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      A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB

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          Abstract

          The lysosome plays a key role in cellular homeostasis by controlling both cellular clearance and energy production to respond to environmental cues. However, the mechanisms mediating lysosomal adaptation are largely unknown. Here, we show that the Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, colocalizes with master growth regulator mTOR complex 1 (mTORC1) on the lysosomal membrane. When nutrients are present, phosphorylation of TFEB by mTORC1 inhibits TFEB activity. Conversely, pharmacological inhibition of mTORC1, as well as starvation and lysosomal disruption, activates TFEB by promoting its nuclear translocation. In addition, the transcriptional response of lysosomal and autophagic genes to either lysosomal dysfunction or pharmacological inhibition of mTORC1 is suppressed in TFEB−/− cells. Interestingly, the Rag GTPase complex, which senses lysosomal amino acids and activates mTORC1, is both necessary and sufficient to regulate starvation- and stress-induced nuclear translocation of TFEB. These data indicate that the lysosome senses its content and regulates its own biogenesis by a lysosome-to-nucleus signalling mechanism that involves TFEB and mTOR.

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          Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2

          Morris E. Feldman, Beth Apsel, Aino Uotila … (2009)
          Introduction The mammalian target of rapamycin (mTOR) is a serine-threonine kinase related to the lipid kinases of the phosphoinositide 3-kinase (PI3K) family. mTOR exists in two complexes, mTORC1 [1,2] and mTORC2 [3,4], which are differentially regulated, have distinct substrate specificities, and are differentially sensitive to rapamycin. mTORC1 integrates signals from growth factor receptors with cellular nutritional status and controls the level of cap-dependent mRNA translation by modulating the activity of key translational components such as the cap-binding protein and oncogene eIF4E [5]. mTORC2 is insensitive to rapamycin, and selective inhibitors of this complex have not been described. Partly because acute pharmacological inhibition of mTORC2 has not been possible, the functions of mTORC2 are less well understood than those of mTORC1. mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt [3,6] and SGK [7] although other kinases, including DNA-PK and Ilk, have also been implicated in Akt hydrophobic motif phosphorylation [8–11]. Growth factor stimulation of PI3K causes activation of Akt by phosphorylation at two key sites: the activation loop (T308) and the C-terminal hydrophobic motif (S473). Active Akt promotes cell survival in many ways, including suppressing apoptosis, promoting glucose uptake, and modifying cellular metabolism [12]; consequently, there is significant interest in identifying the kinase(s) responsible for each activating phosphorylation, the relationship between these phosphorylation sites, and the role of differential Akt phosphorylation on Akt substrate phosphorylation. Of the two phosphorylation sites on Akt, activation loop phosphorylation at T308, which is mediated by PDK1, is indispensable for kinase activity, whereas hydrophobic motif phosphorylation at S473 enhances Akt kinase activity by approximately 5-fold [13]. The disruption of mTORC2 by different genetic and pharmacological approaches has variable effects on Akt phosphorylation. Targeting mTORC2 by RNA interference (RNAi) [6,14], homologous recombination [15–17], or long-term rapamycin treatment [18] results in loss of Akt hydrophobic motif phosphorylation (S473), strongly implicating mTORC2 as the kinase responsible for phosphorylation of this site. RNAi targeting mTORC2 and long-term rapamycin result in loss of Akt phosphorylation on its activation loop (T308), but this phosphorylation remains intact in mouse embryonic fibroblasts (MEFs) lacking the critical mTORC2 component SIN1. It cannot be inferred from this genetic data whether acute pharmacological inhibition of mTORC2 would block the phosphorylation of Akt only at S473, resulting in partial Akt deactivation, or also disrupt phosphorylation at T308, resulting complete Akt inhibition. Several small molecules have been identified that directly inhibit mTOR by targeting the ATP binding site; these include LY294002, PI-103, and NVP-BEZ235 [19–22]. These molecules were originally discovered as inhibitors of PI3Ks and later shown to also target mTOR. Because all of these molecules inhibit PI3Ks and mTOR with similar potency, they cannot be used to selectively inhibit mTOR or PI3Ks in cells. Indeed, because mTORC1 and mTORC2 function downstream of PI3Ks in most settings, it is unclear to what extent the ability of these molecules to block the activation of signaling proteins such as Akt reflects PI3K versus mTOR inhibition. It is possible that some of the functions attributed to PI3Ks using the classical inhibitor LY294002 are a consequence of mTOR inhibition [19,23], but it is has not been possible address this, because small molecules that inhibit mTOR without inhibiting PI3Ks have not been available. We recently reported the synthesis of pyrazolopyrimidines that inhibit members of the PI3K family, including mTOR [24]. Two of these molecules, PP242 and PP30, are the first potent, selective, and ATP-competitive inhibitors of mTOR. Unlike rapamycin, these molecules inhibit both mTORC1 and mTORC2, and, unlike PI3K family inhibitors such as LY294002, these molecules inhibit mTOR with a high degree of selectivity relative to PI3Ks and protein kinases. To distinguish these molecules from the allosteric mTORC1 inhibitor rapamycin, we are calling them “TORKinibs” for TOR kinase domain inhibitors. The dual role of mTOR within the PI3K→Akt→mTOR pathway as both an upstream activator of Akt and the downstream effector of pathway activity on cell growth and proliferation has excited interest in active-site inhibitors of mTOR [25–30]. We describe here the biological activity of these molecules. Another small-molecule ATP-competitive mTOR inhibitor called Torin1 was reported while our manuscript was in the process of publication [56]. Results Specific Active-Site Inhibition of mTOR by the TORKinibs PP242 and PP30 PP242 and PP30 inhibit mTOR in vitro with half-maximal inhibitory concentrations (IC50 values) of 8 nM and 80 nM, respectively. As expected for active-site inhibitors, PP242 and PP30 inhibit mTOR in both mTORC1 and mTORC2 (Table S1). Both compounds are selective within the PI3K family, inhibiting other PI3Ks only at substantially higher concentrations (Figure 1). Testing of PP242 against 219 purified protein kinases at a concentration 100-fold higher than its mTOR IC50 value revealed exceptional selectivity with respect to the protein kinome; most protein kinases were unaffected by this drug, and only four—PKC-alpha, PKC-beta, RET, and JAK2 (V617F)—were inhibited more than 80% [24]. We determined IC50 values for PP242 against these kinases in vitro using purified proteins. In these assays, PP242 was relatively inactive against PKC-beta, RET, or JAK2 but inhibited PKC-alpha with an in vitro IC50 of 50 nM (Figure 1). Importantly, PP30 showed no activity against PKC-alpha or PKC-beta in the same assay (Figure 1). These data indicate that PP242 is a highly selective inhibitor of mTOR and that PP30 can be used to confirm that the effects of PP242 are due to inhibition of mTOR and not PKC-alpha. The availability of a second structurally dissimilar mTOR inhibitor—PP30—provides additional control for unanticipated off-targets of PP242. Figure 1 In Vitro IC50 Values for PP242 and PP30 Determined in the Presence of 10 μM ATP Inhibition of mTORC2 and Akt Phosphorylation by TORKinibs We characterized the effect of PP242 on the PI3K→Akt→mTOR pathway. PP242 and PP30 both inhibited insulin-stimulated phosphorylation of Akt at S473, confirming that mTOR kinase activity is required for hydrophobic motif phosphorylation (Figure 2A). The inhibition of mTOR by PP242 and PP30 also resulted in loss of Akt phosphorylation at T308, but significantly higher doses of PP242 and PP30 were required to inhibit T308 as compared with S473 (Figure 2A and 2B). PP242 inhibited S473-P and T308-P at both early and late time points after insulin stimulation, indicating that the differential sensitivity of these sites to PP242 does not reflect differing kinetics of phosphorylation (Figure S1). By comparison, the PI3K inhibitor PIK-90, which does not inhibit mTOR, inhibited the phosphorylation of both Akt sites equipotently (Figure 2B), as observed previously [21]. Figure 2 Inhibition of mTORC2 by TORKinibs Affects pS473 and pT308 of Akt (A) Serum-starved L6 myotubes were pre-treated with kinase inhibitors prior to stimulation with insulin for 3 min. Lysates were analyzed by Western blotting. (B) PP242 inhibits pS473 (red) of Akt more potently than pT308 (gray). Serum-starved L6 myotubes were treated with kinase inhibitors prior to stimulation with insulin for 10 min. Akt phosphorylation was measured by in-cell Western and is shown relative to serum starvation and insulin stimulation (n = 3 for each inhibitor dose). EC50 values from the best fit curves are plotted. ***p < 0.001, F test. EC50 values for PIK-90 on pS473 and pT308 were not significantly different (p = 0.2, F test). We sought to confirm that the loss of T308-P caused by PP242 and PP30 results from inhibition of mTOR-mediated phosphorylation of S473, rather than from inhibition of an off-target kinase, or from an effect of mTOR inhibition unrelated to S473-P. To do this, we examined the effect of PP242 on T308 phosphorylation in two situations in which Akt could not be phosphorylated on S473. First, we overexpressed S473A mutant Akt and stimulated these cells with insulin (Figure 3A). S473A Akt was phosphorylated on T308 to a similar level as wild-type, yet in contrast to the wild-type, T308-P on S473A Akt was not inhibited by PP242. The lack of effect of PP242 on S473A Akt confirms that PP242 inhibition of pT308 requires S473 and also that PP242 does not inhibit PDK1 in cells, as was suggested by direct testing of PDK1 