Research ArticleCell Biology

The Transcription Factor TFEB Links mTORC1 Signaling to Transcriptional Control of Lysosome Homeostasis

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Science Signaling  12 Jun 2012:
Vol. 5, Issue 228, pp. ra42
DOI: 10.1126/scisignal.2002790

Abstract

Lysosomes are the major cellular site for clearance of defective organelles and digestion of internalized material. Demand on lysosomal capacity can vary greatly, and lysosomal function must be adjusted to maintain cellular homeostasis. Here, we identified an interaction between the lysosome-localized mechanistic target of rapamycin complex 1 (mTORC1) and the transcription factor TFEB (transcription factor EB), which promotes lysosome biogenesis. When lysosomal activity was adequate, mTOR-dependent phosphorylation of TFEB on Ser211 triggered the binding of 14-3-3 proteins to TFEB, resulting in retention of the transcription factor in the cytoplasm. Inhibition of lysosomal function reduced the mTOR-dependent phosphorylation of TFEB, resulting in diminished interactions between TFEB and 14-3-3 proteins and the translocation of TFEB into the nucleus, where it could stimulate genes involved in lysosomal biogenesis. These results identify TFEB as a target of mTOR and suggest a mechanism for matching the transcriptional regulation of genes encoding proteins of autophagosomes and lysosomes to cellular need. The closely related transcription factors MITF (microphthalmia transcription factor) and TFE3 (transcription factor E3) also localized to lysosomes and accumulated in the nucleus when lysosome function was inhibited, thus broadening the range of physiological contexts under which this regulatory mechanism may prove important.

Introduction

The degradation and recycling of macromolecules by the autophagy-lysosome pathway plays a critical role in the regulation of nutrient homeostasis as well as in the normal cellular remodeling associated with development and differentiation (1, 2). This pathway is also critical for protection against multiple disease states including neurodegeneration, pathogen infection, cancer, heart disease, and aging (37). The ability of cells to achieve optimal lysosome function is dependent on multiple parameters that include lysosome number, size, pH, hydrolase content, and intracellular positioning. Improper control of these variables contributes to various human diseases (810). Although much is known about the specific roles played by individual lysosomal proteins in these processes, it is less clear how the functionality of the organelle as a whole is regulated and coordinated. In principle, cells should be able to sense changes in lysosomal status and transduce them into a signal that induces appropriate cellular responses to maintain lysosome homeostasis.

The basic helix-loop-helix leucine zipper transcription factor TFEB (transcription factor EB) has emerged as a master regulator of the expression of genes encoding proteins of the autophagy-lysosome pathway (1113). Furthermore, the nuclear abundance of this transcription factor can be altered to match varying cellular demand for autophagosome-lysosome function (1113). Although phosphorylation plays a role in regulating the nuclear abundance of TFEB (11, 13), the cellular mechanisms that sense lysosomal status and transduce the signals that regulate TFEB localization remain unclear.

The mechanistic target of rapamycin (mTOR), as part of the mTORC1 complex, is a kinase that localizes to lysosomes. This localization is critical for the ability of mTORC1 to integrate signals from growth factor signaling, cellular stress, and nutrient abundance to control various cellular processes including promoting cell growth, regulating metabolism, and repressing autophagy (1418). In addition to serving as a platform for the organization of proteins contributing to mTORC1 activation, the lysosome itself can also potentially influence the activity of mTORC1 (19). This connection between lysosome status and mTORC1 activity makes mTORC1 a candidate for contributing to a feedback mechanism that controls lysosome homeostasis.

Here, we investigated the mechanism by which lysosome status is communicated to TFEB and found a major role for mTORC1 in this process. We observed that TFEB was mainly localized to the cytoplasm with focal concentrations associated with lysosomes under basal conditions, and it translocated to the nucleus when lysosome function was inhibited. We identified 14-3-3 proteins as binding partners of TFEB that prevented its nuclear accumulation under conditions of optimal lysosome function. Furthermore, TFEB was recruited to lysosomes through an interaction with mTORC1, and mTORC1-dependent phosphorylation of TFEB was required for its interaction with 14-3-3 to prevent nuclear translocation. Collectively, these findings support a model for lysosome homeostasis in which lysosome status is communicated to TFEB through mTORC1 to prevent nuclear localization of TFEB when lysosome function is optimal. In response to impaired lysosome function, this pathway promotes translocation of TFEB to the nucleus where it increases the expression of genes encoding lysosomal proteins. Furthermore, our analysis of two closely related transcription factors, MITF (microphthalmia transcription factor) and TFE3 (transcription factor E3), suggests that this regulatory mechanism is conserved within this family of transcription factors.

Results

To investigate the mechanisms linking lysosomal status to the regulation of TFEB subcellular localization, we expressed green fluorescent protein (GFP)–tagged TFEB (TFEB-GFP) in HeLa cells and imaged live cells by spinning disk confocal microscopy. Under basal conditions, the abundance of this protein was higher in the cytoplasm compared to the nucleus (Fig. 1A and movie S1). In addition to the diffuse cytoplasmic signal, there was a distinct enrichment of TFEB on lysosomes (Fig. 1A, fig. S1A, and movie S1).

