off-target effect associated with the shRNA treatment (Figures 2C–D and S2A). Moreover, depletion of β-TRCP resulted in suppression of both mTORC1 and mTORC2 kinase activity, as revealed by the decreases in both pT389-S6K and pS473-AKT (Figures 2B–C). Furthermore, depletion of endogenous DEPTOR impaired shβ-TRCP1-induced downregulation of pS6K, indicating that accumulation of DEPTOR might play an important role in suppressing mTORC kinase activity upon depletion of β-TRCP (Figure 2E). Consistent with the role of SCFβ-TRCP in regulating DEPTOR stability and mTOR activity, DEPTOR specifically interacts with CUL1 (Figure S2B) and depletion of endogenous CUL1 with 4 independent shRNAs resulted in marked accumulation of DEPTOR with concomitant loss of S6K T389 phosphorylation (Figure 2F).
Next, we sought to understand the physiological role of β-TRCP in governing DEPTOR stability. As illustrated in Figure 3A, DEPTOR accumulated in HeLa cells upon growth factor deprivation, and was rapidly degraded following serum re-addition. Importantly, the abundance of DEPTOR was inversely correlated with mTOR activity as assessed by S6K phosphorylation on T389. In agreement with a role for SCFβ-TRCP, depletion of β-TRCP (β-TRCP1 and/or β-TRCP2) resulted in impaired DEPTOR degradation following serum re-addition, and a subsequent reduction in phosphorylation of S6K at T389 (Figure 3A–B). Similar results were observed in T98G cells after depletion of β-TRCP1 (Figure 3C), further supporting the critical role of β-TRCP in promoting DEPTOR destruction. Consistent with this, cycloheximide chase analysis revealed that the increase in DEPTOR abundance following β-TRCP depletion is largely due to increased DEPTOR half-life (Figures 3D–E). On the other hand, depletion of β-TRCP did not significantly increase DEPTOR mRNA levels (Figures 3F–H), implying that the observed DEPTOR accumulation following depletion of β-TRCP primarily occurs through a post-transcriptional mechanism.
Recognition of substrates by β-TRCP typically involves kinase-driven formation of a phosphodegron on the substrate, which is recognized by the WD40-repeat surface of β-TRCP (Cardozo and Pagano, 2004; Frescas and Pagano, 2008; Winston et al., 1999; Wu et al., 2003). As noted earlier, the β-TRCP phosphodegron binding site contributes to its interaction with DEPTOR (Figure 1F), consistent with recognition of DEPTOR via a phosphodegron. To pinpoint upstream signaling pathways controlling DEPTOR stability, we evaluated DEPTOR abundance in serum-stimulated cells pre-treated with a panel of small-molecule kinase or proteasome inhibitors. As shown in Figure 4A and Figure S3A, MG132 treatment efficiently blocked DEPTOR destruction while ERK (U0126), PKC or GSK3 inhibitors had minimal effects on DEPTOR abundance. These results indicate that while DEPTOR contains putative ERK, GSK3 and PKC sites as predicted by the Scansite program (data not shown), these kinases are unlikely to participate in DEPTOR turnover. On the other hand, consistent with a previous report (Peterson et al., 2009), inhibition of the PI3K/mTOR pathway with either LY294002 or rapamycin blocked DEPTOR destruction to an extent equivalent to that observed with MG132. To further dissect the possible role of either the mTORC1 or mTORC2 pathway in this process, we utilized PP242, an inhibitor that can suppress both mTORC1 and mTORC2 (Feldman et al., 2009), or high and low levels of rapamycin that may differentially inhibit mTORC1 and mTORC2. We found that both low and high concentrations of rapamycin led to increased DEPTOR abundance (Figure 4B). Similarly, inhibition of mTOR by PP242 efficiently blocked DEPTOR destruction, which supports a model that both mTORC1 and mTORC2 are involved in this process (Figure 4B). Moreover, depletion of either RICTOR or RAPTOR resulted in DEPTOR stabilization during the response to serum stimulation (Figures 4C and S3B–C). However, it remained unclear whether mTORC1 or mTORC2 directly phosphorylates DEPTOR or indirectly regulates DEPTOR through activating its downstream kinases such as AKT or S6K.