in vitro (Figure 1). Figure 3 PP242 Does Not Directly Inhibit Phosphorylation of Akt at T308 (A) pT308 is not inhibited by PP242 in cells overexpressing S473A Akt. HEK293 cells were transfected with wild-type Akt, S473A Akt, or not transfected (Mock) and were treated with 2.5 μM PP242 or 625 nM PIK-90 as indicated prior to insulin stimulation. Lysates were analyzed by Western blotting. Quantitation of pT308 relative to insulin treated cells overexpressing wild-type Akt (lane 2) is shown below that blot. Data are representative of two independent experiments. (B) pT308 is not inhibited by PP242 in SIN1−/− MEFs, which lack pS473. Primary wild-type (WT) and SIN1−/− MEFs were pre-treated with 625 nM PIK-90, 10 μM BX-795, or 100 nM rapamycin for 24 h, 100 nM rapamycin for 30 min, or the indicated concentrations of PP242 prior to stimulation with insulin. Lysates were analyzed by Western blotting. As a further test of the specificity of PP242 and the requirement for functional S473 phosphorylation in order for PP242 to inhibit T308-P, we examined the effect of PP242 on the phosphorylation of Akt in primary MEFs from embryos that lack SIN1 [16] (Figure 3B). SIN1 is a component of mTORC2, and knockout of SIN1 compromises the physical integrity of mTORC2 leading to a complete loss of Akt phosphorylation at S473 without affecting its phosphorylation at T308. Consistent with our results from L6 cells, PP242 inhibited the phosphorylation of Akt at both S473 and T308 in wild-type MEFs. By contrast, PP242 had no effect on the phosphorylation of T308 in SIN1−/− MEFs that lack mTORC2. Furthermore, PP242 had no effect on the constitutive phosphorylation of the turn motif of Akt at T450 [16,31]. As a further comparison, we examined the effect of long-term rapamycin, which is known to block the assembly of mTORC2 is some cell lines [18]. Similar to PP242, long-term rapamycin treatment of wild-type MEFs inhibited S473-P and reduced the phosphorylation of T308-P, as was seen previously [18]. Importantly, the PI3K inhibitor PIK-90 and the PDK1 inhibitor BX-795 [32] blocked phosphorylation of T308 in SIN1−/− MEFs, indicating that the failure of PP242 to block T308 in SIN1−/− MEFs does not reflect a general resistance of T308 to dephosphorylation in cells that lack mTORC2. From these data, we conclude that PP242′s effect on T308-P is dependent on its inhibition of Akt phosphorylation by mTOR at S473. It remains unclear why mTORC2 knockout cells, but not cells treated with RNAi or pharmacological inhibitors of mTORC2, are able to retain T308 phosphorylation in the absence of phosphorylation at S473. However, there are a growing number of examples in which genetic deletion of a kinase results in compensatory changes that mask relevant phenotypes observed with the corresponding small molecule inhibitor [33]. Akt Substrate Phosphorylation Is Only Modestly Inhibited by PP242 Akt requires phosphorylation at both S473 and T308 for full biochemical activity in vitro [13], but it is unclear whether all of the cellular functions of Akt require it to be dually phosphorylated. Singly phosphorylated (T308-P) Akt from SIN1−/− MEFs is competent to phosphorylate the cytoplasmic Akt substrates GSK3 and TSC2, but not the nuclear target FoxO [16]. Because low concentrations of PP242 inhibit the phosphorylation of S473 and higher concentrations partially inhibit T308-P in addition to S473-P, we used PP242 to examine whether some substrates of Akt are especially sensitive to loss of S473-P (Figure 4). We compared PP242 to the PI3K inhibitor PIK-90 and the allosteric Akt inhibitor Akti-1/2 [34], which inhibit the phosphorylation of Akt at both sites. In contrast to PIK-90 and Akti-1/2, which completely inhibited the phosphorylation of Akt and its direct substrates, PP242 only partially inhibited the phosphorylation of cytoplasmic and nuclear substrates of Akt. This suggests that phosphorylation of the Akt substrates we examined is only modestly sensitive to loss of S473-P. A caveat of comparing Akt substrates in Sin1−/− MEFs with PP242-treated cells is the different turn motif (T450-P) status in these two conditions (Figure 3B). Figure 4 Phosphorylation of the Akt Substrates GSK3α/β, TSC2, and FoxO1/O3a Is Not Potently Inhibited by PP242 Lysates from L6 myotubes treated with kinase inhibitors and stimulated with insulin were analyzed by Western blotting. Quantitation of pAkt and pTSC2 relative to the insulin control (lane 2) is show below these blots. In contrast to Akt, which maintains T308-P, SGK activity is completely inhibited by genetic disruption of mTORC2 [7]. Because SGK can phosphorylate FoxO and its activity is completely inhibited by disruption of mTORC2, it was suggested that the loss of FoxO phosphorylation in SIN1−/− MEFs indicates that FoxO is primarily phosphorylated by SGK rather than Akt [7]. Because Akti-1/2 does not inhibit SGK [34] but inhibits FoxO1/O3a phosphorylation at T24/T32 in L6 myotubes (Figure 4), our data suggests that the major kinase for T24/T32 of FoxO1/O3a in L6 myotubes is Akt and not SGK. PP242 Does Not Have an Obvious Effect on Actin Stress Fibers TORC2 is required for the generation of a polarized actin cytoskeleton in yeast [35]. Previous analysis of mTORC2 function using RNAi revealed a role for mTORC2 in the control of the actin cytoskeleton [3,4], yet these findings were not confirmed in primary MEFs lacking mTORC2 [15,17]. We examined actin stress fibers in NIH 3T3 cells (Figure 5) and in primary MEFs (unpublished data) treated with PP242. After 8 h of treatment with PP242, we found no obvious effect on the morphology or abundance of actin stress fibers (Figure 5), suggesting that mTORC2 activity is not required for the maintenance of actin stress fibers in these cells. That PP242 didn't obviously affect the morphology or abundance of actin stress fibers, does not rule out a role for mTOR in the control of the actin cytoskeleton, but it does show that pharmacological inhibition of mTORC2 does not affect the obvious changes in actin structure seen with RNAi. Figure 5 PP242 Inhibits Proliferation without Affecting Actin Stress Fibers (A) NIH 3T3 cells were stained for actin with Alexa 488-phalloidin (green) and for DNA with DAPI (blue). Images are representative of greater that 100 cells. (B) Differential inhibition of cell proliferation by PP242 and rapamycin does not require mTORC2. Proliferation of primary MEFs cultured for 3 d in the presence of kinase inhibitors was assayed by resazurin fluorescence (RF) and is shown in arbitrary units. PP242 Inhibits Proliferation More Completely Than Rapamycin We next measured the effect of dual mTORC1/mTORC2 inhibition by PP242 on the proliferation of primary MEFs (Figure 5B). For this analysis, we compared PP242 to selective mTORC1 inhibition by rapamycin. Rapamycin was tested at concentrations above its mTOR IC50, and at all concentrations tested, it inhibited growth to the same extent. By contrast, PP242 had a dose-dependent effect on proliferation and at higher doses was much more effective than rapamycin at blocking cell proliferation. The ability of PP242 to block cell proliferation more efficiently than rapamycin could be a result of its ability to inhibit mTORC1 and mTORC2, because rapamycin can only inhibit mTORC1. To test this possibility, we measured the effects of both compounds on the proliferation of SIN1−/− MEFs, which lack mTORC2. In SIN1−/− MEFs, rapamycin was also less effective at blocking cell proliferation than PP242. That PP242 and rapamycin exhibit very different anti-proliferative effects in SIN1−/− MEFs suggests that the two compounds differentially affect mTORC1. Rapamycin-Resistant mTORC1 mTORC1 regulates protein synthesis by phosphorylating the hydrophobic motif of p70S6-Kinase (S6K) at T389 and the eIF4E-binding-protein, 4EBP1, at multiple sites. Our proliferation experiments suggest that rapamycin and PP242 have distinct effects on mTORC1. We compared the effects of acute treatment with rapamycin and PP242 on S6K, ribosomal protein S6 (S6), and 4EBP1 phosphorylation (Figure 6A) to see if these inhibitors differentially affect the phosphorylation of these canonical substrates of mTORC1. Both rapamycin and PP242 inhibited the phosphorylation of S6K and its substrate S6, and neither rapamycin nor PP242 affected the phosphorylation of 4EBP1 on T70 (Figure S2A). In contrast, PP242 fully inhibited the phosphorylation of 4EBP1 at T36/45 and S65, whereas rapamycin only had a modest affect on these same phosphorylations. Treatment of cells with PP30 was also effective at reducing the phosphorylation of 4EBP1 at T36/45 (Figure S3), indicating that the block of T36/45 phosphorylation by PP242 is due to its inhibition of mTOR and not PKC-alpha. PIK-90 did not reduce the phosphorylation of 4EBP1 at T36/45, demonstrating that inhibition of PI3K and Akt activation alone is not sufficient to block the phosphorylation of 4EBP1 at T36/45 (Figure S3). Figure 6 PP242 Inhibits Rapamycin-Resistant mTORC1 (A) Western blots of lysates from L6 myotubes treated with kinase inhibitors and stimulated with insulin for 10 min. (B) Western blots of lysates from Figure 3B. Actin loading control is repeated here for clarity. The enhanced dephosphorylation of 4EBP1 caused by PP242 as compared with rapamycin could be due to incomplete inhibition of mTORC1 by rapamycin or involvement of mTORC2 in the phosphorylation of 4EBP1. To examine these alternatives, we analyzed the effect of PP242 and rapamycin on the phosphorylation of 4EBP1 in SIN1−/− MEFs that lack mTORC2 (Figure 6B). SIN1−/− MEFS showed higher levels of p4EBP1, suggesting that due to the lack of mTORC2, these cells have more mTORC1 activity, although stronger S6K phosphorylation in wild-type cells contradicts this simple interpretation. Despite an increase in p4EBP1 in SIN1−/− compared with wild-type MEFs, shorter exposures of the p4EBP1 blots (Figure S2B) show that PP242 inhibits p4EBP1 with the same potency in both cells. The fuller inhibition of p4EBP1 by PP242 than by rapamycin in wild-type and SIN1−/− MEFs indicates that the presence of mTORC2 is not required for rapamycin and PP242 to have distinct effects on 4EBP1 phosphorylation, and suggests that PP242 is a more complete inhibitor of mTORC1 than rapamycin. Inhibition of