Fig. 1

TFEB localizes to lysosomes and accumulates in the nucleus in response to perturbation of lysosomal function. (A) Live imaging (spinning disk confocal) of TFEB-GFP (green) and DQ-BSA (red, a lysosomal marker) in HeLa cells shows an enrichment of the TFEB-GFP signal on lysosomes. Insets show higher-magnification views. (B) TFEB-GFP localization ±chloroquine (CQ) treatment. (C) Percentage of cells exhibiting lysosomal localization of TFEB-GFP under the indicated conditions (n = 3 experiments; 30 to 40 cells per condition per experiment). (D) Percentage of cells showing nuclear enrichment for TFEB-GFP under the indicated conditions (n = 3 experiments; 30 to 40 cells per condition per experiment). (E) Western blotting of total cytoplasmic and nuclear subcellular fractions obtained from HeLa cells stably expressing TFEB-GFP ± CQ treatment. Lamin A/C and tubulin represent control proteins for the nuclear and cytoplasmic fractions, respectively (n = 3 blots). (F) Effect of CQ on TFEB-GFP abundance. P < 0.01 by t test. n = 3 experiments. (G) Chloroquine treatment causes nuclear enrichment of TFEB-GFP. P < 0.01 by t test. n = 3 experiments. (H) Western blot for TFEB-GFP from cells grown under basal conditions ± phosphatase treatment of the lysates. Arrows indicate the relative positions of phosphorylated (upper arrow) and dephosphorylated (lower arrow) TFEB (n = 4 blots). (I) Localization of wild-type TFEB-GFP compared to that of the Δ30TFEB-GFP mutant (n = 3 experiments; >20 cells per condition per experiment; most cells observed exhibited this phenotype). Scale bars, 10 μm.

The localization of TFEB to lysosomes suggested that regulatory mechanisms may link TFEB activity to lysosomal status and was intriguing given the previous observations that TFEB accumulates in the nuclei of cells that contain mutations that compromise the digestive ability of lysosomes (12). We tested the relationship between TFEB localization and lysosomal status by incubating cells stably expressing TFEB-GFP with chloroquine, a weak base that impairs lysosome function by accumulating in lysosomes and raising their pH (20). In response to chloroquine, TFEB lost its lysosomal localization and accumulated in the nucleus (Fig. 1, B to D). A similar response was observed after treatment with bafilomycin A, which prevents lysosome acidification by inhibiting the vacuolar H+ pump (fig. S1B).

In addition, subcellular fractionation revealed that blocking lysosomal function resulted in decreased overall abundance of TFEB (Fig. 1, E and F) and increased nuclear abundance of TFEB (Fig. 1, E and G), an observation that parallels the link between nuclear localization of MITF and its degradation (21). The altered migration of TFEB on SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gels after chloroquine treatment (Fig. 1E) suggested a change in phosphorylation status. Indeed, a comparable mobility shift for TFEB was induced by phosphatase treatment of control lysates (Fig. 1H). Because essentially all of the TFEB runs at the higher molecular size in untreated samples, we concluded that a substantial fraction of TFEB is phosphorylated under basal conditions.

We next used mutagenesis to characterize the determinants for lysosomal localization of TFEB. Forms of TFEB lacking the first 30 N-terminal amino acids (Δ30TFEB) or with targeted mutation of highly conserved amino acids within this region showed loss of lysosomal localization and increased nuclear abundance of TFEB (Fig. 1I and fig. S1C). Thus, essential determinants for lysosomal localization of TFEB reside within the N terminus of the protein.

To identify additional proteins that contribute to the regulated subcellular localization of TFEB, we used a combination of stable isotope labeling with amino acids in cell culture (SILAC), affinity chromatography, and quantitative proteomics. This strategy identified 14-3-3 proteins (all seven isoforms were present) as major binding partners of TFEB (Fig. 2A). Consistent with the ability of TFEB to heterodimerize with the closely related transcription factors TFE3 and MITF (22), these proteins also copurified with TFEB (Fig. 2A). We detected 14-3-3 proteins in TFEB-GFP immunoprecipitations using Coomassie staining (fig. S2A) and immunoblotting with a pan–14-3-3 antibody (fig. S2B). Interactions with 14-3-3 had previously been reported to regulate the nuclear abundance of MITF (23), and TFE3 was identified as a 14-3-3 binding protein in a proteomic screen for 14-3-3 binding proteins (24). Thus, 14-3-3 interactions are a shared property within this family of transcription factors.

Fig. 2

Phosphorylation-dependent interaction of TFEB with 14-3-3 proteins. (A) Affinity purification and mass spectrometry analysis of heavy-labeled HeLa cells stably expressing TFEB-GFP compared to control light-labeled HeLa cells. Averaged peptide intensities are plotted against heavy/light (H/L) SILAC ratios. Significant outliers are colored as indicated in the legend; other identified proteins are shown in dark blue. Representative of results from two independent experiments. (B) Western blotting of anti-GFP immunoprecipitations (IPs) from cells expressing the indicated TFEB-GFP constructs (n = 3 blots). WT, wild type. (C) Effect of the S211A mutation on the subcellular location of TFEB-GFP (n = 4 experiments; >10 cells per condition per experiment; most cells exhibited this phenotype). Scale bar, 10 μm. (D) Western blotting of anti–GFP IPs from cells expressing WT compared to Δ30TFEB-GFP (n = 3 blots).

14-3-3 proteins typically interact with their targets through short phosphoserine-containing motifs (25). The 14-3-3 binding site on MITF had been mapped to Ser173, which aligns with Ser211 of TFEB (23) (fig. S2C). This site closely conforms to the RSxpSxP consensus 14-3-3 binding motif (25). A form of TFEB with a Ser211→Ala (S211A) mutation did not interact with 14-3-3 proteins (Fig. 2B), whereas mutation of Ser142 (a nearby mitogen-activated protein kinase phosphorylation site) (13) had no effect on the 14-3-3 interaction (Fig. 2B). Likewise, immunoblotting with an antibody specific for phosphorylated 14-3-3 binding motifs revealed a signal on TFEB that was selectively reduced with the S211A mutant (Fig. 2B). We used this selective recognition of Ser211 phosphorylation by the anti–14-3-3 binding motif antibody in subsequent experiments to determine the phosphorylation status of Ser211 in TFEB immunoprecipitates.