Inhibition of S6K with a specific small molecule inhibitor, PF-4708671 (Figure 4B), or depletion of S6K with 2 independent shRNAs did not affect DEPTOR abundance, dispelling a possible role of S6K in DEPTOR turnover (Figures 4B–C). However, specific inhibition of AKT led to a significant increase in DEPTOR expression (Figures 4A–B). On the other hand, specific depletion of AKT1 did not affect DEPTOR expression (Figure 4C), suggesting that inhibition of all three AKT isoforms might be required to induce DEPTOR expression. Careful examination of DEPTOR’s protein sequence did not reveal a canonical AKT phosphorylation consensus site (data not shown). Furthermore, ectopic expression of a myristoylated version of either AKT1 or AKT2 did not affect DEPTOR abundance (Figure S3D). These results suggest that AKT might indirectly regulate DEPTOR stability through its downstream targets. One candidate is TSC2, a well-characterized AKT target that negatively regulates mTORC1 kinase activity (Inoki et al., 2002). In support of this indirect role of AKT in DEPTOR stability control, we found that in TSC2-depleted HeLa cells, the AKT inhibitor failed to impair β-TRCP/DEPTOR interaction (Figure S3E), and subsequently failed to stabilize DEPTOR (Figure 4D). These results suggest that AKT indirectly activates mTORC1 to influence DEPTOR destruction. Moreover, it further supports a model in which mTOR directly phosphorylates DEPTOR to govern its stability.
A role for mTOR in β-TRCP-dependent turnover of DEPTOR would predict that inhibition of mTOR in vivo would affect the β-TRCP-DEPTOR interaction. Consistent with this hypothesis, we found that the mTORC1/mTORC2 inhibitor PP242 abolished the interaction between HA-DEPTOR and Flag-β-TRCP1 (Figure 4E). Inhibition of mTORC1 by rapamycin reduced the β-TRCP1-DEPTOR interaction, but did not abolish it (Figure 4E), likely due to the presence of mTORC2 activity under the conditions used.
We noticed that D4476, the small molecule inhibitor of CKI, resulted in accumulation of DEPTOR in serum stimulated cells to an extent similar to that seen with proteasome or mTOR inhibition (Figure 4B), suggesting that additional kinases may collaborate with mTOR to promote DEPTOR turnover. An additional CKI inhibitor IC261 behaved similarly (Figure 5A). CKI isoforms are encoded by several genes. We found that depletion of CKIα in HeLa cells with 3 independent shRNAs resulted in accumulation of DEPTOR without affecting the abundance of mTOR, RAPTOR, or RICTOR (Figure 5B). In contrast, depletion of CKIδ and CKIε isoforms had minimal effect on DEPTOR abundance, suggesting that CKIα selectively contributes to DEPTOR turnover.
We reasoned that if CKIα was also involved in the interaction of DEPTOR with β-TRCP, then overexpression of CKIα might promote the β-TRCP1-DEPTOR interaction. As shown in Figure 5C, expression of GST-CKIα promoted the association of FLAG-β-TRCP1 with HA-DEPTOR. If this effect of CKIα required mTOR, we would anticipate that inhibition of mTOR would abolish the effect of CKIα overexpression on the β-TRCP-DEPTOR interaction. Indeed, treatment of cells with the mTOR inhibitor PP242 greatly reduced, and rapamycin substantially reduced, the interaction between β-TRCP1 and DEPTOR (Figure 5C). These pharmacological experiments indicate that mTOR and CKI might collaborate to promote the association of β-TRCP with DEPTOR.