Translation by TORKinibs While the precise role of S6K in translation control is still poorly understood, it is known that the hypophosphorylated 4EBP1 protein acts as negative regulator of the major cap-binding protein eIF4E. We directly assessed the effect of PP242 on cap-dependent translation downstream of mTOR activation. The phosphorylation of 4EBP1 by mTOR in response to growth factor and nutrient status causes it to dissociate from eIF4E allowing eIF4G and associated factors to bind to the 5' cap, recruit the 40S subunit of the ribosome, and scan the mRNA for the start codon to initiate translation. The phosphorylation of 4EBP1 by mTOR is complicated in that it occurs at multiple sites, and not all sites are equally effective at causing dissociation of 4EBP1 from eIF4E [36]. Furthermore, a hierarchy is thought to exist whereby the N-terminal threonine phosphorylations at 36/45 precede and are required for the C-terminal phosphorylations at S65 and T70 [37,38]. Phosphorylation at S65 causes the greatest decrease in affinity of 4EBP1 for eIF4E [39,40], and S65 is probably the most important site in cells for dissociation of 4EBP1 from eIF4E [41], but other sites are also important [36,42]. We examined the effect of PP242 on the active eIF4E initiation complex of translation by using a cap-binding assay. eIF4E binds tightly to beads coated with the cap analogue 7-methyl GTP (m7GTP), allowing proteins bound to eIF4E to be examined. Rapamycin caused partial inhibition of the insulin-stimulated release of 4EPB1 from eIF4E (Figure 7A), consistent with its partial inhibition of S65 phosphorylation (Figure 6A). The rapamycin-induced retention of 4EBP1 was accompanied by a loss of recovery of eIF4G, because the binding of 4EBP1 and eIF4G to eIF4E are mutually exclusive. In contrast, treatment with PP242 caused a much larger retention of 4EBP1, raising the retention of 4EBP1 above the level seen in unstimulated serum-starved cells, which are known to have low levels of protein translation [43]. Figure 7 PP242 Inhibits Cap-Dependent Translation (A) Cap-binding proteins in lysates from Figure 6A were purified by 7-methyl GTP (m7GTP) affinity and analyzed by Western blotting. (B) Primary MEFs were transfected with a bicistronic reporter vector. The ratio of renilla (cap-dependent) to firefly (IRES-dependent) luciferase activity was measured after incubation overnight in either 10% serum (steady state) or with the indicated inhibitors in the presence of 10% serum (n = 3). *p < 0.05, **p < 0.01, ANOVA with Tukey's post test. (C) Primary MEFs were incubated overnight as in (B) prior to labeling new protein synthesis with 35S. Newly synthesized proteins were separated by SDS-PAGE, transferred to nitrocellulose and visualized by autoradiography. (D) Newly synthesized protein from three experiments as in (C) was quantified. *p < 0.05, ANOVA with Tukey's post test. Translation initiation depending on eIF4E activity is the rate-limiting step in cap-dependent protein translation [44]. PP242 caused a higher level of binding between 4EBP1 and eIF4E than rapamycin (Figure 6A), suggesting that cap-dependent translation will be more highly suppressed by PP242 than by rapamycin. To quantify the efficiency of cap-dependent translation in the presence of PP242 and rapamycin, we used the well-established bicistronic reporter assay where translation initiation of the first cistron is dependent on the 5′ cap, whereas initiation of the second cistron depends on a viral internal ribosome entry site (IRES) that bypasses the need for cap-binding proteins such as eIF4E [45]. PP242 caused a significant decrease in cap-dependent, but not IRES-dependent (Figure S4), translation, whereas rapamycin did not have a statistically significant effect on cap-dependent translation (Figure 7B), consistent with the modest effect of rapamycin on 4EBP1 phosphorylation (Figure 6A). Based on this assay, inhibition of mTOR and p4EBP1 reduces cap-dependent translation by about 30%, suggesting that cap-dependent translation is only partially inhibited by hypophosphorylated 4EBP1. The majority of protein synthesis is thought to be cap-dependent [44], and consistent with this we find that PP242 also reduces total protein synthesis by about 30%, whereas rapamycin does not have a significant effect (Figure 7C and 7D). Inhibition of mTORC1 and mTORC2 In Vivo Mouse knock-outs of mTORC1 or mTORC2 result in embryonic lethality and thus it has been difficult to examine the effects of loss of mTOR in animals. To begin to explore the tissue specific roles of mTORC1 and mTORC2 and confirm the pathway analysis from cell culture experiments, we treated mice with PP242 and rapamycin and examined the acute effect of these drugs on insulin signaling in fat, skeletal muscle, and liver tissue (Figure 8). In fat and liver, PP242 was able to completely inhibit the phosphorylation of Akt at S473 and T308, consistent with its effect on these phosphorylation sites observed in cell culture. Surprisingly, PP242 was only partially able to inhibit the phosphorylation of Akt in skeletal muscle and was more effective at inhibiting the phosphorylation of T308 than S473, despite it's ability to fully inhibit the phosphorylation of 4EBP1 and S6. These results will be confirmed by in vivo dose-response experiments, but, consistent with the partial effect of PP242 on pAkt in skeletal muscle, a muscle-specific knockout of the integral mTORC2 component rictor resulted in only a partial loss of Akt phosphorylation at S473 [46]. These results suggest that a kinase other than mTOR, such as DNA-PK [8,9], may contribute to phosphorylation of Akt in muscle. Figure 8 PP242 Inhibits mTORC2 and Rapamycin-Resistant mTORC1 In Vivo Rapamycin (5 mg/kg), PP242 (20 mg/kg), or vehicle were injected into the intraperitoneal (IP) cavity of mice, followed by IP injection of 250 mU insulin or saline. Lysates were prepared from perigenital fat, leg muscle, and liver and analyzed by Western blotting. Rapamycin often stimulates the phosphorylation of Akt [47,48], probably by relieving feedback inhibition from S6K to the insulin receptor substrate 1 (IRS1) [49], a key signaling molecule that links activation of the insulin receptor to PI3K activation. In all tissues examined, and especially in fat and muscle, acute rapamycin treatment activated the phosphorylation of Akt at S473 and T308 (Figure 8). In contrast to rapamycin, by inhibiting both mTORC2 and mTORC1, PP242 suppresses rather than enhances Akt activation. As was seen in cell culture, rapamycin and PP242 also differentially affect the mTORC1 substrates S6K and 4EBP1 in vivo. S6 phosphorylation was fully inhibited by rapamycin and PP242 in all tissues examined. While PP242 was effective at blocking the phosphorylation of 4EBP1 on both T36/45 and S65 in all tissues examined, rapamycin did not block 4EBP1 phosphorylation as completely as PP242. Further experiments will be required to identify the mechanism by which 4EBP1 phosphorylation is partially resistant to rapamycin. Discussion Rapamycin has been a powerful pharmacological tool allowing the discovery of mTOR's role in the control of protein synthesis. Since the discovery of a rapamycin-insensitive mTOR complex, there has been a significant effort to develop pharmacological tools for studying this complex. We have used two structurally distinct compounds to pharmacologically dissect the effects of mTOR kinase inhibition toward mTORC1 and mTORC2 activity. We have shown through the use of these inhibitors that the inhibition of mTOR kinase activity is sufficient to prevent the phosphorylation of Akt at S473, providing further evidence that mTORC2 is the kinase responsible for Akt hydrophobic motif phosphorylation upon insulin stimulation. We also find that phosphorylation at T308 is linked to phosphorylation at S473, as had been observed in experiments where mTORC2 was disabled by RNAi and long-term rapamycin, but not homologous recombination. Surprisingly however, inhibition of mTORC2 does not result in a complete block of Akt signaling, as T308P is partially maintained and Akt substrate phosphorylation is only modestly affected when S473 is not phosphorylated. Despite its modest effect on Akt substrate phosphorylation, PP242 was a strikingly more effective anti-proliferative agent than rapamycin. These results were reproduced even in cells lacking mTORC2 (SIN1−/−), suggesting that downstream mTORC1 substrates might be responsible for PP242′s strong anti-proliferative effects. Interestingly, we observe that phosphorylation of the mTORC1 substrate 4EBP1 is partially resistant to rapamycin treatment at concentrations that fully inhibit S6K, whereas PP242 completely inhibits both S6K and 4EBP1. Because rapamycin can only partially inhibit the phosphorylation of 4EBP1, but it can fully in inhibit the phosphorylation of S6K, rapamycin appears to be a substrate-selective inhibitor of mTORC1. Consistent with this finding, experiments with purified proteins have shown that rapamycin/FKBP12 only partially inhibits the in vitro phosphorylation of 4EBP1 at Ser 65 by mTOR but can fully inhibit the in vitro phosphorylation of S6K [50]. By contrast, LY294002, a direct inhibitor of many PI3K family members including mTOR, was equally effective at inhibiting the phosphorylation of S6K and 4EBP1 by mTOR in vitro [50] and in cells [23], although this finding is complicated by LY294002′s inhibition of multiple lipid and protein kinases [51] including PIM, a kinase potentially upstream of 4EBP1 phosphorylation [52,53]. These results argue that PP242, in addition to being useful for investigating mTORC2, can reveal rapamycin-resistant components of mTORC1 function. Indeed, proliferation of SIN1−/− MEFs is more sensitive to PP242 than rapamycin (Figure 5B), suggesting that rapamycin-resistant functions of mTORC1, including the aspects of translation initiation highlighted in Figure 7, are key to the anti-proliferative effects of PP242. Furthermore, our findings suggest that the inhibition of translational control and the anti-proliferative effects of PP242 require inhibition of 4EBP1 phosphorylation and eIF4E activity. Using TORKinibs to acutely inhibit mTOR has surprisingly led to the identification of outputs from mTORC1 that are rapamycin-resistant. These