We next characterized the localization of the S211A mutant. Live cell imaging revealed that the TFEB-S211A mutant localized to the nucleus to a greater extent than did the wild-type TFEB (Fig. 2C). Although overall cytoplasmic abundance was reduced, a punctate distribution was still observed, which suggests that this mutation does not eliminate the ability of TFEB to associate with lysosomes. Therefore, we conclude that phosphorylation of Ser211 and the resulting interaction with 14-3-3 proteins have a major role in regulating the nuclear abundance of TFEB, whereas the lysosomal recruitment of TFEB is 14-3-3–independent. The Δ30TFEB mutant, which does not target to lysosomes, showed reduced phosphorylation of Ser211 and 14-3-3 binding (Fig. 2D). These results demonstrate the importance of lysosomal localization in controlling the phosphorylation state of Ser211 in TFEB and, by extension, in promoting the 14-3-3 interactions that retain a large pool of TFEB in the cytoplasm.

The accumulation of TFEB in the nucleus under conditions of starvation-induced autophagy has been linked to the TFEB-mediated regulation of genes encoding proteins important for autophagy (13). Given that interactions of phosphorylated Ser211 with 14-3-3 resulted in cytoplasmic retention of TFEB, we suspected that the signaling pathway responsible for phosphorylation of Ser211 should be inhibited when autophagy is induced. Thus, we focused our attention on the kinase mTOR because (i) mTOR localizes to the cytoplasmic surface of lysosomes as part of the mTORC1 complex (2628) and (ii) the loss of lysosomal localization and activity of mTOR under starvation conditions promotes autophagy (15, 17, 27). Thus, we investigated the localization of TFEB under conditions of starvation or mTOR inhibition. Starvation resulted in the accumulation of TFEB in the nucleus, which was accompanied by the loss of punctate distribution of TFEB, consistent with loss of lysosomal localization (Fig. 3A). The allosteric mTORC1 inhibitor rapamycin (29) had minimal effects on TFEB localization (Fig. 3A). However, the ability of rapamycin to inhibit mTORC1 is cell type– and substrate-dependent (2931). Therefore, we also tested the effect of torin 1, an ATP (adenosine 5′-triphosphate)–competitive inhibitor that blocks the activity of mTOR toward all substrates (31), and observed increased nuclear translocation and lysosome association of TFEB and a reduction in the diffuse cytoplasmic pool (Fig. 3A). The loss of lysosomal localization of TFEB after starvation but not mTOR inhibition was surprising because both treatments (as well as chloroquine treatment) inhibited mTORC1 activity (fig. S3A). Analysis of the time course of TFEB nuclear accumulation in response to mTOR inhibition showed that the effect was significant within 30 min and was maximal after ~1 hour of treatment (Fig. 3B). This change in subcellular localization was paralleled by the dephosphorylation of the native TFEB protein (Fig. 3C and fig. S3D), and the time course for TFEB nuclear accumulation and dephosphorylation was comparable to that observed for the mTORC1 substrate 4E-BP1 (14) (Fig. 3C). To further investigate how these different TFEB localization patterns related to phosphorylation of Ser211 and 14-3-3 interactions, we immunoprecipitated TFEB from starved and torin 1–treated cells and compared them to untreated controls and chloroquine-treated samples. Similar to chloroquine treatment, starvation appeared to decrease 14-3-3 binding and Ser211 phosphorylation, and these effects were more robust in response to torin 1 (Fig. 3D). Because mTOR inhibition by torin 1 eliminated the detection of TFEB by the anti–14-3-3 binding site antibody (Fig. 3D), whereas expression of the S211A mutant reduced but did not abolish this signal (Fig. 2B), there must be additional mTOR-dependent phosphorylation sites on TFEB. However, given that the effects of mTOR inhibition on TFEB localization, phosphorylation, and 14-3-3 interaction phenocopied those of the S211A mutation (Fig. 2, B and C), we conclude that phosphorylation of Ser211 is a major mechanism for mTOR-dependent regulation of TFEB.

Fig. 3

Regulation of TFEB by mTORC1. (A) Live cell imaging of TFEB-GFP after starvation or treatment with rapamycin or torin 1. n = 3 experiments; >30 cells per condition per experiment; images are representative of most cells observed. (B) Quantification of the changes in the nuclear/cytoplasmic ratio for TFEB-GFP after torin 1 treatment for the indicated periods of time. n = 3 experiments; average of 321 cells per condition per experiment. *P < 0.01 by analysis of variance (ANOVA) with Bonferroni post test. (C) Time course showing the change in the electrophoretic mobility of native TFEB and the decrease in phosphorylation of 4E-BP1 at Thr37 and Thr46 in HeLa cells treated with torin 1 for the indicated times (n = 2 experiments). Arrows indicate the relative positions of phosphorylated TFEB (t = 0) and dephosphorylated TFEB (t = 60 and beyond). (D) Western blots of anti-GFP IPs from TFEB-GFP–expressing cells subjected to treatment with CQ, starvation (Earl’s buffered saline solution), and torin 1 (n = 3 blots).

To further test the role of mTOR in regulating TFEB localization, we performed small interfering RNA (siRNA)–mediated knockdowns of mTOR and RagC, a guanosine triphosphatase (GTPase) that recruits the mTORC1 complex to lysosomes (26, 28). Knockdown of either RagC or mTOR (fig. S3C) resulted in increased nuclear abundance of TFEB (Fig. 4, A and B), reduced punctate localization of TFEB (which is consistent with reduced lysosomal localization of TFEB) (Fig. 4A), reduced association with 14-3-3 (Fig. 4C), and diminished TFEB phosphorylation (Fig. 4C).

