Our study demonstrates that TFEB, a master gene for lysosomal biogenesis, is regulated by the lysosome via the mTOR pathway. mTORC1 and TFEB meet on the lysosomal membrane where mTORC1 phosphorylates TFEB.
We previously reported that ERK2 phosphorylates TFEB and, in cells treated with an MEK inhibitor, the TFEB nuclear fraction was increased (Settembre et al, 2011
). In the same study, we reported that the mTOR inhibitor rapamycin had little or no effects on TFEB subcellular localization. Here, we compared three different types of kinase inhibitors—MEK inhibitor U0126 and mTOR inhibitors rapamycin, Torin 1, and Torin 2—in their ability to cause a shift in TFEB molecular weight and to induce TFEB nuclear translocation. As shown in , Torin 1 and Torin 2 induced TFEB nuclear translocation more efficiently compared to U0126. The more pronounced shift of TFEB molecular weight, which was observed in cells treated with Torin 1, suggests that mTORC1 induces TFEB phosphorylation at multiple sites, either directly or indirectly.
In a recent high throughput mass spectrometry study, TFEB was predicted to be phosphorylated at 11 different residues, thus suggesting a complex regulation of its activity with several phosphorylation sites potentially involved (Dephoure et al, 2008
). Here, we have used an mTORC1 in-vitro
kinase assay and a phosphoantibody to demonstrate that serine S142, which we previously found to be phosphorylated by ERK2, is also phosphorylated by mTOR and that this phosphorylation has a crucial role in controlling TFEB subcellular localization and activity. In addition, we have mutated 12 different serines, which were candidate mTOR phosphorylation sites, into alanines, thus abolishing the corresponding TFEB phosphorylation sites. Testing the effects of each of these mutations on TFEB subcellular localization led to the identification of an additional residue, serine S211, which plays a role in TFEB subcellular localization, confirming the predicted complexity of TFEB regulation by phosphorylation.
Phosphorylation of TFEB by mTOR had already been reported in a previous study (Pena-Llopis et al, 2011
). However, in that study the authors concluded that mTOR promoted, rather than inhibited, TFEB activity. Several lines of evidence indicate that mTOR inhibits TFEB activity. First, TFEB is entirely nuclear when cells are either starved or treated with Torin1, both conditions in which mTOR activity is profoundly inhibited. Second, treatment of starved cells with nutrients, a condition that boosts mTORC1 activity, resulted in TFEB cytoplasmic accumulation, with TFEB being undetectable in the nuclear fraction. Third, treatment with drugs such as chloroquine or SalA, which inhibit mTORC1 function, induced TFEB nuclear accumulation. Fourth, transfection of mutant Rag proteins that inhibit mTORC1 resulted in nuclear accumulation of TFEB and, conversely, mutant Rags that constitutively activate mTORC1 prevented TFEB nuclear accumulation upon starvation, chloroquine and SalA treatment. Fifth, TFEB is in the nucleus in its low-phosphorylated form, an observation that is consistent with a model in which inhibition, rather than activation, of a kinase induces TFEB nuclear translocation. It is difficult to explain the discrepancy between our observations and those reported by Pena-Llopis et al.
We considered the possibility that the TSC2-deficient cells that were used in that study may behave differently to other cellular systems in the assays performed. To test this possibility, we analysed TFEB regulation by amino acids, chloroquine and Torin 1 in TSC2−/− cells but obtained the same results that we observed in other cell types both on exogenous TFEB–GFP and on endogenous TFEB (Supplementary Figures S8 and S9
Our data indicate that mTORC1 negatively regulates TFEB via the amino acid/Rag GTPase pathway. The phosphorylation status of TFEB and its subcellular localization were entirely determined by the activation state of the Rag GTPases, which regulate mTORC1 activity downstream of amino acids (Kim et al, 2008
; Sancak et al, 2008
). In particular, constitutively active Rags rescued nuclear translocation of TFEB caused by starvation and lysosomal stress, while inactive Rag mutants caused TFEB to accumulate in the nucleus even in fully fed cells. These results imply that, among the many regulatory inputs to mTORC1, the amino acid pathway is particularly important in controlling TFEB activity and plays not only a permissive but also an instructive role. This idea is further supported by our observation that constitutive activation of the growth factor inputs to mTORC1 that occurs in TSC2−/− cells cannot prevent TFEB nuclear accumulation caused by starvation and lysosomal stress. Future work will be required to address how each upstream input to mTOR contributes to TFEB regulation. Nonetheless, compounded with recent evidence showing that amino acid sensing by the v-ATPase/Rag GTPase/mTORC1 may begin in the lysosomal lumen (Zoncu et al, 2011a
) our findings substantiate the role of TFEB as the end point of a lysosome-sensing and signalling pathway.