observations should motivate further studies aimed at understanding how rapamycin is able to selectively affect different outputs downstream of mTORC1. As active site inhibitors of mTOR join rapamycin and its analogs in the clinic [22,27,30], it will be important to understand the distinct effects of these pharmacological agents on cellular and organismal physiology and to evaluate their efficacy in the treatment of disease and cancer caused by hyperactivation of the PI3K→Akt→TOR pathway. Materials and Methods Ethics statement. Mice were handled in accordance with protocols approved by the committee for animal research at the University of California San Francisco, United States of America. Cell culture. Cells were grown in DMEM supplemented with 10% FBS, glutamine, and penicillin/streptomycin. Confluent L6 myoblasts were differentiated into myotubes by culturing them for 5 d in medium containing 2% FBS. L6 myotubes were maintained in medium containing 2% FBS until use. Primary wild-type MEFs used in Figure 7 were isolated at embryonic day 13.5 as previously described [54]. Primary SIN1−/− MEFs and matching wild-type controls were provided by B. Su and isolated as previously described [16]. Cell lysis and Western blotting. Except where indicated otherwise, cells were serum starved overnight and incubated with inhibitors or 0.1% DMSO for 30 min prior to stimulation with 100 nM insulin for 10 min. All inhibitors were either synthesized as previously described [21,24,55] or were from Calbiochem (rapamycin and Akti-1/2). Cells were lysed by scraping into ice cold lysis buffer followed by brief sonication. Lysates were cleared by centrifugation, resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies from Cell Signaling Technology. Unless otherwise indicated, cells were lysed in 300 mM NaCl, 50 mM Tris pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.02% NaN3, 20 nM microcystin (Calbiochem), Sigma phosphatase inhibitor cocktails 1 and 2, Roche protease inhibitor cocktail, and 2 mM PMSF. For Figures 6A and 7A, and Figure S2A, cells were lysed in cap lysis buffer (140 mM KCl, 10 mM Tris pH 7.5, 1 mM EDTA, 4 mM MgCl2, 1 mM DTT, 1% NP-40, 20 nM microcystin, Sigma phosphatase inhibitor cocktails 1 and 2, Roche protease inhibitor cocktail without EDTA and 2 mM PMSF). Cap pull-down assay. L6 myotubes from one well of a six-well plate were lysed in 300 μl of cap lysis buffer as described above. 50 μl of detergent-free cap lysis buffer and 20 μl of pre-washed cap beads were added to 150 μl of cleared lysate and incubated at 4 °C overnight with tumbling. The beads were washed twice with 400 μl of cap wash buffer (cap lysis buffer with 0.5% NP-40 instead of 1% NP-40) and twice with 500 μl of PBS. The beads were boiled in SDS-PAGE sample buffer and the retained proteins analyzed by Western blot. All antibodies were from Cell Signaling Technologies except for the anti-eIF4E antibody, which was from BD Biosciences. Kinase assays. Phosphorylation of histone H1 (4 μM) by PKC was assayed in a buffer containing 200 ng/ml recombinant kinase, 25 mM HEPES pH 7.5, 10 mM MgCl2, 5 mM ß-glycerol phosphate, 0.05 mg/ml phosphatidylserine, 0.03% Triton X-100, 0.5 mg/ml BSA, 2.5 mM DTT, 100 μM CaCl2, 1 μM PMA, 10 μM ATP, and 15 μCi/ml of γ-32P-ATP. Inhibitors were tested in a four-fold dilution series from 10 μM to 600 pM, and four measurements were made at each concentration. The kinase reaction was terminated by spotting onto nitrocellulose, which was washed 5 times with 1 M NaCl/1% phosphoric acid. The radioactivity remaining on the nitrocellulose sheet was quantified by phosphorimaging, and IC50 values were determined by fitting the data to a sigmoidal dose-response curve using the Prism software package. PDK1, mTORC1, and mTORC2 were assayed as previously described [21]. In-cell Western. L6 myotubes were grown and differentiated in 96-well plates. The outside wells of the plate were not used for the experiment, but were kept filled with media. Following stimulation, cells were fixed for 15 min with 4% formaldehyde in PBS with Ca++ and Mg++. The cells were washed three times with PBS and the blocked and permeabilized with 5% goat serum in PBS with 0.3% Triton X-100 (PBS-GS-TX). Primary antibodies to S473 (Cell Signaling #4060) and T308 (Cell Signaling #2965) were added at 1:1000 and 1:500, respectively, in PBS-GS-TX, and the plates were incubated at 4 °C overnight. The plates were then washed three times with PBS, and goat anti-rabbit secondary antibody (Pierce Biotechnology) was added at 0.01 μg/ml in PBS-GS-TX. After 1 h at room temperature, plates were washed three times with PBS. ELISA chemiluminescent reagent (Femto, Pierce Biotechnology) was added to each well and after 1 min, the plate was read in a luminescence plate reader using a 100-ms integration time. The pAkt signal from pT308 and pS473 was normalized to control wells, so that 0 represents the level of pAkt in serum starved cells and 1 represents the level upon insulin stimulation. EC50 values were determined by fitting the data to a sigmoidal dose-response curve using the Prism software package. The significance of differences between EC50 values was evaluated using the F test. Akt transfection. Akt was transfected into HEK293 cells using Lipofectamine 2000 according the manufacturers protocol. Two days after transfection, cells were serum starved overnight and the next day they were treated with inhibitors and processed for western blotting as described above. Actin cytoskeleton staining. NIH 3T3 cells were plated on poly-lysine coated coverslips at 30% confluence the day before the experiment. Following treatment with PP242 or 0.1% DMSO for 8 h in 10% serum growth medium, the actin cytoskeleton was stained as previously described [24]. Bicistronic reporter assay. Primary MEFs were transfected with a bicistronic reporter [54] containing a viral IRES using Lipofectamine 2000 according to the manufacturers protocol. At 2 d post transfection, cells were treated overnight with compounds as indicated or starved of serum. The next day, Renilla and Firefly luciferase activity were measured using the Dual-Luciferase kit (Promega). Differences in the ratio of Renilla to Firefly luciferase signals were analyzed for statistical significance by one-way ANOVA with Tukey's post test using the Prism software package. 35S labeling of new protein synthesis. Primary MEFs grown to 70% confluence in six-well plates were incubated overnight in either 10% Serum (Steady State), kinase inhibitors in 10% serum, or 0.1% serum (starved). Cells were then washed once with DMEM lacking cysteine and methionine (DMEM-noS), and the medium was replaced with DMEM-noS including dialyzed serum and kinase inhibitors as indicated. After incubation for 1 h, 50 μCi of Expre35S35S (NEN) was added to each well and the cells were labeled for 4 h. Cells were washed once with ice-cold PBS, and lysed as described above for Western blotting. Following separation by SDS-PAGE, and transfer to nitrocellulose, 35S-labeled proteins were visualized by autoradiography with film. For quantitation, the membrane was exposed to a phosphorimager screen and the resulting image was quantified in ImageJ. Differences in 35S incorporation were analyzed for statistical significance by one-way ANOVA with Tukey's post test using the Prism software package. In vivo drug treatment and Western blotting. Drugs were prepared in 100 μl of vehicle containing 20% DMSO, 40% PEG-400, and 40% saline. Six-wk-old male C57BL/6 mice were fasted overnight prior to drug treatment. PP242 (0.4 mg), rapamycin (0.1 mg), or vehicle alone was injected IP. After 30 min for the rapamycin-treated mouse or 10 min for the PP242 and vehicle-treated mice, 250 mU of insulin in 100 μl of saline was injected IP. 15 min after the insulin injection, the mice were killed by CO2 asphyxiation followed by cervical dislocation. Tissues were harvested and frozen on liquid nitrogen in 200 μl of cap lysis buffer. The frozen tissue was thawed on ice, manually disrupted with a mortar and pestle, and then further processed with a micro tissue-homogenizer (Fisher PowerGen 125 with Omni-Tip probe). Protein concentration of the cleared lysate was measured by Bradford assay and 5–10 μg of protein was analyzed by Western blot as described above. Cell proliferation assay. Wild-type and SIN1−/− MEFs were plated in 96-well plates at approximately 30% confluence and left overnight to adhere. The following day cells were treated with PP242, rapamycin, or vehicle (0.1% DMSO). After 72 h of treatment, 10 μl of 440 μM resazurin sodium salt (Sigma) was added to each well, and after 18 h, the florescence intensity in each well was measured using a top-reading florescent plate reader with excitation at 530 nm and emission at 590 nm. Supporting Information Figure S1 PP242 Inhibits Akt Phosphorylation over the Course of 1 h L6 myotubes were pre-treated with PP242 or DMSO for 30 min and stimulated with insulin for the indicated times prior to lysis and analysis by Western blotting. (4.31 MB AI). Click here for additional data file. Figure S2 Additional Analysis of 4EBP1 Phosphorylation (A) 4EBP1 phosphorylation at T70 is not inhibited by either PP242 or rapamycin. L6 myotube lysates from Figure 6A were analyzed by Western blotting. (B) 4EBP1 phosphorylation is inhibited by PP242 with similar potency in SIN1−/− and wild-type (WT) MEFs. Western blotting from Figure 6B is shown with shorter exposures of p4EBP1. (4.97 MB AI). Click here for additional data file. Figure S3 Rapamycin-Resistant Phosphorylation of 4EBP1 Is Sensitive to the TORKinibs PP30 and PP242, but Not the PI3K Inhibitor PIK-90 L6 myotube lysates from Figure 2A were analyzed by Western blotting. (2.44 MB AI). Click here for additional data file. Figure S4 PP242 Inhibits Cap, but Not IRES-Dependent, Translation (A) Renilla (cap-dependent) luciferase activity from samples in Figure 7B. (B) Firefly (IRES-dependent) luciferase activity from samples in Figure 7B. Firefly luciferase activity of the PP242 treated sample is not significantly different from control (p = 0.4, ANOVA). (1.55 MB AI). Click here for additional data file. Table S1 In Vitro IC50 Determinations Using Three Forms of mTOR (19 KB DOC) Click here for additional data file.