Fig. 4

RagC and mTOR are required for regulation of TFEB localization through phosphorylation-dependent control of interaction with 14-3-3. (A) Live cell imaging of TFEB-GFP localization after RagC and mTOR knockdowns. Scale bar, 10 μm. More than 30 cells per condition per experiment. (B) Quantification of the effects of RagC and mTOR knockdowns on the nuclear/cytoplasmic ratio of TFEB-GFP. n = 3 experiments; average of 169 cells analyzed per condition per experiment. *P < 0.001 by ANOVA with Bonferroni post test. (C) Western blots of anti-GFP IPs from TFEB-GFP cells after RagC and mTOR knockdowns (n = 3 blots).

The contrasting results from mTOR inhibition compared to that of siRNA knockdown on the lysosomal localization of TFEB suggested that the recruitment of TFEB to lysosomes was dependent on the physical presence of mTOR but not necessarily its kinase activity, implying that the mTORC1 complex could participate in the recruitment of TFEB to lysosomes. Consistent with this hypothesis, the amount of mTOR on lysosomes parallels that of TFEB because it is reduced in response to starvation (27) and increased in response to torin 1 (32). Thus, although we had not detected an interaction between TFEB and mTORC1 components in the SILAC experiment described above (Fig. 2A), we reasoned that because the localization of TFEB to lysosomes was enhanced in response to mTOR inhibition (Fig. 5, A and B) and that mTOR and TFEB colocalized on lysosomes under such conditions (Fig. 5C), potential interactions between TFEB and mTORC1 components could also be enhanced. Indeed, mTOR and raptor co-immunoprecipitated to a greater extent with wild-type TFEB from torin 1–treated cells (Fig. 5D) than with the Δ30TFEB mutant, which does not localize to lysosomes (Fig. 5E). In addition, mTOR inhibition increased the nuclear abundance of endogenous TFEB in both HeLa (Fig. 5, F and G) and ARPE-19 cells (fig. S3D).

Fig. 5

An interaction between TFEB and mTOR on the cytoplasmic surface of lysosomes. (A) Immunofluorescent staining showing the colocalization of TFEB-GFP and LAMP1 ± torin 1 treatment. Cells were permeabilized with saponin before fixation to extract the diffuse cytoplasmic pool of TFEB. (B) Quantification of the intensity ratios for LAMP1 and TFEB-GFP in cells treated ± torin 1. n = 3 experiments; average of 21,874 lysosomes analyzed per condition per experiment. *P < 0.05 by t test. (C) Immunofluorescence images showing extensive colocalization of TFEB and mTOR on lysosomes after torin 1 treatment. n = 3 experiments; >30 cells per condition per experiment; most cells observed exhibited the indicated phenotype. (D) Western blot of anti-GFP IPs from control HeLa cells compared to those from a TFEB-GFP stable line ± torin 1 treatment (n = 4 blots). (E) Western blots of anti-GFP IPs from torin 1–treated cells that demonstrate reduced interaction between Δ30TFEB-GFP with mTOR and raptor (n = 2 blots). (F) Detection of native TFEB after subcellular fractionation of HeLa cells ± torin treatment. Arrows indicate the relative positions of phosphorylated (upper) and dephosphorylated (lower) TFEB (n = 3 blots). (G) Quantification of the nuclear abundance of TFEB in the preceding fractionation experiments. n = 3 experiments. *P < 0.05, significantly different from 1 by one-sample t test. Scale bars, 10 μm.

Collectively, our results support a model wherein mTORC1-dependent Ser211 phosphorylation of TFEB results in interactions with 14-3-3 that promote the cytoplasmic retention of TFEB. We searched for nuclear localization signals (NLSs) (33, 34) in TFEB and identified a candidate sequence between amino acids 241 and 252 (fig. S2C). To test the hypothesis that binding of 14-3-3 to the nearby Ser211-containing motif occludes this NLS, we mutated basic residues (Arg245 to Arg248) within the predicted NLS to alanine. Treatment with torin 1 failed to stimulate an increase in the nuclear abundance of this TFEB mutant (Fig. 6A), although it still inhibited 14-3-3 binding and Ser211 phosphorylation (Fig. 6B). These results indicate that the loss of 14-3-3 interactions after mTOR inhibition triggers nuclear accumulation through unmasking of this adjacent NLS.

Fig. 6

Mutation of a predicted NLS in TFEB blocks nuclear accumulation in response to mTOR inhibition. (A) Live imaging of WT TFEB-GFP and TFEBΔNLS-GFP localization after torin 1 treatment (2 μM, 2 hours). n = 3 experiments; >10 cells per condition per experiment. Images are representative of the phenotype observed in most cells. Scale bar, 10 μm. (B) Immunoblots of TFEBΔNLS-GFP immunoprecipitates ± torin 1 treatment (2 μM, 2 hours). n = 2 blots.

The close sequence conservation between TFEB, MITF, and TFE3 (fig. S2C), the ability of these proteins to form functional heterodimers (22), and evidence of interactions between both MITF and TFE3 with 14-3-3 proteins (23, 24) led us to consider the possibility that the localization of MITF and TFE3 might also be regulated by lysosome status. Indeed, both MITF (isoforms A and D) and TFE3 also exhibited a predominantly cytoplasmic signal with focal concentration on lysosomes under basal cell culture conditions (Fig. 7, A and B, and fig. S4). As observed for TFEB, both MITF and TFE3 translocated to the nucleus in response to chloroquine treatment (Fig. 7C and fig. S4, B to D). Furthermore, like the Δ30TFEB mutant (Fig. 1I), the MITF-M isoform, which is predominantly present in melanocytes and naturally has a truncated N terminus due to alternative promoter usage (35) (fig. S4A), also did not localize to lysosomes and was enriched in the nucleus under basal conditions (Fig. 7D). Based on these findings, the regulatory mechanisms that we initially uncovered in our investigation of TFEB regulation appear to be broadly conserved within this family of transcription factors.

