MAP4K3 mediates amino acid-dependent regulation of autophagy via phosphorylation of TFEB

Abstract

Autophagy is the major cellular pathway by which macromolecules are degraded, and amino acid depletion powerfully activates autophagy. MAP4K3, or germinal-center kinase-like kinase, is required for robust cell growth in response to amino acids, but the basis for MAP4K3 regulation of cellular metabolic disposition remains unknown. Here we identify MAP4K3 as an amino acid-dependent regulator of autophagy through its phosphorylation of transcription factor EB (TFEB), a transcriptional activator of autophagy, and through amino acid starvation-dependent lysosomal localization of MAP4K3. We document that MAP4K3 physically interacts with TFEB and MAP4K3 inhibition is sufficient for TFEB nuclear localization, target gene transactivation, and autophagy, even when mTORC1 is activated. Moreover, MAP4K3 serine 3 phosphorylation of TFEB is required for TFEB interaction with mTORC1-Rag GTPase-Ragulator complex and TFEB cytosolic sequestration. Our results uncover a role for MAP4K3 in the control of autophagy and reveal MAP4K3 as a central node in nutrient-sensing regulation.

Fig. 1

Knockout of MAP4K3 promotes autophagy induction and flux. a Validation of MAP4K3 knockout (k.o.) cell lines. Wild-type (WT) and HEK293A cells gene-edited with either of two different sgRNAs (M1 and M4) were lysed, and protein lysates were immunoblotted for MAP4K3. Immunoblotting of β-actin served as a loading control. b, c Knockout of MAP4K3 promotes autophagy flux. WT HEK293A cells, M1-1 MAP4K3 k.o. cells, and M4-6 MAP4K3 k.o. cells (not shown) were cultured in complete media (CM) or subjected to amino acid starvation (– AA), and remained untreated or were treated with ammonium chloride. Protein lysates were immunoblotted for LC3 and β-actin, which served as a loading control b. The ratio of LC3-II:actin was determined by densitometry using ImageJ and normalized to WT CM, which was arbitrarily set to 1 c. One-way ANOVA with post-hoc Tukey’s test; *P < 0.05. d, e Knockout of MAP4K3 promotes autophagy induction. LC-3 immunostaining of WT HEK293A cells and three different MAP4K3 k.o. cell lines, all cultured in CM d. Quantification of LC3 puncta area per cell area was determined using ImageJ. n > 100 cells per genotype. One-way ANOVA with post-hoc Tukey’s test; **P < 0.01, ***P < 0.001. f–h Knockout of MAP4K3 promotes autophagy flux. WT HEK293A cells and MAP4K3 k.o. cells were cultured in CM or amino acid starved, and were transfected with a GFP-mCherry-LC3 expression construct (f). Note the predominance of red puncta indicative of autolysosomes in MAP4K3 k.o. cells. Quantification of autophagosome number per cell was determined by counting yellow puncta GFP-mCherry-LC3-expressing cell (g). Quantification of autolysosome number per cell was determined by counting red puncta/GFP-mCherry-LC3-expressing cell (h). n > 50 cells per condition. One-way ANOVA with post-hoc Tukey’ test; **P < 0.01, ***P < 0.001. All experiments were performed in triplicate. Error bars = SEM. Scale bars = 10 μm

Fig. 2

MAP4K3 regulates TFEB subcellular localization. a Knockout of MAP4K3 yields TFEB nuclear localization. WT HEK293A cells and MAP4K3 k.o. cells were transfected with a TFEB-FLAG expression construct and cultured in complete media (CM) or starved of amino acids for 120 min, and then restimulated with amino acids for 10 min. Here we see representative images of cells immunostained with anti-FLAG antibody. b Quantification of cells with predominantly TFEB nuclear localization from experiment shown in a. n > 100 cells per condition. One-way ANOVA with post-hoc Tukey’s test; ***P < 0.001. c TFEB nuclear localization in MAP4K3 k.o. cells is rescued by MAP4K3 expression. WT HEK293A cells and MAP4K3 k.o. cells were transfected with a TFEB-FLAG expression construct and cultured in CM or amino-acid starved, and MAP4K3 k.o. cells were co-transfected with a MAP4K3-mCherry expression construct. Cells were stained with DAPI and immunostained with anti-FLAG antibody to permit visualization of nuclei, TFEB, and MAP4K3, as indicated. TFEB remains in the cytosol in CM in WT HEK293A cells and in MAP4K3 k.o. cells transfected with the MAP4K3 vector (white arrows); however, TFEB exhibits nuclear localization in untransfected MAP4K3 k.o. cells (orange arrows). Under conditions of amino acid starvation, TFEB translocates to the nucleus in WT cells, MAP4K3-transfected MAP4K3 k.o. cells (white arrow), and untransfected MAP4K3 k.o. cells (orange arrow). d Quantification of cells with predominantly TFEB nuclear localization from experiment shown in c. n > 50 cells per condition. One-way ANOVA with post-hoc Tukey’s test; *P < 0.05, **P < 0.01. All experiments were performed in triplicate. Error bars = SEM. Scale bars = 10 μm