The best-characterized phosphodegron recognized by β-TRCP contains the DpSGΦXpS motif (where Φ is a hydrophobic residue) in which both serine residues are phosphorylated. Additional bona fide substrates of SCFβ-TRCP such as PERIOD and BIM proteins contain phosphodegrons that diverge significantly from the canonical motif, with a phosphoserine or phosphothreonine replacing the aspartic acid in the canonical motif (Figure 6A). Previous studies identified a serine rich region in DEPTOR, and mutation of 13 phosphorylation sites in this region was sufficient to block serum-dependent DEPTOR degradation (Peterson et al., 2009). Importantly, 4 sites in this region displayed reduced phosphorylation in the presence of the mTOR inhibitor TORIN (Peterson et al., 2009) (Figure 6A). This region contains sequences that are reminiscent of previously identified phosphodegrons in PERIOD and BIM proteins (Figure 6A), wherein the SSGYFS sequence most closely corresponds to the PERIOD and BIM non-canonical degron. Phosphorylation of S286 and S287, as seen previously in DEPTOR (Peterson et al., 2009), would create 2 negatively charged residues mimicking DpSG in the canonical β-TRCP phosphodegron. We initially performed proteomics on DEPTOR purified from cells treated with MG132 to accumulate DEPTOR in a form wherein the phosphodegron was extensively phosphorylated (Figure S4A). This led to the identification of 19 phosphorylation sites, including all but two residues (S241 and S282) observed by Peterson et al., (2009) in the central region of DEPTOR (Figure S4B) and 2 sites (Ser6 and Thr7) in the N-terminus of DEPTOR (Figure S4A). We then examined whether the N-terminus or the central serine-rich region was required for the interaction between DEPTOR and β-TRCP. DEPTOR lacking the first 20 residues associated with β-TRCP1 to an extent similar to that seen with the wild-type protein while the previously reported 13A mutant failed to interact with β-TRCP (Figure 6C). As expected, the DEPTOR 13A mutant failed to undergo serum stimulation-dependent turnover (Figure 6D). Consistent with a role for this region in CKI-dependent association between β-TRCP and DEPTOR, the 13A mutant was unable to associate with β-TRCP1 even when CKI was overexpressed (Figure 6E).
Given the critical role of mTOR and CKI in governing DEPTOR destruction, we next tested whether CKI and mTOR could phosphorylate DEPTOR in vitro. The DEPTOR 13A mutant showed reduced phosphorylation with both CKI and mTOR in vitro (Figures S4F–G). We then utilized mass spectrometry analysis to further define both mTOR and CKI-mediated phosphorylation sites on DEPTOR. We identified phosphopeptides containing either pS286 or pS287 in CKI-treated DEPTOR samples (Figure S4D). In mTOR treated samples, we identified peptides containing pS265, pS286, pS293, pT295, and pS299 (Figure S4C). Furthermore, we found that mutation of S286, S293 and T295/S299, but not S244 and S265, to alanine in DEPTOR significantly reduced mTOR-dependent DEPTOR phosphorylation in vitro (Figure S4I). Interestingly, S286, S293, T295 and S299 are located either within or in close proximity to the candidate non-canonical degron sequence (Figure 6A).