Fig. 7

MITF and TFE3 localize to lysosomes and accumulate in the nucleus in response to inhibition of lysosome function. (A) Live imaging of MITF-GFP [“D” isoform (35), which is most similar to TFEB (fig. S4A)] and TFE3-GFP reveals an enrichment on lysosomes (labeled by DQ-BSA) and relatively low amounts in the nucleus under basal conditions (n = 3 experiments; quantified in fig. S4, C and D). (B) Both MITF-GFP and TFE3-GFP lose their lysosomal association and accumulate in the nucleus in response to CQ (n = 3 experiments; quantified in fig. S4, C and D). (C) Subcellular fractionation and immunoblotting show that CQ treatment of HeLa cells increases the nuclear abundance of endogenous MITF (n = 2 experiments). (D) Live cell imaging of the localization of the MITF-M isoform fused to GFP under basal conditions (n = 3 experiments; >30 cells per condition per experiment; most cells observed exhibited this localization). See also fig. S4 for further MITF isoform–specific results and quantification. Scale bars, 10 μm.

Discussion

Our study identifies TFEB as a target of mTOR signaling and defines how the regulation of TFEB abundance in the nucleus is linked to lysosomal status (Fig. 8). This regulation occurs on the surface of lysosomes through mTOR-dependent phosphorylation of TFEB on Ser211. Phosphorylated TFEB binds to 14-3-3 in the cytoplasm, which results in the occlusion of a nearby NLS. When mTOR is inactive, the balance shifts toward dephosphorylation of Ser211, resulting in reduced interaction with 14-3-3 and enhanced accumulation of TFEB in the nucleus. An independent study has also reported a role for mTORC1-dependent phosphorylation in the lysosomal localization of TFEB (36).

Fig. 8

Regulation of TFEB subcellular localization by mTOR interactions. This diagram summarizes how the localization of TFEB to lysosomes through interaction with mTORC1 results in phosphorylation of Ser211 and subsequent cytoplasmic sequestration by interactions with 14-3-3 proteins.

Our data point to the lysosome as the site where mTORC1-dependent phosphorylation of TFEB occurs. This finding builds upon the rapidly growing understanding of the mechanisms by which the mTORC1 complex is activated by growth factors and amino acids on the surface of this organelle (14, 19, 2628). Specific motifs have been described previously for mTOR substrates, such as p70 S6 kinase and 4E-BP1, that mediate their interactions with mTORC1 through direct interactions with raptor (3739). However, because such motifs are not evident in TFEB, it will be necessary to elucidate the specific mechanisms that recruit TFEB to mTORC1.

Our study has revealed a specific role for phosphorylation of TFEB at Ser211 in preventing the nuclear accumulation of TFEB, which occurs through 14-3-3 binding to phosphorylated Ser211, an interaction that masks the nearby NLS on TFEB. Other phosphorylation sites on TFEB have been reported to influence the nuclear abundance of this transcription factor (11, 13), and phosphoproteomics efforts have identified more sites with unknown functions (40). Additional biologically relevant phosphorylation sites on TFEB can further be inferred from identifying sequences that are conserved with MITF (21, 41). Given that we observed additional mTOR-dependent phosphorylation on TFEB that remained in the S211A mutant (compare Figs. 2D and 3D), it is possible that mTOR can exert distinct regulatory effects through phosphorylation of other sites on TFEB. Indeed, phosphoproteomic studies of mTOR-dependent phosphorylation sites have identified multiple such candidate sites in TFEB (Thr330, Thr331, Ser332, and Ser334) (42) as well as a site in the C terminus of TFE3 (43) that is conserved and that corresponds to Ser455 of TFEB (fig. S2C).

Ser142 of TFEB was previously identified as a site phosphorylated by extracellular signal–regulated kinase 2 (ERK2), which has been implicated in limiting the nuclear abundance of TFEB (13), and the equivalent site on MITF (Ser73) is a well-characterized ERK phosphorylation site that simultaneously reduces its stability and promotes its transcriptional activity (21). Although the mechanisms by which ERK-mediated phosphorylation of Ser142 controls TFEB localization have not been elucidated, it is interesting that a pool of ERK is present on lysosomes and it shares with mTOR some of the machinery responsible for its recruitment to this organelle (44, 45). Thus, lysosomal enrichment of TFEB may also be relevant to its regulation by ERK.

mTORC1 was reported to promote the nuclear localization of TFEB by enhancing the dephosphorylation (through an as yet unidentified phosphatase) of multiple sites in the TFEB C terminus (11), a finding that contrasts with the inhibitory role for mTORC1 that we have uncovered. A possible explanation is the differences in the experimental conditions. First, Peña-Llopis et al. used rapamycin, which may have resulted in incomplete inhibition of mTORC1 and thus prevented detection of the mTORC1-mediated phosphorylation of Ser211 of TFEB reported here. Second, the link between enhanced mTORC1 activity and increased TFEB nuclear localization was revealed in the context of tuberous sclerosis complex 2 (TSC2) knockout or knockdown (11). TSC2 is a GTPase-activating protein (GAP) for Rheb, a small GTPase that promotes the activation of mTORC1 (46, 47). Because of the tight regulation of mTORC1 signaling and the cross talk between mTOR and other signaling proteins (48), it is possible that cells could compensate for excessive mTORC1 activity in such a way that diminishes its ability to regulate TFEB. Further studies are required to elucidate how TFEB is recruited to mTORC1 and how this process is potentially altered in response to the prolonged absence of TSC2. The results of such studies could prove interesting for understanding the potential contributions of TFEB dysregulation to the pathology of tuberous sclerosis.

