Multistep regulation of TFEB by MTORC1

Abstract

The master regulator of lysosome biogenesis, TFEB, is regulated by MTORC1 through phosphorylation at S211, and a S211A mutation increases nuclear localization. However, TFEB^S211A localizes diffusely in both cytoplasm and nucleus and, as we show, retains regulation by MTORC1. Here, we report that endogenous TFEB is phosphorylated at S122 in an MTORC1-dependent manner, that S122 is phosphorylated in vitro by recombinant MTOR, and that S122 is important for TFEB regulation by MTORC1. Specifically, nuclear localization following MTORC1 inhibition is blocked by a S122D mutation (despite S211 dephosphorylation). Furthermore, such a mutation inhibits lysosomal biogenesis induced by Torin1. These data reveal a novel mechanism of TFEB regulation by MTORC1 essential for lysosomal biogenesis.

Keywords: Lysosomal biogenesis; MTOR; TFEB; YWHA; subcellular localization.

Figure 1.

Torin1 enhances TFEB^S211A nuclear localization. (A) Validation of the newly generated p-TFEB (S211) antibody. HeLa cells were transfected with the indicated MYC-tagged plasmids (WT, wild-type TFEB; S211A TFEB mutant or EV, empty vector), lysed, and treated or not with lambda phosphatase (“λ”) before MYC immunoprecipitation (IP) and western blot analysis. (B) p-TFEB (S211) analysis of Torin1-treated HeLa cells (250 nM). Endogenous TFEB was immunoprecipitated and analyzed by western blot. (C) HeLa cells transfected with the indicated MYC-tagged TFEB constructs were subjected to immunoprecipitation with YWHA or MYC antibodies and analyzed by western blot. Torin1 (250 nM) for 6 h. Where indicated, immunoprecipitates were treated with lambda phosphatase (λ). (D) Quantification of TFEB-GFP or TFEB^S211A-GFP subcellular localization in HeLa cells transfected as indicated (C, exclusively cytoplasmic; C and N, cytoplasmic and nuclear; N, exclusively nuclear). Graph illustrates average (n = 2; ≥100 cells per experiment) ± SD. *, P < 0.05 for comparison between TFEB-WT and TFEB^S211A for each of the fractions. (E) HeLa cells were transfected with the indicated plasmids and biochemical fractionation was performed after Torin1 treatment (250 nM for 3 h). (F) HeLa cells stably expressing TFEB-GFP (WT vs. S211A) were treated with vehicle (DMSO) or Torin1 (250 nM for 6 h) and analyzed by immunofluorescence (left panel, representative images; middle panel, schematic representation; right panel, quantification). Two-factor ANOVA was used to compare the global effect of the TFEB^S211A mutation. ***, P < 0.001; ns, nonsignificant. Scale bar: 20 μm.

Figure 2.

TFEB serine 122 is regulated by MTORC1. (A) Validation of the p-TFEB (S122) antibody. HeLa cells were transfected with the indicated MYC-tagged TFEB constructs followed by MYC-IP and western blot. λ, lambda-phosphatase treatment of immunoprecipitates. HeLa cells were treated with Torin1 (250 nM) (B), or starved for amino acids (C), serum (D), or glucose (E). (F, G) HeLa cells were transfected with active (RRAGB^GTP/D^GDP) or inactive (RRAGB^GDP/D^GTP) RRAG-GTPases and its effects on TFEB localization and phosphorylation were assessed. (H) MTOR in vitro kinase assay of epitope tag immunoprecipitates from cells transfected with empty vector (EV) vs. MYC-TFEB and incubated with or without recombinant MTOR. Scale bar: 10 μm.

Figure 3.

Serine 122 regulation is essential for TFEB-mediated lysosome biogenesis. (A, B) Subcellular localization of transfected full-length-TFEB or S122D mutant in HeLa cells analyzed by confocal microscopy or biochemical subcellular fractionation in cells treated or not with Torin1 (250 nM, 3 h). Graph represents average ± SD (n = 6; ≥ 100 cells per experiment; *, P < 0.05). (C, D) HeLa cells depleted of TFEB were transfected with the indicated constructs and treated with Torin1 for 36 h. Cells were stained with LysoTracker Red (C) or an antibody against LAMP1 (D) and analyzed by FACS. Graphs indicate mean ± SE (n = 3). Asterisks illustrate statistically significant differences in LysoTracker Red or LAMP1 intensity. **, P < 0.01. (E) qPCR analysis of TFEB-target genes in HeLa cells depleted of TFEB and transfected with the indicated constructs (Torin1 250 nM, 36 h). ***, P < 0.001.

Figure 4.

Serine 122 contributes to TFEB nuclear enrichment in the context of S211A mutation (A-C) Subcellular localization of transfected full-length-TFEB, S122A, S211A or S122A;S211A mutants in HeLa cells by biochemical fractionation (A) or confocal microscopy (B, C) following (or not) treatment with Torin1 (250 nM, 3 h). Graph represents average ± SD (n = 3; ≥ 100 cells per experiment; ***, P < 0.01). (D) YWHA protein immunoprecipitation from HeLa cells transfected with the indicated TFEB constructs. (E) Western blot for S211 phosphorylation of wild-type or mutant MYC-TFEB immunoprecipitated from transfected HeLa cells. (F) Proposed model for TFEB regulation by MTORC1. Scale bar: 20 μm.