In order to evaluate the contribution of individual phosphorylation sites to DEPTOR stability, we performed binding experiments with point mutations in several residues in and around the non-canonical degron, focusing primarily on S293, T295, and S299 identified as in vitro mTOR sites and S286 which was found to be phosphorylated in vitro with either mTOR or CKI by mass spectrometry (Figures S4C–D). DEPTOR S286A, S293A, T295A/S299A and S286A/T295A/S299A(3A) mutants showed negligible or greatly reduced binding to β-TRCP1 in transfected cells (Figures 6F and S4E). In contrast, alanine-replacement mutants in S244 and S265 in DEPTOR, sites found to be phosphorylated in vivo (Figures S4A–B), did not affect their association with β-TRCP1 (Figure 6F). In keeping with these results, mTOR-mediated phosphorylation of wild-type (WT), but not S286A or S286A/T295A/S299A DEPTOR led to increased interaction with GST-β-TRCP1 in vitro (Figures 6G and S4H). Consistent with the binding data, S286A, T295A/S299A, and S286A/T295A/S299A DEPTOR mutants displayed an extended half-life in HeLa cells upon serum stimulation, compared with wild-type, S244A, and S265A DEPTOR (Figure 6H). To further examine the relationship between mTOR and CKI in degron generation, we employed the CKI-overexpression assay described above and tested the impact of mutations in the candidate degron on the binding of β-TRCP with DEPTOR. While transient CKI expression could promote association of WT-DEPTOR with β-TRCP, CKI could not promote association of either S286A-DEPTOR or S286A/T295A/S299A-DEPTOR with β-TRCP (Figure 6I). In contrast, CKI could promote partial binding of a T295A/S299A-DEPTOR mutant to β-TRCP (Figure 6I). These data suggests that CKI overexpression may overcome a requirement for phosphorylation at the major mTOR sites in DEPTOR for formation of the degron and are consistent with our finding that CKI can phosphorylate S286 and S287 in DEPTOR in vitro in the absence of mTOR.
Previous studies indicate that both S293 and S299 phosphorylation in vivo are suppressed by the mTOR inhibitor TORIN (Peterson et al., 2009) (Figure 6A). Using a specific anti-pSer299-DEPTOR antibody (Figure S4J), we showed that mTOR but not CKI could promote DEPTOR S299 phosphorylation in vitro (Figure S4K) and depletion of various mTOR components resulted in decreased pS299-DEPTOR in vivo (Figures S4L–M). These results support the notion that S299 is a physiological mTOR phosphorylation site. Moreover, although depletion of either RAPTOR or RICTOR reduced pS299-DEPTOR, depletion of mTOR led to a stronger reduction in pS299-DEPTOR, indicating that both mTORC1 and mTORC2 could phosphorylate DEPTOR. Currently, it is unclear whether mTOR phosphorylation of S286 in DEPTOR is physiologically significant or represents relaxed specificity of the kinase in vitro. As discussed below, the simplest conclusion of the in vitro and in vivo data is that mTOR-dependent phosphorylation of S293, T295, and/or S299 promotes CKI-dependent phosphorylation of S286 and S287. Consistent with this notion, we found that pre-phosphorylation of GST-DEPTOR by mTOR significantly stimulated DEPTOR phosphorylation by CKI (Figure 6J), and promoted in vitro DEPTOR ubiquitination mediated by SCFβ-TRCP (Figure S4N).
Stabilization of DEPTOR during serum-stimulated cell cycle re-entry would be expected to result in mTOR inhibition and a reduction in phosphorylation of downstream mTOR targets. To address this question, the non-degradable DEPTOR S286A/T295A/S299A (3A) mutant was transiently transfected into HeLa cells, the cells were subjected to serum deprivation, and then re-stimulated to enter the cell cycle. Wild-type DEPTOR was degraded on schedule with concomitant S6K phosphorylation (Figure 7A). In contrast, the non-degradable DEPTOR mutant caused prolonged inhibition of S6K phosphorylation (Figure 7A). Thus, DEPTOR turnover is required for proper control of S6K phosphorylation and activation.