TFEB promotes the expression of genes encoding autophagosomal and lysosomal proteins (1113), a function that could have effects at the organismal level because these organelles are ubiquitous to all cells. Thus, it is interesting that the TFEB knockout mice develop to midgestation and die because of impaired vascularization in the placenta (49). It has not yet been established whether this embryonic lethality is related to a defect in lysosome function. Likewise, human chromosomal translocations that result in TFEB overexpression can cause renal carcinoma (50), but it is also not yet known whether this arises because of changes in lysosome activity or because of other unappreciated roles for TFEB in such cells.

The finding that MITF (with the exception of the melanocyte-specific M isoform) and TFE3 also exhibit lysosome localization and nuclear accumulation in response to perturbation of lysosome function suggests that the mTORC1- and 14-3-3–dependent regulatory mechanism that we have defined for TFEB also applies to these closely related transcription factors. Although MITF and TFE3 have not previously been linked to regulation of lysosome gene expression, they are important for osteoclast development and function (51), at least in part through the regulation of genes encoding proteins in the specialized lysosome-related organelles that enable osteoclasts to degrade bone through regulated exocytosis and release of hydrolytic enzymes (52, 53). Additional but similar roles have been identified for these transcription factors in relation to the specialized lysosome-related organelles of mast cells and natural killer cells (53). It will thus be interesting to determine whether the status of lysosome-related organelles is also communicated to the nucleus through the mechanisms that we have defined in this study and, if so, the relevance of such a pathway to the physiology of these specialized cells.

It is possible that the mTOR-TFEB pathway of converting lysosomal status to a transcriptional signal could be therapeutically enhanced to promote the clearance of damaged organelles or protein aggregates in neurodegenerative diseases (5456). Indeed, our findings provide a mechanism to explain previous observations that mTOR inhibitors reduce pathology in models of neurodegenerative disease by enhancing lysosome function (57). Likewise, TFEB-dependent gene transcription has been proposed to protect cells from lysosomal storage disorders (58), and thus, enhancing TFEB activity through mTOR inhibition warrants consideration for the treatment of such diseases. Conversely, excessive activity of TFEB and related transcription factors causes renal carcinoma (59), and a better understanding of the cellular mechanisms that inhibit their activity has implications for the treatment of this cancer.

Materials and Methods

Cell culture and transfection

HeLa cells [American Type Culture Collection (ATCC); or HeLa M subline provided by P. De Camilli, Yale University] were grown in Dulbecco’s modified Eagle’s medium (DMEM) (+l-glutamine), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin supplement (all from Invitrogen). ARPE-19 cells (ATCC) were grown in DMEM/F12 (Invitrogen), 10% FBS, and 1% penicillin/streptomycin supplement. Transfections were performed with either Fugene 6 or ExtremeGene 9 from Roche with 500 ng of plasmid DNA, 1.5 μl of transfection reagent, and 100 μl of Opti-MEM (Invitrogen) per 35-mm dish of subconfluent cells. For transfection of larger dishes, the volumes were scaled up proportionally as per the manufacturer’s directions. For generation of stable cell lines, selection was performed with G418 (500 μg/ml), and colonies were visually screened for uniform GFP-tagged protein expression. A pcDNA–TFEB-GFP plasmid was provided by A. Ballabio (Telethon Institute of Genetics and Medicine, Naples, Italy). This TFEB complementary DNA (cDNA) was further subcloned into pEGFP-N1 from Clontech through Hind III and Kpn I restriction enzyme sites. Deletions of the N terminus of TFEB were performed by polymerase chain reaction (PCR) amplification of the truncated fragments and their ligation into pEGFP-N1 through Hind III and Kpn I sites. MITF and TFE3 cDNAs were generated by PCR amplification from human brain cDNA (Clontech) and cloned into pEGFP-N1 through Hind III and Kpn I restriction sites. Site-directed mutagenesis was performed with the QuikChange strategy (Agilent Technologies).

Lysosome labeling

Lysosomes were visualized by preloading cells overnight with Bodipy-conjugated bovine serum albumin (DQ-BSA, 5 μg/ml, Invitrogen) or Alexa594-conjugated dextran (125 μg/μl, Invitrogen).

Drug treatments

Chloroquine was purchased from Sigma and used at a final concentration of 50 μg/ml (a concentration that was found in preliminary experiments to yield maximal effects on both nuclear translocation of TFEB and inhibition of lysosomal function as assessed by DQ-BSA labeling) for 15 hours. Cells were incubated in bafilomycin A (100 nM; Calbiochem) for 15 hours. Cells were incubated with rapamycin (200 nM; EMD) and torin 1 (2 μM; Tocris) for 2 hours. Cells were starved in Earl’s buffered saline solution for 2 hours.

siRNA transfections

siRNA was transfected with the RNAiMAX transfection reagent (Invitrogen). mTOR siRNAs (SignalSilence mTOR siRNAs I and II) were purchased from Cell Signaling Technology; RagC siRNA (ON-TARGETplus SMARTpool) and TFEB siRNA (D-009798-03, target sequence: AGACGAAGGUUCAACAUCA) were from Dharmacon.

Antibodies

The following antibodies were used in our experiments: anti–GFP-HRP (horseradish peroxidase; Miltenyi and Rockland Immunochemicals); anti–lamin A/C and anti–pan–14-3-3 (Santa Cruz Biotechnology); anti-tubulin (Sigma); anti-MITF (clone C5; Millipore); anti-dynamin (clone 41; Millipore); anti-TFEB (Bethyl); and anti–phospho–14-3-3 binding motif, anti-mTOR, anti-raptor, anti–S6 kinase, anti–phospho–S6 kinase, anti–4E-BP1, anti–phospho–4E-BP1, and anti-RagC (Cell Signaling Technology). The anti-LAMP1 monoclonal antibody developed by J. T. August and J. T. and J. E. K. Hildreth was obtained from the Developmental Studies Hybridoma Bank.