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            Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling.

            Yonghao Yu, Sang-Oh Yoon, George Poulogiannis … (2011)
            The evolutionarily conserved serine-threonine kinase mammalian target of rapamycin (mTOR) plays a critical role in regulating many pathophysiological processes. Functional characterization of the mTOR signaling pathways, however, has been hampered by the paucity of known substrates. We used large-scale quantitative phosphoproteomics experiments to define the signaling networks downstream of mTORC1 and mTORC2. Characterization of one mTORC1 substrate, the growth factor receptor-bound protein 10 (Grb10), showed that mTORC1-mediated phosphorylation stabilized Grb10, leading to feedback inhibition of the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated, mitogen-activated protein kinase (ERK-MAPK) pathways. Grb10 expression is frequently down-regulated in various cancers, and loss of Grb10 and loss of the well-established tumor suppressor phosphatase PTEN appear to be mutually exclusive events, suggesting that Grb10 might be a tumor suppressor regulated by mTORC1.
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              Regulation of TFEB and V-ATPases by mTORC1

              Samuel Peña-Llopis, Silvia Vega-Rubín-de-Celis, Jacob Schwartz … (2011)
              Introduction Target of rapamycin (TOR) complex 1 (TORC1) is a pivotal regulator of cell growth conserved from yeast to humans and implicated in cancer (Wullschleger et al, 2006; Abraham and Eng, 2008). Mammalian TORC1 (mTORC1) includes the atypical serine/threonine kinase mTOR and an adaptor protein, regulatory-associated protein of mTOR (Raptor) (Hara et al, 2002; Kim et al, 2002; Loewith et al, 2002). mTORC1 regulates cell growth, at least in part, by promoting cap-dependent translation and its best-characterized substrates are the eukaryotic initiation factor 4E binding protein-1 (4E-BP1) (Sonenberg and Hinnebusch, 2009) and S6 kinase 1 (S6K1) (Fingar and Blenis, 2004). mTORC1 is specifically inhibited by rapamycin (also called sirolimus) (Jacinto et al, 2004; Sarbassov et al, 2004) and rapamycin analogues are used clinically for many applications including cancer treatment (Abraham and Eng, 2008). Indeed, two rapamycin analogues, temsirolimus (Hudes et al, 2007) and everolimus (Motzer et al, 2008), are approved by the FDA for the treatment of advanced RCC (renal cell carcinoma). mTORC1 is regulated by a complex formed by the proteins tuberous sclerosis complex 1 (TSC1) and 2 (TSC2), which is essential for the relay of signals from oxygen (Brugarolas et al, 2004; Connolly et al, 2006; Kaper et al, 2006; Liu et al, 2006; DeYoung et al, 2008), energy stores (Inoki et al, 2003b; Corradetti et al, 2004; Shaw et al, 2004) and growth factors (Jaeschke et al, 2002; Kwiatkowski et al, 2002; Zhang et al, 2003a). mTORC1 regulation by hypoxia involves the protein regulated in development and DNA damage response 1 (REDD1), a conserved protein with a novel fold (Vega-Rubin-de-Celis et al, 2010), which, when overexpressed is sufficient to inhibit mTORC1 in a TSC1/TSC2-dependent manner (Brugarolas et al, 2004). Interestingly, in some settings, hypoxia signals are transduced via AMP-activated protein kinase (Liu et al, 2006; Wolff et al, 2011), which phosphorylates TSC2 and is normally involved in the relay of energy signals (Inoki et al, 2003b; Gwinn et al, 2008). mTORC1 regulation by growth factors involves TSC2 phosphorylation by Akt (Dan et al, 2002; Inoki et al, 2002; Manning et al, 2002; Potter et al, 2002), extracellular signal-regulated kinase (Erk) (Ma et al, 2005) and ribosomal S6 kinase (Rsk) (Roux et al, 2004). TSC2 functions as a GTPase-activating protein towards Ras homologue enriched in brain (Rheb) (Castro et al, 2003; Garami et al, 2003; Tee et al, 2003; Inoki et al, 2003a; Zhang et al, 2003b), a small GTPase that directly interacts with and activates mTORC1 (Sancak et al, 2007; Avruch et al, 2009). mTORC1 regulation by nutrients is independent of TSC1/TSC2 (Zhang et al, 2003a; Smith et al, 2005; Roccio et al, 2006) and involves its localization to a cellular compartment where it becomes receptive to activation by Rheb (Sancak et al, 2008, 2010). Amino-acid stimulation drives mTORC1 to the surface of the late endosome/lysosome in a manner that depends on a Rag GTPase heterodimer (RagA [or B] bound to RagC [or D]) and a multimeric complex termed the Ragulator (Kim et al, 2008; Sancak et al, 2010). Importantly, constitutive mTORC1 targeting to this compartment renders mTORC1 insensitive to amino-acid withdrawal (Sancak et al, 2010). The significance of the late endosome/lysosome in mTORC1 activation has been recently established in experiments manipulating endosome maturation (Flinn et al, 2010; Li et al, 2010). Disrupting endosome maturation with a constitutively active Rab5 or through depletion of hVps39 inhibited the activation of mTORC1 by growth factors and amino acids (Flinn et al, 2010; Li et al, 2010). A critical component of the endosome required for its acidification and maturation is the vacuolar H+-ATPase (V-ATPase) (Marshansky and Futai, 2008). V-ATPases are multisubunit complexes formed by a membrane-embedded V0 domain (a, d, e, c, c′ and c″ subunits) responsible for proton translocation, and a cytosolic V1 domain (A–H subunits), which provides energy through ATP hydrolysis (Forgac, 2007). V-ATPases are present in virtually every eukaryotic cell and V-ATPase function is essential for survival under a variety of conditions (Beyenbach and Wieczorek, 2006). However, little is known about the regulation of V-ATPases and in particular about how their expression is controlled. Here, we have uncovered a regulatory network linking V-ATPases and endocytosis to mTORC1 that involves the transcription factor EB (TFEB). TFEB is a basic helix-loop-helix (bHLH) leucine zipper transcription factor of the Myc family, microphthalmia transcription factor subfamily (Steingrimsson et al, 2004), that is translocated in a subset of renal tumours (Davis et al, 2003; Kuiper et al, 2003; Srigley and Delahunt, 2009). Results V-ATPase enrichment among mTORC1-regulated genes We identified V-ATPase genes in an unbiased screen for genes whose expression was tightly regulated by mTORC1. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic. In Tsc2 +/+ immortalized mouse embryo fibroblasts (MEFs), but not Tsc2 −/−, mTORC1 is inhibited by serum deprivation (0.1% fetal bovine serum, FBS) (Figure 1A). In Tsc2 −/− cells, abnormally increased mTORC1 activity can be corrected by treatment with rapamycin (Figure 1B). By contrast, rapamycin has little effect on mTORC1 in Tsc2 +/+ cells in which mTORC1 is already inhibited by low serum (Figure 1B). Thus, under serum-deprived conditions, mTORC1 activity is low in Tsc2 +/+ cells (untreated or rapamycin treated), high in Tsc2 −/− cells, but lowered by rapamycin; a pattern referred to as a ‘low/low/high/low' or ‘LLHL' (Figure 1B). This experimental setup creates an ‘asymmetric' response to rapamycin (in Tsc2 −/− versus Tsc2 +/+ cells), which, as illustrated hereafter, can be harnessed to great advantage. Using an Affymetrix MOE430A array platform and duplicate samples, genes were identified with an LLHL expression pattern (upregulated in untreated Tsc2 −/− cells compared with a baseline formed by Tsc2 +/+ cells, untreated or rapamycin treated, and Tsc2 −/− rapamycin-treated cells). With a false discovery rate-corrected P-value (FDR Q) cutoff of 0.05 and a threshold of 1.5-fold, we identified 78 probe sets (henceforth referred to as probes). They corresponded to 75 genes (Figure 1C and D). We hypothesized that if the association of probes with the LLHL pattern was random, there would be similar numbers of probes with the three alternate patterns (HLLL, LHLL and LLLH). Strikingly, however, no probes fitted an HLLL or LHLL pattern, and only 22 probes conformed to an LLLH pattern (Figure 1C). Given these results and assuming a binomial distribution with equal probabilities for all patterns, the probability of finding 78 probes with an LLHL pattern by chance alone was below 1.6 × 10−28. To validate the microarray findings, qRT–PCR was performed on a subset of genes with the weakest LLHL association (highest P-values). In every instance, the LLHL pattern was confirmed and the fold change was even greater than expected (Figure 1E). Among the LLHL probes, we found six for V-ATPase genes (Figure 1D; Supplementary Table S1). The probability that there would be six V-ATPase probes among the 78 probes identified by chance alone was 20% reduction in mean expression levels in Tsc2 −/− MEFs by Tfeb knockdown), including several V-ATPases (Supplementary Table S3). LLHL ‘flattening' was confirmed for several V-ATPase genes by qRT–PCR (Figure 4E). Interestingly, the decrease in the number of genes with an LLHL pattern after Tfeb knockdown was specific for the LLHL pattern. In fact, Tfeb knockdown resulted in an increase in the number of genes with a LLLH pattern (35 versus 323; Figure 4C; Supplementary Table S4). These results suggest that Tfeb is an important effector downstream of mTORC1 involved in the regulation of a substantial number of genes. Regulation of endocytosis by V-ATPases, TFEB and mTORC1 Having determined that mTORC1 regulated V-ATPase expression in cells in culture and in mice and having established that TFEB was required for mTORC1-induced V-ATPase expression, we sought to examine the functional significance of V-ATPase regulation by mTORC1. Because V-ATPases are involved in vesicle trafficking (Marshansky and Futai, 2008), we evaluated the effects of mTORC1 on endocytosis. For these experiments, we used albumin, the most abundant protein in