Next, we asked how manipulation of β-TRCP-mediated DEPTOR destruction affects the mTOR signaling pathway and its cellular functions in response to various environmental stresses. We found that in response to certain stresses such as glucose or serum starvation, DEPTOR was induced and this led to suppression of the mTOR signaling pathway and induction of autophagy, as indicated by the increased presence of the lipidated species of the LC3 autophagy marker (referred to as LC3-II) (Figure 7B). Depletion of β-TRCP resulted in accumulation of DEPTOR and induction of LC3-II even in favorable nutrient conditions. Furthermore, induced expression of a non-degradable form of DEPTOR resulted in a dose-dependent increase in LC3-II concomitant with a decrease in p62 abundance (Figure 7C), as expected for cells experiencing an increase in autophagic flux. Moreover, analysis of cells expressing non-degradable DEPTOR with the autophagic vesicle marker monodansylcadaverine, revealed a substantial increase in their number, consistent with the status of LC3-II and p62 (Figure S5A–C). In a reciprocal set of experiments, we used a Cherry-GFP-LC3B fusion protein to measure autophagic flux in HeLa cells depleted of DEPTOR. The Cherry-GFP-LC3B protein produces yellow foci when associated with autophagosomes but upon fusion with lysosomes and a reduction in pH, the GFP signal, but not the Cherry signal, is quenched, allowing a determination of the extent of transit from the autophagosome to the lysosomes (Pankiv et al., 2007). In the absence of Glucose, we observed an increase in the percentage of Cherry-positive/GFP-negative vesicles, consistent with activation of autophagy (Figure 7D–E). In contrast, depletion of DEPTOR reduced the increase in autophagic flux observed upon Glucose deprivation, as seen by a decrease in the percentage of Cherry-positive/GFP-negative vesicles (Figure 7D–E). The reduced number of autophagosome in the shDEPTOR-treated HeLa cells was further supported by the increased p62 levels concomitant with slightly reduced LC3-II levels in the DEPTOR-depleted samples (Figure 7F).
These results indicate that the β-TRCP/DEPTOR/mTOR pathway is an integrated sensor mechanism that allows the cells to better respond to environmental cues. Therefore, impaired DEPTOR destruction might contribute to deregulated mTOR activity in many types of diseases. In support of this contention, we found that in a panel of breast cancer cell lines, DEPTOR expression levels varied dramatically, which inversely correlated with mTOR kinase activities as evidenced by both pSer2481-mTOR and pSer473-Akt signals (Figure S5D). Interestingly, we found that multiple myeloma cell lines bearing low expression of DEPTOR were significantly more sensitive to bortezomib than those with relatively high DEPTOR expression (Figure S5E–G), which may reflect a reduction in mTOR activity due to DEPTOR accumulation upon proteasome inhibition.
mTOR serves as a critical sensor of metabolic and nutrient stresses by receiving and interpreting environmental inputs and transducing them to downstream signaling pathways to control cellular metabolism, cellular growth, and survival (Efeyan and Sabatini, 2010; Reiling and Sabatini, 2006; Sengupta et al., 2010; Yang and Guan, 2007). Recent studies implicate the mTOR inhibitor DEPTOR as a critical regulator of the process (Peterson et al., 2009). In particular, conditions that lead to increased DEPTOR abundance correlate with reduced mTOR activity and the transition from low to high mTOR activity in response to nutrients correlates with a reduction in the abundance of DEPTOR in a proteasome dependent manner (Figure 1B) (Peterson et al., 2009). The data present here provide a molecular explanation for how mTOR is rapidly switched from the OFF (low activity) state to the ON (high activity) state via a positive feedback mechanism involving mTOR and CKI-dependent DEPTOR phosphorylation and ensuing SCFβ-TRCP-dependent DEPTOR ubiquitination and subsequent destruction by the proteasome (Figure 7G).