Immunoprecipitations

TFEB-GFP was immunoprecipitated with GFP-Trap agarose beads (Allele Biotechnology). Cells were lysed by scraping in phosphate-buffered saline (PBS) with 1% Triton X-100 (TX100), Complete Protease Inhibitor Cocktail (Roche), and PhosSTOP phosphatase inhibitors (Roche). Lysates were cleared by centrifugation for 10 min at 20,000g before incubation with the beads [1:2 mix of GFP-Trap and unconjugated agarose beads (Promega)]. After an hour of gentle rotation at 4°C, the beads were washed four times with lysis buffer before elution in 2× Laemmli sample buffer.

Immunoblotting

Immunoblotting was performed by standard methods with 7.5% or 4 to 15% Mini-PROTEAN TGX precast polyacrylamide gels and nitrocellulose membranes (Bio-Rad). The 7.5% gels were better for detecting phosphorylation-dependent shifts in TFEB mobility. One exception to the precast gels was the use of homemade 15% polyacrylamide gels (4% stacking layer) for the 4E-BP1 immunoblots. Ponceau S staining of membranes was routinely used to assess equal sample loading and transfer efficiency. Blocking and antibody incubation were performed with 5% milk or BSA in PBS or tris-buffered saline with 0.1% Tween 20. Signals were detected with HRP-conjugated secondary antibodies (Bio-Rad) and either Super Signal West Pico or Femto chemiluminescent detection reagents (Thermo Scientific) on a VersaDoc imaging system (Bio-Rad). ImageJ was used to measure band intensities.

Gel staining

For visualization of TFEB-GFP and interacting proteins, SDS-PAGE gels were stained with the Coomassie-based Imperial Protein Stain (Thermo Scientific).

Immunofluorescence

Cells were grown on 12-mm No. 1.5 coverslips (Carolina Biological Supply) and were fixed with 4% paraformaldehyde (Electron Microscopy Sciences)–0.1 M sodium phosphate (pH 7.2). Where indicated, 0.1% saponin was used to permeabilize and extract cells for 10 s before fixation. This strategy washes out unanchored cytoplasmic proteins and thus facilitates visualization and quantification of the lysosomal signal for TFEB. Coverslips were washed with 50 mM NH4Cl (pH 7.2) and then blocked and permeabilized with PBS with 3% BSA and either 0.1% TX100 or 0.1% saponin. Subsequent primary and secondary antibody incubations used this buffer. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole;1 μg/μl; Invitrogen) during one of the post–secondary antibody washes. Alexa488- and Alexa594-conjugated secondary antibodies were obtained from Invitrogen. Coverslips were finally mounted in ProLong Gold mounting medium (Invitrogen). Images were acquired with a Zeiss LSM 710 laser scanning confocal microscope with a 63× Plan Apo [numerical aperture (NA) = 1.4] oil immersion objective and Zeiss Efficient Navigation software.

Live cell imaging

Spinning disk confocal microscopy was performed with the Improvision UltraVIEW VoX system including a Nikon Ti-E Eclipse inverted microscope (equipped with 60× CFI Plan Apo VC, NA 1.4, and 100× CFI Plan Apo VC, NA 1.4 objectives) and a spinning disk confocal scan head (CSU-X1, Yokogawa) driven by Volocity (Improvision) software. Images were acquired without binning with a 14-bit (1000 × 1000) Hamamatsu electron multiplying charge-coupled device. Illumination was provided by Coherent solid-state 488-nm/50-mW diode and Cobolt solid-state 561-nm/50-mW diode lasers. Emission filters for GFP and Bodipy/Alexa594 were the following: a 527-nm single bandpass center wavelength, 55-nm half-power bandwidth, and a double bandpass 500 to 548 nm and 582 to 700 nm, respectively. Typical exposure times and acquisition rates were 100 to 500 ms and 0.25 Hz, respectively. Cells were imaged at room temperature (~22°C). Post-acquisition image analysis was performed with Volocity and ImageJ software.

Image analysis

The presence of lysosome localization in Figs. 1 and 7 was determined by visual inspection of photographs. If TFEB-positive lysosomes were observed, then the cell was scored as positive for lysosomal localization; cells were visually scored as having nuclear localization if the nuclear abundance of TFEB exceeded those in the cytoplasm. Thirty to 40 cells were scored per condition per experiment.

The absolute ratio of nuclear/cytoplasmic TFEB was quantified with CellProfiler (60) (http://www.cellprofiler.org) on images acquired by spinning disk confocal microscopy with either a 20× Plan Apochromat (NA 0.75, air) or a 40× Plan Apochromat (NA 1.0, oil immersion) objective. Nuclei were identified by DAPI staining, and nuclear edges were uniformly expanded to form a halo that defined the surrounding cytoplasmic compartment in each cell. The mean intensities of the nuclear and cytoplasmic regions were measured and used to calculate nuclear/cytoplasmic ratios on a cell-by-cell basis. The number of cells analyzed per experiment is presented in the respective figure legends.

The relative enrichment of TFEB to lysosomes was also quantified with CellProfiler, which allowed us to analyze an average of ~21,874 lysosomes per condition per experiment. The lysosomes were first identified by the LAMP1 immunofluorescence signal. This signal was then used to create a mask wherein the mean intensity of the TFEB or LAMP1 signal was measured.