plasma and a protein that is taken up by macrophages and hydrolyzed to amino acids, which are then released in the circulation and are utilized by different cells (Guyton and Hall, 1996). While albumin uptake occurs through different pathways in different cell types (Mayor and Pagano, 2007), albumin is taken up by MEFs through a caveolin-dependent mechanism (Razani et al, 2001). FITC-conjugated bovine serum albumin (FITC-BSA) was efficiently taken up by MEFs, where it was found in punctate structures reminiscent of vesicles, which also contained V-ATPases (Figure 5A). In keeping with the idea that mTORC1 regulates endocytosis, FACS analyses showed higher levels of albumin uptake in Tsc2 −/− than Tsc2 +/+ cells (Figure 5B and C). In addition, treatment of Tsc2 −/− cells with rapamycin downregulated albumin uptake (Figure 5C). Albumin uptake required V-ATPases as determined by experiments using the V-ATPase inhibitor bafilomycin A1 (Bowman and Bowman, 2002) (Figure 5B and C). In addition, albumin uptake was downregulated by siRNA-mediated depletion of essential components of either the V0 or the V1 domain (alone or in combination) in highly transfectable HeLa cervical carcinoma cells (Figure 5D and E). Next, we tested whether Tfeb may be similarly involved in endocytosis. Indeed, Tfeb knockdown resulted in a statistically significant decrease in albumin uptake (Figure 5F). The effects of Tfeb knockdown on albumin uptake in Tsc2 −/− cells were similar to those achieved by treatment with rapamycin (Figure 5F). Taken together, these data show that mTORC1 promotes endocytosis in a Tfeb- and V-ATPase-dependent manner. Regulation of Tfeb phosphorylation and subcellular localization by the Tsc1/Tsc2 complex and rapamycin Having established the importance of Tfeb downstream of mTORC1, we explored the mechanism whereby mTORC1 regulated Tfeb. We observed that not only the expression levels of Tfeb appeared to be upregulated by mTORC1, but also that mTORC1 affected Tfeb electrophoretic mobility on SDS–PAGE (Figures 4B and 6A). In Tsc2 −/− cells, Tfeb was prominently found in a fast-migrating form(s) (Figures 4B and 6A). Treatment of Tsc2 −/− cells with rapamycin shifted Tfeb up giving rise to a pattern similar to that observed in wild-type cells (Figure 6A). Similar results were observed in Tsc1 +/+ and Tsc1 −/− cells (Figure 6B). To determine how quickly Tfeb mobility was affected by rapamycin, a time course was performed. As shown in Figure 6C, rapamycin treatment for 1 h was sufficient to change the distribution of Tfeb. Changes in Tfeb migration may reflect changes in phosphorylation, and phosphoproteomic studies have shown TFEB to be phosphorylated in >10 sites (Dephoure et al, 2008; Mayya et al, 2009). To determine whether Tfeb migration was affected by phosphorylation, we evaluated the effects of λ phosphatase treatment. As shown in Figure 6D, the migration of Tfeb was accelerated by treatment of cell lysates with λ phosphatase. Parenthetically, by comparison to similarly processed samples incubated without λ phosphatase for the same amount of time, phosphatase exposure not only accelerated Tfeb migration, but also led to a downregulation of total Tfeb protein levels (Figure 6D). Notably, even fast-migrating Tfeb in Tsc2 −/− cells was subject to a downshift following phosphatase treatment (Figure 6D). These results indicate that even in Tsc2 −/− cells, Tfeb retained some level of phosphorylation. We considered that the changes in Tfeb phosphorylation may be linked to changes in its subcellular localization and performed subcellular fractionation experiments. Whereas in Tsc2 +/+ cells, nuclear Tfeb was undetectable, Tfeb was found in nuclear fractions of Tsc2 −/− cells (Figure 6E). In the nucleus, Tfeb appeared to be present almost exclusively in a fast-migrating form (Figure 6E). To determine whether nuclear Tfeb retained some level of phosphorylation, nuclear extracts were treated with λ phosphatase. As shown in Figure 6F, this resulted in a further shift, indicating that nuclear Tfeb was also phosphorylated. Since phosphatase treatment collapsed both nuclear and cytosolic Tfeb onto an undistinguishable band, the differences in migration likely reflect differences in phosphorylation. Next, we sought to determine how rapamycin affected nuclear Tfeb in Tsc2 −/− cells. Rapamycin treatment caused a shift up in nuclear Tfeb mobility and a progressive decline in nuclear Tfeb (Figure 6G). Taken together, these data show that the migration and nuclear localization of Tfeb are coordinately regulated by Tsc1/Tsc2 and rapamycin and suggest that mTORC1 controls the phosphorylation state and nuclear localization of Tfeb. Regulation of TFEB phosphorylation and nuclear localization by mTORC1 To extend these studies, we examined the regulation of TFEB by mTORC1 in HeLa cells. HeLa cells were transduced with lentiviruses encoding a TSC2 shRNA or a scrambled control and, as for MEFs, cultured for 24 h in 0.1% FBS with or without rapamycin. The results were remarkably similar to those observed in MEFs. TSC2 inactivation led to a downshift in TFEB and this was reverted by rapamycin (Figure 7A and B). As in MEFs, phosphatase treatment accelerated TFEB migration even in cells depleted of TSC2 (Figure 7C). Subcellular fractionation studies showed the presence of fast-migrating TFEB in nuclear fractions of cells depleted of TSC2, but not in controls (Figure 7D) and nuclear TFEB retained some level of phosphorylation (Figure 7E). Thus, the regulation of TFEB phosphorylation and nuclear localization by TSC1/TSC2 and rapamycin was conserved across cell types. To unequivocally establish that TFEB regulation by TSC1/TSC2 and rapamycin was indeed mTORC1 dependent, we inactivated mTORC1 by knocking-down Raptor. HeLa cells were transduced with a Raptor shRNA (or a scrambled control) and, following selection, were subjected to TSC2 depletion using siRNA. As shown in Figure 7F, in cells depleted of Raptor, TSC2 RNAi failed to accelerate TFEB mobility. In a complementary set of experiments, Raptor siRNA (like rapamycin) retarded TFEB mobility in HeLa cells depleted of TSC2 (Figure 7G). Regardless of the means to deplete Raptor, Raptor inactivation prevented the accumulation of faster-migrating TFEB (Figure 7F and G). Taken together, our data indicate that mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization in multiple cell types. mTORC1-dependent nuclear localization of TFEB requires C-terminal serine-rich motif To dissect TFEB regulation by mTORC1, we first identified conditions in which ectopically expressed TFEB behaved like endogenous TFEB. Epitope-tagged TFEB, when expressed at low levels in HeLa cells, preserved its regulation by mTORC1; it was enriched in the nuclei of TSC2-depleted cells and the levels were decreased by rapamycin (Figure 8A). To determine what regions were important for TFEB regulation by mTORC1, we examined a series of deletions (Figure 8B). TFEB233–476 and TFEB290–476 behaved like full-length TFEB, indicating that the N-terminal half of TFEB was dispensable for mTORC1-dependent nuclear localization (Figure 8C). Within this region, mutation of a serine residue (S142) proposed to be phosphorylated by ERK2 and to regulate TFEB nuclear localization (Settembre et al, 2011) had no appreciable effect (Figure 8D and E). Deletion of residues 1–320 resulted in a protein that was enriched in nuclei (Figure 8C). Interestingly, deletion of just 15 amino acids from the C-terminus abrogated mTORC1-dependent nuclear localization (Figure 8C). By contrast, deletion of seven amino acids had no effect (Figure 8C). These data indicated that there were critical residues between 462–469. This corresponded to a serine-rich region (462SSRRSSFS469). To further evaluate, this serine-rich motif, we mutated all S to A (5 × SA). Interestingly, TFEB5 × SA was largely cytoplasmic and failed to be driven into the nucleus in cells with active mTORC1 (Figure 8D and E). Thus, a serine(s) within this motif is necessary for mTORC1-dependent nuclear TFEB localization. Conversely, mutation of the same residues to aspartic acid resulted in an enrichment of TFEB in the nucleus (Figure 8D and E). TFEB5 × SD localized to the nucleus even in cells with inactive mTORC1. Interestingly, the percentage of cells with TFEB5 × SD in the nucleus was the same as for wild-type TFEB in mTORC1-active cells (Figure 8E; see also Figure 8A). These data show that the presence of phosphomimetic residues in the serine-rich motif obviates the mTORC1 requirement for TFEB nuclear localization. The simplest explanation for these data is that a serine(s) in the C-terminal serine-rich motif is phosphorylated in an mTORC1-dependent manner and that phosphorylation is both necessary and sufficient to drive TFEB into the nucleus (see model in Figure 8F). Complex TFEB regulation beyond mTORC1 by multiple signals Next, we examined TFEB in wild-type MEFs and HeLa cells grown under standard conditions (10% FBS). TFEB was found in both a slow and fast-migrating form(s) in MEFs, but predominantly in a slow migrating form(s) in HeLa cells (Figure 9A). We evaluated the effects of multiple interventions on TFEB mobility. Glucose withdrawal resulted in a downshift in TFEB in both MEFs and HeLa cells (Figure 9A). Amino acid withdrawal induced acute changes in TFEB migration in HeLa cells and subsequently led to the accumulation of slow migrating TFEB, which was also observed in MEFs (Figure 9A). The mobility of TFEB was also affected by serum deprivation in both MEFs and HeLa cells (Figure 9A). Finally, changes in TFEB mobility were also induced by treatment with a reducing agent, DTT. DTT led to a progressive shift in TFEB to a fast-migrating form(s) in both HeLa and MEFs (Figure 9A). Overall, these data show that TFEB is