Several lines of evidence support this model. First, depletion of β-TRCP or CUL1 stabilizes DEPTOR during the cellular response to serum, with concomitant suppression of S6K phosphorylation by mTORC1. Second, β-TRCP associates with DEPTOR in vitro and in vivo in an mTOR dependent manner, as assessed using pharmacological inhibitors of mTOR as well as depletion of either RICTOR or RAPTOR. Previous studies have demonstrated that mutation of 13 serine/threonine residues in DEPTOR located between the N-terminal DEP domains and the C-terminal PDZ domain resulted in stabilization of DEPTOR (Peterson et al., 2009). Our data suggests that this region is targeted by mTOR and CKI to form a non-canonical β-TRCP degron. We identified 4 sites in this region that can be phosphorylated in vitro by mTOR (S286, S293, T295, and S299) and mutation of S286 or S293 to alanine, or mutation of T295 and S299 to alanine, abolished the association of DEPTOR with β-TRCP in vivo and in vitro. Moreover, we found that CKI can phosphorylate S286 and S287 in vitro and inhibition of mTOR in vivo resulted in a reduction in the extent of CKI-driven association of DEPTOR with β-TRCP. The sequence encompassing S286 is reminiscent of non-canonical β-TRCP phosphodegrons found in PERIOD and BIM proteins in that the characteristic aspartate of the DpSGΦXpS motif is replaced by serine. Phosphorylation of this serine (S286) by either CKI or mTOR would create a negative charge at this position, likely serving the function of the aspartate in the canonical phosphodegron that binds to R474 and R521 in the WD40 domain of β-TRCP (Wu et al., 2003). While we identified phosphorylation of both S286 and S287 in DEPTOR purified from human cells, we did not identify S291, the candidate phosphoacceptor at position 6 in the degron. We favor a model in which mTOR phosphorylates S293, T295, and S299 to either facilitate phosphorylation by CKI on S286 and S287 directly, or alternatively, these phosphorylation events may open up the structure to allow access of CKI to the S286 and S287 sites. In this regard, CKI is thought to be constitutively active, and therefore, the timing of DEPTOR destruction is controlled by mTOR activity, which provides an initiating event in formation of the phosphodegron. We cannot exclude the possibility that mTOR phosphorylates S286 and CKI phosphorylates S287 in vivo to form the degron. Consistent with this model, Duan et al. (2011) have demonstrated that CKI can promote phosphorylation of the SSGYFS motif in vitro using a phosphospecific antibody that recognizes all three phosphoserines within the motif, and this phosphorylation is enhanced by mTOR. Furthermore, using in vitro kinase assays, we also found that pre-phosphorylation of DEPTOR by mTOR greatly facilitated CKI-mediated phosphorylation of DEPTOR in vitro (Figure 6J). These events appear to be sufficient to support in vitro ubiquitination of DEPTOR by SCFβ-TRCP (Figure S4N and Duan et al., 2011). Moreover, replacement of S286, S287, and S291 with aspartic acid rendered DEPTOR association with β-TRCP independent of phosphorylation of S293 and S299 by mTOR (Duan et al., 2011), consistent with the idea that mTOR cooperates with CKI to phosphorylate the non-canonical degron in DEPTOR (Figure 7G).
Deregulated mTOR kinase activity contributes to many types of diseases including cancer and diabetes (Zoncu et al., 2010). Therefore, a clearer understanding of how mTOR activity is governed will facilitate the development of strategies to control mTOR activity. In the presence of serum, DEPTOR is constitutively unstable, thereby maintaining mTOR in an active state. Upon depletion of β-TRCP, DEPTOR levels were increased (Figure 7B). Under nutrient poor conditions when mTOR activity is low, DEPTOR levels are elevated and there is no further accumulation upon β-TRCP depletion (Figure 7B). Thus, DEPTOR levels are under physiological control by β-TRCP. In a panel of breast cancer cell lines, we found that DEPTOR levels inversely correlate with the activity of the mTOR signaling pathway (Figure S5D). More interestingly, our finding that multiple myeloma cells expressing low DEPTOR levels are typically sensitive to proteasome inhibition (Figure S5E–F) suggests that inappropriate DEPTOR accumulation may promote cell death via a decrease in mTOR activity. Alterations in mTOR signaling levels have also been reported to be involved in regulating cellular metabolism including glycolysis and central carbon metabolism (Choo et al., 2010; Duvel et al., 2010). Using metabolic profiling, we identified 42 distinct metabolite changes upon β-TRCP depletion relative to the control (shGFP) in various pathways including intermediate glycolysis (Figure S5H) but not in the TCA cycle (Figure S5I). This finding is consistent with the idea that reduced mTOR activity upon DEPTOR stabilization would lead to alterations in glycolysis.