Our data shed light into the logic that underlies the control of TFEB localization. In fully fed cells, a fraction of TFEB could always be found on lysosomes, although the majority appeared to freely diffuse in the cytoplasm. The lysosomal localization of TFEB is associated with its ability to physically bind mTORC1, as shown by co-immunoprecipitation assays. Moreover, time-lapse analysis of TFEB–GFP in cells treated with Torin 1 showed that TFEB clustered on lysosomes shortly after the onset of drug treatment, and then progressively appeared in the nucleus (Supplementary Movies S2 and S3). Together, these results suggest the following model of control of TFEB subcellular localization and activity (). At any given time, a fraction of TFEB rapidly and transiently binds to the lysosomal surface, where it is phosphorylated by mTORC1 and thus kept in the cytoplasm. Nutrient withdrawal, v-ATPase inhibition, and lysosomal stress inactivate the Rag GTPases, causing loss of mTORC1 from the lysosome and resulting in failure to re-phosphorylate TFEB. Unphosphorylated TFEB progressively accumulates in the nucleus, where it activates lysosomal gene expression programs aimed at correcting the defective nutrient and/or pH status of the lysosome. In this model, the lysosome represents a bottleneck where mTORC1 tightly regulates the amount of TFEB that is allowed to reach the nucleus.
Figure 7 Model of lysosomal sensing and lysosome-to-nucleus signalling by TFEB and mTOR. (A) (Left) Under full nutrients and in the absence of lysosomal stress, the complex formed by v-ATPase, Ragulator, and Rag GTPases is in the active state and recruits mTORC1 (more ...)
mTORC1 may regulate a yet undiscovered TFEB function at the lysosome. This possibility is supported by the observation that blocking mTORC1 activity with Torin 1 resulted in a dramatic accumulation of TFEB not only in the nucleus but also on lysosomes, which was visible as increased binding to mTORC1 in co-IP assays, as well as reduced mobility in FRAP experiments. Future work will address what function, if any, TFEB performs on the lysosomal surface. Interestingly, recent evidence indicating that TFEB regulates multiple aspects of lysosomal dynamics, including the propensity of lysosomes to fuse with the plasma membrane (Medina et al, 2011
), suggests that the range of biological functions of TFEB still needs to be fully elucidated.
Our data further emphasize the emerging role of the lysosome as a key signalling centre. In particular, a molecular machinery that connects the presence of amino acids in the lysosomal lumen to the activation of mTORC1 indicates a new role for the lysosome in nutrient sensing and cellular growth control (Rabinowitz and White, 2010
; Singh and Cuervo, 2011
; Zoncu et al, 2011a
). It also suggests that mTORC1 participates in a lysosomal adaptation mechanism that enables cells to cope with starvation and lysosomal stress conditions (Yu et al, 2010
). This mechanism responds to a wide range of signals that relay the metabolic state of the cell, as well as the presence of various stress stimuli. For instance, loss of lysosomal proton gradient, caused by either energy depletion or pathological conditions, may suppress mTORC1 activity, either by blocking the proton-coupled transport of nutrients to and from the lysosome, or by directly affecting the v-ATPase (Marshansky, 2007
). Similarly, lysosomal membrane permeabilization observed in certain LSDs and neurodegenerative diseases may result in nutrient leakage and suppression of mTORC1 (Dehay et al, 2010
; Kirkegaard et al, 2010
We found that the transcriptional response of lysosomal and autophagy genes to starvation and mTOR inhibition by Torin 1 was hampered in hepatocytes from mice carrying a liver-specific conditional knockout of TFEB, demonstrating that TFEB is a main mediator of this response. Therefore, TFEB translates a lysosomal signal into a transcriptional program.
This lysosome-to-nucleus signalling mechanism, which operates a feedback regulation of lysosomal function, presents intriguing parallels with the sterol sensing pathway in the endoplasmic reticulum, where cholesterol depletion and ER stress cause the nuclear translocation of the Sterol Responsive Element Binding Protein (SREBP) transcription factor, which then activates gene expression programs that enhance cholesterol synthesis and ER function (Wang et al, 1994
; Peterson et al, 2011
). Another example is represented by the mitochondria retrograde signalling pathway, in which mitochondrial dysfunction activates factors such as NFκB, NFAT, and ATF, through altered Ca2+
dynamics (Butow and Avadhani, 2004
Finally, as TFEB overexpression was able to promote substrate clearance and to rescue cellular vacuolization in LSDs (Medina et al, 2011
), the identification of a lysosome-based, mTOR-mediated, mechanism that regulates TFEB activity offers a new tool to promote cellular clearance in health and disease.