extensively and dynamically regulated by multiple stimuli. While post-translational modifications other than phosphorylation may be involved in the changes observed in TFEB migration, TFEB could be down-shifted by phosphatase treatment in every instance (data not shown). The dynamic changes in TFEB mobility induced by the various interventions could not be explained by their expected effects on mTORC1 suggesting that TFEB regulation involves multiple pathways and is likely to be rather complex. This is consistent with the finding that TFEB is phosphorylated in >10 sites (Dephoure et al, 2008; Mayya et al, 2009; Yu et al, 2011). Finally, since TFEB is an oncogene (Davis et al, 2003; Kuiper et al, 2003; Srigley and Delahunt, 2009), we tested whether TFEB was implicated in the regulation of cell proliferation. As shown in Figure 9B, stable depletion of TFEB in both MEFs and HeLa cells lowered the rates of cell expansion. Discussion Using a robust experimental paradigm, we uncovered a novel regulatory network linking endocytosis to TFEB and mTORC1. An unbiased gene expression analysis coupled with a design amenable to refined statistics led us to identify a connection between mTORC1 and V-ATPase genes. Publicly available data sets confirmed the link and experiments in mice showed that mTORC1 regulated V-ATPase expression also in vivo. A recent study had established that TFEB regulated lysosomal biogenesis (Sardiello et al, 2009) and among the genes regulated by TFEB we found several V-ATPases. These results led us to hypothesize that V-ATPase regulation by mTORC1 may be mediated by TFEB. Indeed, TFEB was necessary for V-ATPase upregulation by mTORC1. mTORC1 regulated TFEB phosphorylation and nuclear localization and a C-terminal serine-rich motif was identified that is essential for mTORC1-dependent TFEB nuclear localization. Our data suggest that mTORC1 induces the phosphorylation of a serine(s) within this motif driving thereby TFEB to the nucleus. TFEB regulates the expression of V-ATPases and other lysosomal genes and TFEB was required for mTORC1-induced endocytosis. To our knowledge this is the first study to show that endogenous TFEB is regulated at the subcellular localization level and that mTORC1 promotes TFEB nuclear localization. These data link an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis. An increasing amount of evidence implicates TORC1 in endocytosis. mTOR was identified in an RNAi screen for kinases involved in clathrin-mediated endocytosis (Pelkmans et al, 2005) and Tsc2-deficient cells were previously shown to have increased fluid-phase endocytosis (Xiao et al, 1997). In Drosophila melanogaster, a sensitized screen for genes that modified a tissue-specific dTOR overexpression phenotype identified an important regulator of endocytosis, Hsc70-4, a clathrin uncoating factor, and the authors showed that dTOR was involved in fat body endocytosis (Hennig et al, 2006). Interestingly, besides V-ATPases, mTORC1 regulated the expression of multiple lysosomal genes (Figure 1D; see also Supplementary Table S5). These effects may be mediated, at least in part, by TFEB, which regulates lysosome biogenesis (Sardiello et al, 2009). Thus, one mechanism whereby mTORC1 and TFEB may affect endocytosis is by regulating V-ATPase levels and lysosomes. The lysosome is the final destination not only for endosomal cargo but also for autophagosomes. In response to starvation, mTORC1 is inhibited leading to the formation of autophagosomes that engulf intracellular components for recycling and sustenance (He and Klionsky, 2009; Kroemer et al, 2010). Autophagosomes and their content are delivered to lysosomes, which accumulate in the perinuclear area (Korolchuk et al, 2011). Autophagosomes fuse with lysosomes to generate autolysosomes, a process that rapidly depletes the lysosomal pool (Yu et al, 2010). The degradation of macromolecules and release of their constituents into the cytosol reactivates mTORC1 (Yu et al, 2010). mTORC1 reactivation terminates autophagy and through a poorly understood mechanism repletes the lysosome pool (Yu et al, 2010). However, how the pool of lysosomes is repleted is unknown. Our results suggest that lysosomal reformation in response to autophagy, which is mTORC1 dependent (Yu et al, 2010), may be mediated, at least in part, by TFEB. mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization. mTORC1 promoted TFEB nuclear localization and this process required a C-terminal serine-rich motif. Serine substitution to non-phosphorylatable residues abrogated TFEB regulation by mTORC1. More importantly, mutation to phosphomimetic amino acids was sufficient to reproduce the effect of mTORC1 on TFEB nuclear localization obviating the need for mTORC1. These data support a model in which mTORC1 (or an effector kinase) directly phosphorylates a serine(s) within the C-terminal serine-rich motif driving thereby TFEB to the nucleus. Consistent with this notion, nuclear TFEB was phosphorylated. This model may seem at odds with the observation that nuclear TFEB is fast-migrating and possibly hypo-phosphorylated by comparison to cytoplasmic (presumably cytosolic) TFEB. However, as our data suggest, nuclear TFEB may be phosphorylated on sites that are not phosphorylated on cytosolic TFEB, and thus no inferences can be drawn about the degree of phosphorylation based on SDS–PAGE migration. Furthermore, while we have identified a critical motif involved in mTORC1 regulation, other sites in TFEB appear to be regulated by mTORC1 (Yu et al, 2011). Our results further emphasize the link between mTORC1 and the late endosome/lysosome and provide evidence for the existence of bidirectional regulatory loops. mTORC1 localizes to the surface of the late endosome/lysosome in a highly choreographed manner (Korolchuk et al, 2011; Narita et al, 2011). The late endosome/lysosome is necessary for mTORC1 activation (Flinn et al, 2010; Li et al, 2010; Sancak et al, 2010) and mTORC1 is reactivated under conditions of persistent starvation following macromolecule degradation in autolysosomes (Yu et al, 2010). However, not only is mTORC1 downstream of the lysosome, but, inasmuch as mTORC1 regulates TFEB and V-ATPases, mTORC1 is also upstream. mTORC1 reactivation by autolysosomes requires Spinster, a lysosomal efflux sugar transporter. Spinster has been proposed to act by regulating lysosomal pH (Rong et al, 2011), which is controlled primarily by the V-ATPase. Interestingly, not only has luminal pH been implicated in mTORC1 regulation, but changes in cytosolic pH occur in response to alterations in nutrient conditions (Korolchuk et al, 2011) and have been implicated in the regulation of lysosome topology and mTORC1 (Korolchuk et al, 2011). Recently, TFEB was found to regulate autophagy gene expression (Settembre et al, 2011). Starvation led to ERK2 inactivation, which in turn, decreased TFEB phosphorylation and increased its nuclear localization. This model is in principle compatible with our results. TFEB is phosphorylated in over 10 sites (Dephoure et al, 2008; Mayya et al, 2009; Yu et al, 2011), and mechanisms likely exist that drive TFEB into the nucleus independently of the activation state of mTORC1. However, the studies do differ in one aspect. Whereas mutation of the putative ERK2 phosphorylation site, S142, to A is reported to be sufficient to drive TFEB into the nucleus (Settembre et al, 2011), we find that, at least under low serum conditions, the subcellular localization of TFEB is not appreciably altered by a S142A mutation. The experimental conditions studied by Settembre et al (2011), which involve evaluating ectopically expressed TFEB in cells in the absence of glucose, amino acids and serum, are quite different from ours. Individual deprivation of glucose, amino acids or serum has profound, dynamic and diverse effects on TFEB (see Figure 9A). Given these observations and the complexity of TFEB phosphorylation (Dephoure et al, 2008; Mayya et al, 2009; Yu et al, 2011), how starvation of all three sources will affect TFEB is unclear. Consistent with this complexity, the changes in TFEB migration observed following deprivation of individual factors could not be explained by the predicted changes on mTORC1 activity. In fact, we speculate that the discovery that mTORC1 regulates TFEB may have been possible only because of the tight experimental system we used. Besides V-ATPases and lysosomal genes, many other genes downstream of mTORC1 were regulated in a Tfeb-dependent manner. Approximately 25% of genes induced by mTORC1 required Tfeb. Experiments are ongoing to determine how many of these genes are directly regulated by Tfeb. A similar experimental setup previously identified Hif-1α as an mTORC1 effector (Brugarolas et al, 2003), and Hif-1α is an important mediator of a metabolic gene expression programme (Duvel et al, 2010). Along with sterol regulatory element-binding protein-1 (Porstmann et al, 2008), a transcription factor whose nuclear localization is regulated by mTORC1, and Hif-1α, TFEB is an important mediator of mTORC1 effects on gene expression. The discovery that mTORC1 regulates TFEB has clinical implications. It may explain the expression of melanocyte lineage markers in tumours of patients with TSC (Martignoni et al, 2008), a syndrome arising from mutations in either the TSC1 or TSC2 genes, resulting in constitutive mTORC1 activation (El-Hashemite et al, 2003; Kenerson et al, 2007). Patients with TSC are predisposed to develop renal angiomyolipomas, which express melanocytic markers such as HMB45 and Melan-A (Martignoni et al, 2008), and since expression of these proteins in melanomas is thought to be driven by TFEB (or related family members with whom TFEB can heterodimerize) (Steingrimsson et al, 2004), these markers may be