Collectively, our results provide insight into a positive feedback mechanism used to control the activity status of mTOR during cell cycle entry in response to serum stimulation. Our data reveal critical roles for mTOR itself as well as CKI in generating a degron in DEPTOR that is recognized by β-TRCP, and promotes DEPTOR turnover by the proteasome. The initiation of mTOR activity sets in motion a feed-forward loop that facilitates the concerted turnover of DEPTOR (Figure 7G). A critical question for the future is whether defects in the DEPTOR turnover pathway contribute to disease progression.
Cell culture conditions including serum starvation and transfection, have been described previously (Wei et al., 2004; Wei et al., 2005). Lentiviral shRNA virus packaging and subsequent infection of various cell lines were performed according to the protocol described previously (Boehm et al., 2005). Glucose or glutamine deprivation was performed as described before (Choo et al., 2010). For cell viability assays, cells were plated at 10,000 per well in 96-well plates, and incubated with appropriate medium containing bortezomib for the indicated time period. Assays were performed with CellTiter-Glo Luminescent Cell Viability Assay Kit according to the manufacturer’s instructions (Promega). Cycloheximide (CHX) experiments were performed as described previously (Gao et al., 2009b). Human primary foreskin fibroblast was cultured as described previously (Gao et al., 2009a).
Total RNA was extracted using the Qiagen RNeasy mini kit, and the reverse transcription reaction was performed using the ABI Taqman Reverse Transcriptional Reagents (N808-0234). After mixing the generated cDNA template with primers and ABI Taqman Fast Universal PCR Master Mix (4352042), the real-time RT-PCR was performed with the ABI-7500 machine. β-TRCP1 (Hs00182707_m1), β-TRCP2 (Hs00362667_m1), DEPTOR (Hs00961900_m1) and GAPDH (Hs99999905_m1) primers were purchased from ABI.
β-TRCP1 binding to immobilized GST-fusion proteins was performed as described previously (Inuzuka et al., 2010). Where indicated, GST-DEPTOR proteins were incubated with mTOR in the presence of ATP for 1 hour before the binding assays.
In vitro Ub assay was performed as described previously (Inuzuka et al., 2010). To purify SCFβ-TRCP complex, 293T cells were transfected with vectors encoding GST-β-TRCP1, Myc-Cul-1, Myc-Skp1, and HA-Rbx1. Then SCFβ-TRCP (E3) complexes were purified by GST affinity precipitation. Before the ubiquitination assay, GST-DEPTOR proteins (wild type or 3A mutant) were first incubated with mTOR kinase and Casein Kinase 1 sequentially in the presence of ATP. Afterwards, phosphorylated DEPTOR protein was incubated with purified SCFβ-TRCP E3 complex together with E1, E2 (UbcH5a and UbcH3) and ubiquitin. The ubiquitination reactions were stopped by addition of SDS sample buffer and resolved by SDS-PAGE for immunobloting.
We thank Alan Lau, Christoph Schorl, Zhiwei Wang and Susan Glueck for critical reading of the manuscript, Tamar Melman for help with data analysis, Jianping Jin, William Hahn and James DeCaprio for providing reagents; Michele Pagano and Yi Sun for sharing unpublished data, and members of the Wei, Harper and Cantley labs for useful discussions. W.W. is a MLSC New Investigator. This work was supported in part by the DOD Prostate New Investigator award to W.W., and by grants from the National Institutes of Health (A.T., CA122099; W.W., GM089763; J.W.H., AG011085 and GM054137). D.G. is supported by Lady Tata Memorial Trust International Awards for Research in Leukemia Postdoctoral Fellowship. M.T. is supported by an A-STAR Pre-doctoral Fellowship. C.A.L. is the Amgen fellow of the Damon Runyon Cancer Research Foundation.
Conflict of Interest Statement: J.W.H. is a consultant for Millennium Pharmaceuticals.
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