Subcellular fractionation

Cells were plated on 100-mm dishes at a density of 800,000 cells per dish. The next day, the cells were treated overnight with 50 μM chloroquine or for 2 hours with 2 μM torin 1. At the end of the treatment, the cells were washed twice in PBS, harvested in the presence of 500 μl of ice-cold hypotonic buffer [10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.15% NP-40], and homogenized with 20 strokes of a Dounce homogenizer. SDS (1% final) and 25 U of Benzonase (Novagen) were added to 100 μl of the homogenate, and the rest of the homogenate was spun at 4°C for 5 min at 14,000 rpm. The supernatant (cytoplasmic fraction) was transferred to a new tube, and the pellet (nuclear fraction) was resuspended in 200 μl of high-salt buffer (20 mM Hepes, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5% NP-40) and solubilized with SDS (1% final) in the presence of 25 U of Benzonase. The protein concentration was measured with the BCA reagent (Thermo Scientific), and samples were subsequently analyzed by immunoblotting.

Stable isotope labeling with amino acids in cell culture

Cells were grown for greater than six passages in DMEM lacking arginine and lysine (PAA Laboratories), 10% dialyzed FBS (Invitrogen), l-glutamine (Invitrogen), and penicillin/streptomycin (Invitrogen) supplemented with either normal/“light” lysine and arginine (light condition) or “heavy” lysine and arginine [l-lysine-U-13C6,15N2 and l-arginine-U-13C6,15N4] (Cambridge Isotope Laboratories; heavy condition) before immunoprecipitation experiments (see methods above). Cells from one near-confluent 150-mm dish were used per experiment to yield ~1 ml of lysate at ~3 mg of protein per milliliter. At the end of the immunoprecipitation, the beads were washed two times with PBS with 1% TX100 and four times with PBS before elution in 8 M urea and 25 mM tris (pH 8). Eluates were mixed, reduced for 20 min at room temperature (22°C) in 1 mM DTT, and alkylated for 30 min by 5.5 mM iodoacetamide in the dark. Samples were digested for 3 hours with LysC at room temperature and diluted four times with 10 mM ammonium bicarbonate (ABC) buffer (pH 8). Trypsin was added to a final concentration of 1 μg/50 μg of protein, and samples were incubated at room temperature overnight. Digestion was stopped by acidification with trifluoroacetic acid. Samples were desalted and concentrated with C18 reverse-phase stop and go extraction tips (STAGE tips). Peptides were separated on-line with an Easy nLC system (Thermo Fisher Scientific). Samples (5 μl) were loaded as described (61). Peptides were eluted with a segmented gradient of 10 to 60% solvent B over 102 min with a constant flow of 250 nl/min. The high-performance liquid chromatography (HPLC) system was coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) through a nanoscale LC interface (Proxeon Biosystems; Thermo Fisher Scientific). The spray voltage was 2 kV, and the temperature of the heated capillary was 180°C. Survey full-scan spectra [mass/charge ratio (m/z) = 300 to 1750] were acquired in positive ion mode with a resolution of 30,000 at m/z = 400 after accumulation of 1,000,000 ions. Up to 10 most-intense ions were sequenced by HCD (higher energy collisional dissociation) in the Orbitrap. Precursor ion charge-state screening was enabled, and all unassigned charge states as well as singly charged peptides were rejected. The dynamic exclusion list was restricted to a maximum of 500 entries with a maximum retention period of 90 s and a relative mass window of 10 parts per million (ppm). Orbitrap measurements were performed, enabling the lock mass option for survey scans to improve mass accuracy. Data were acquired with the Xcalibur software (version 2.1, Thermo Fisher Scientific) and MaxQuant (version 1.2.2.5; http://maxquant.org/downloads.htm) (62). The data were searched against the human database concatenated with reversed copies of all sequences. Carbamidomethylated cysteines were set as fixed, whereas oxidation of methionine, N-terminal acetylation, and phosphorylation of serine, threonine, and tyrosine were set as variable modifications. Maximum allowed mass deviation for MS/MS (tandem mass spectrometry) peaks and missed cleavages were 20 ppm and 3, respectively. Maximum false discovery rates were 0.01 both on the peptide and on the protein levels. Minimum required peptide length was six residues. Proteins with at least two peptides were considered identified. Plots were generated with the open-source R software package (http://www.r-project.org/).

Statistical analysis

Data were analyzed by Prism (GraphPad Prism), with the tests specified in the figure legends. All error bars represent the SEM.

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/5/228/ra42/DC1

Fig. S1. Regulation of TFEB localization by lysosomal status.

Fig. S2. 14-3-3 proteins are major binding partners of TFEB.

Fig. S3. Relationship between TFEB, mTOR, and lysosomes.

Fig. S4. MITF and TFE3 localization is sensitive to lysosome status.

Movie S1. Time-lapse imaging of TFEB-GFP.

References and Notes

Acknowledgments: We thank H. Shen, M. Caplan, and P. De Camilli for their insightful comments, guidance, and advice and A. Ballabio for the original TFEB-GFP plasmid. Technical support with reagent and assay development was provided by M. Krak, A. Goldberg, and N. Roy. Funding: C.S.P. was supported by an Anderson Fellowship from Yale University. This study was supported by an NIH grant (GM095982) to T.C.W. Author contributions: A.R.-F., C.S.P., F.F., B.A., T.C.W., and S.M.F. designed the experiments; F.F. and T.C.W. analyzed the SILAC samples; A.R.-F., C.S.P., S.Q., J.K., B.A., and S.M.F. performed all other experiments and analyzed their data; and S.M.F. wrote the paper with input from A.R.-F. and T.C.W. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The data associated with this manuscript may be downloaded from the ProteomeCommons.org Tranche network using the following hash: ke+4fiwp5emKdVgZyUJkxpkWvmzKJ1FTaj4PHYwEBuk7NR/4ZhExJTjdWFMSDnGU8lCk+ynTy6omRLRkn4Xf7WNPH9MAAAAAAAAKaQ==.
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