similarly driven by TFEB in renal angiomyolipomas. Interestingly, TFEB functions as an oncogene in the kidney. The TFEB gene is translocated in a subset of renal tumours where it is subjected to a robust heterologous promoter (Davis et al, 2003; Kuiper et al, 2003). Translocation carcinomas tend also to aberrantly express melanocytic markers (Srigley and Delahunt, 2009), which may be similarly driven by TFEB. TFEB activation may also account for the expression of cathepsin-K, a lysosomal protease that has been recently proposed as a marker for this tumour type (Martignoni et al, 2009). Interestingly, translocation carcinomas are often recognized by immunohistochemical studies showing high levels of nuclear TFEB (Srigley and Delahunt, 2009). Because TFEB nuclear localization is regulated by mTORC1 and as we show, sirolimus excludes TFEB from the nucleus, sirolimus, or its derivatives, temsirolimus and everolimus, may be effective against this tumour type. While this study focused on a small subset of genes, many genes were identified whose expression was regulated by mTORC1 not only positively but also negatively (Supplementary Figures S8–S10; Supplementary Table S1). Moreover, genes were also found whose expression was regulated by Tsc2 independently of mTORC1 (LLHH and HHLL patterns). As previously reported (Brugarolas et al, 2003), this would point towards mTORC1-independent functions of the Tsc1/Tsc2 complex (Supplementary Figure S8; Supplementary Table S1). In addition, the expression of some genes appeared to be regulated by rapamycin to a significantly greater extent than by Tsc2 (HLHL and LHLH patterns) (Supplementary Figure S8; Supplementary Table S1). Importantly, in all these settings, analyses of alternative patterns showed the associations to be statistically significant (Supplementary Figure S8). Incidentally, among the genes with an HLHL pattern, there was enrichment for genes implicated in glycolysis, pentose phosphate pathway and fatty acid biosynthesis (Supplementary Table S5). These pathways were recently reported to be regulated by mTORC1 (Duvel et al, 2010) and our results suggest that they are affected by rapamycin to a greater extent than by Tsc1/Tsc2. In summary, our work uncovered a novel regulatory network connecting mTORC1 to a master regulator of lysosome biogenesis, TFEB, which is essential for mTORC1-induced endocytosis. Materials and methods Cell culture, drug and hypoxia treatments MEFs Tsc2 +/+; p53 −/− (Tsc2 +/+) and Tsc2 −/−; p53 −/− (Tsc2 −/−) were a gift of DJ Kwiatkowski (Brigham and Women's Hospital, Harvard Medical School, Boston, MA). Cells were grown in high glucose Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% FBS (HyClone) and 1% (v/v) penicillin/streptomycin (P/S) (Gibco) in a humidified incubator at 37°C and 5% CO2. For experiments, cells were plated to achieve ∼70% confluency at the time of treatment. For low-serum experiments, cells were washed twice in PBS and medium was changed to the same base medium but supplemented with 0.1% FBS and cells were harvested after 24 h. Where indicated, rapamycin (LC Laboratories) was used at 25 nM in methanol and methanol was used as vehicle. Where indicated, cells were treated with actinomycin D (Sigma) at 10 μg/ml, bafilomycin A1 (LC Laboratories) at 100 nM, or DTT (10 mM). For growth curves, cells were plated on day 0, maintained in 10% FBS and triplicates were counted every 24 h. For hypoxia experiments, cells were exposed to 1% O2 and 5% CO2 at 37°C in a hypoxia chamber (Coy Laboratory Products). For starvation time courses, cells were washed with PBS twice and starved of amino acids (amino acid-free media), glucose (glucose-free media) or serum (0.1% FBS) for different amounts of time. Hif-1α, Tfeb, Raptor and TSC2 shRNAs Recombinant lentiviruses were generated in HEK293T cells by transient transfection using calcium phosphate with the envelope plasmid pMD2.G (Database ID p486), packaging plasmid psPAX2 (p485) and the specific plasmid of interest. pMD2.G, psPAX2 and pLKO.1 or pGIPZ vectors were transfected at a ratio of 3:8:10. Supernatants were collected for 2 days, passed through a 0.45-μm filter, and used to infect MEFs or HeLa cells for 24 h in medium containing 8 μg/ml polybrene (Sigma). MEFs and HeLa cells were selected with 2 μg/ml puromycin for at least 1 week and HeLa cells were further enriched by FACS. The following vectors were used: pLKO.1 vectors with a Hif-1α shRNA (5′-AGAGGTGGATATGTCTGGG-3′) (p481) generously provided by LB Gardner (Nemetski and Gardner, 2007), a mouse Tfeb shRNA from Open Biosystems (sequence A, TRCN0000085548, 5′-CGGCAGTACTATGACTATGAT-3′) (p633) or a designed Tfeb shRNA (sequence B, 5′-GGCAGTACTATGACTATGATG-3′) (p652), a human TFEB shRNAs from Open Biosystems (sequence 110, TRCN0000013110, 5′-GAGACGAAGGTTCAACATCAA-3′) (p700) or (sequence 111, TRCN0000013111, 5′-GAACAAGTTTGCTGCCCACAT-3′) (p701), a raptor shRNA from Addgene (Addgene plasmid 1858) (p741) or a scrambled sequence (5′-GGGTCTGTATAGGTGGAGA-3′) (p480). pGIPZ vectors containing a TSC2 (p496) or a scrambled (p483) shRNA were from Open Biosystems. Western blotting and antibodies Cell lysates and western blotting was performed as previously described (Vega-Rubin-de-Celis et al, 2010), using antibodies from the following sources: S6K1, phospho-S6K1 (T389), S6, phospho-S6 (S235/236), total 4E-BP1, phospho-4E-BP1 (T37/46), phospho-4E-BP1 (S65) from Cell Signaling; HIF-1α, TSC1 and Menin from Bethyl laboratories; TSC2, ATF4 and c-Myc from Santa Cruz; Raptor from Millipore; Erk1/2, phospho-Erk1/2 (T183/Y185) and Tubulin from Sigma; Cyclophilin B from Abcam; GLUT-1 from Novus Biologicals; and Grp78 from BD Biosciences. Atp6v1a and Atp6v1b2 antibodies were a generous gift from D Brown (Massachusetts General Hospital, Boston, MA); and Atp6v0a1 and Atp6v0a3 antibodies were kindly provided by V Marshansky (Massachusetts General Hospital, Boston, MA). Tfeb antibodies were developed in collaboration with Bethyl Laboratories. For the remaining Materials and methods, please see the Supplementary data section. Accession numbers Microarrays were deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE27982 and GSE28021. Supplementary Material Supplementary Data Supplementary Table S1 Supplementary Table S3 Supplementary Table S4 Review Process File
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                Author and article information

                Journal
                Journal ID (nlm-ta): EMBO J
                Title: The EMBO Journal
                Publisher: Nature Publishing Group
                ISSN (Print): 0261-4189
                ISSN (Electronic): 1460-2075
                Publication date (Print): 07 March 2012
                Publication date (Electronic): 17 February 2012
                Volume: 31
                Issue: 5
                Pages: 1095-1108
                Affiliations
                [1 ]Telethon Institute of Genetics and Medicine (TIGEM) , Naples, Italy
                [2 ]Department of Molecular and Human Genetics, Baylor College of Medicine , Houston, TX, USA
                [3 ]Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital , Houston, TX, USA
                [4 ]Whitehead Institute for Biomedical Research, Nine Cambridge Center , Cambridge, MA, USA
                [5 ]Department of Biology, Massachusetts Institute of Technology (MIT) , Cambridge, MA, USA
                [6 ]David H Koch Institute for Integrative Cancer Research at MIT , Cambridge, MA, USA
                [7 ]Department of Genetics and Development, College of Physicians and Surgeons, Columbia University , New York, NY, USA
                [8 ]BioMarin Pharmaceutical Inc , Novato, CA, USA
                [9 ]Department of Immunology, Center for Cancer Immunology Research, The University of Texas MD Anderson Cancer Center , Houston, TX, USA
                [10 ]Seven Cambridge Center, Broad Institute , Cambridge, MA, USA
                [11 ]Howard Hughes Medical Institute, MIT , Cambridge, MA, USA
                [12 ]Medical Genetics, Department of Pediatrics, Federico II University , Naples, Italy
                Author notes
                [a ]Telethon Institute of Genetics and Medicine (TIGEM), Via Pietro Castellino 111, Naples 80131, Italy. Tel.: +39 081 6132207; Fax: +39 081 579 0919; E-mail: ballabio@ 123456tigem.it
                [*]

                These authors contributed equally to this work

                Article
                Publisher Item ID: emboj201232
                DOI: 10.1038/emboj.2012.32
                PMC ID: 3298007
                PubMed ID: 22343943
                SO-VID: ca458d16-7d58-4d8f-861e-c2ebd996b611
                Copyright © Copyright © 2012, European Molecular Biology Organization
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                This is an open-access article distributed under the terms of the Creative Commons Attribution Noncommercial Share Alike 3.0 Unported License, which allows readers to alter, transform, or build upon the article and then distribute the resulting work under the same or similar license to this one. The work must be attributed back to the original author and commercial use is not permitted without specific permission.

                History
                Date received : 22 December 2011
                Date accepted : 27 January 2012
                Categories
                Subject: Article

                ScienceOpen disciplines: Molecular biology
                Keywords: autophagy,cellular clearance,endocytosis,starvation
                Data availability:
                ScienceOpen disciplines: Molecular biology
                Keywords: autophagy, cellular clearance, endocytosis, starvation

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