Fig. 3

MAP4K3 regulation of TFEB is upstream of mTORC1. a Knockout of MAP4K3 promotes TFEB-mediated transactivation of its target genes. WT HEK293A cells, untreated, or treated with Torin1, and two different MAP4K3 k.o. cell lines were cultured in CM. Quantitative RT-PCR of isolated RNAs for these cell lines was performed for six TFEB target genes. One-way ANOVA with post-hoc Tukey’s test; *P < 0.05, **P < 0.01. b Activation of mTORC1 does not alter TFEB localization in MAP4K3 k.o. cells. WT HEK293A cells and MAP4K3 k.o. cells were transfected with an expression construct for constitutively active Rheb, epitope-tagged with myc, and starved of amino acids for 120 min. Although untransfected WT HEK293A cells exhibit TFEB nuclear localization (orange arrows), many WT HEK293A cells expressing constitutively active Rheb display TFEB cytosolic localization (white arrows). Although untransfected MAP4K3 k.o. cells also exhibit TFEB nuclear localization (orange arrows) as expected, most MAP4K3 k.o. cells expressing constitutively active Rheb show that TFEB still localizes to the nucleus (white arrows). Scale bar = 10 μm. Quantification of cells with predominantly TFEB nuclear localization for this experiment is shown in the adjacent graph. n > 50 cells per condition. **P < 0.01; two-tailed t-test. c Activation of mTORC1 does not prevent TFEB-mediated target gene activation in MAP4K3 k.o. cells. WT HEK293A cells, mock transfected, or transfected with constitutively active Rheb, and two different MAP4K3 k.o. cell lines, each transfected with constitutively active Rheb, were cultured in CM. Quantitative RT-PCR of isolated RNAs for these cell lines was performed for seven TFEB target genes. One-way ANOVA with post-hoc Tukey’s test; *P < 0.05, **P < 0.01. All experiments were performed in triplicate. Error bars = SEM

Fig. 4

MAP4K3 and TFEB serine 3 phosphorylation are required for interaction of TFEB with the mTORC1-Rag GTPase complex. a TFEB serine 3 is required for its interaction with the mTORC1-Rag GTPase complex. HEK293A cells were transfected with either WT TFEB, TFEB-S3A, or TFEB-Δ30, each FLAG-tagged, and cell lysates and FLAG immunoprecipitates were subjected to immunoblotting. b MAP4K3 is required for TFEB interaction with the mTORC1-Rag GTPase complex. WT HEK293A cells and MAP4K3 k.o. cells were transfected with either WT TFEB or TFEB-S3A, each FLAG-tagged, and cell lysates and FLAG immunoprecipitates were subjected to immunoblotting. c TFEB interaction with the mTORC1-Rag GTPase complex is rescued by MAP4K3 in MAP4K3 k.o. cells. WT HEK293A cells, M1-1 MAP4K3 k.o. cells, and M4-6 MAP4K3 k.o. cells were transfected with TFEB-FLAG alone, or co-transfected with TFEB-FLAG and MAP4K3, and cell lysates and FLAG immunoprecipitates were subjected to immunoblotting. d TFEB phosphomimetic S3E enhances TFEB interaction with the mTORC1-Rag GTPase complex in MAP4K3 k.o. cells. WT HEK293A cells, M1-1 MAP4K3 k.o. cells, and M4-6 MAP4K3 k.o. cells were transfected with either WT TFEB or TFEB-S3E, each FLAG-tagged, and cell lysates and FLAG immunoprecipitates were subjected to immunoblotting. All experiments were performed in triplicate

Fig. 5

MAP4K3 phosphorylates TFEB on serine 3. a MAP4K3 physically interacts with TFEB. WT HEK293A cells were transfected with an expression vector for either kinase dead (KD)-MAP4K3-FLAG, WT-MAP4K3-FLAG, or HDAC6-FLAG, and cell lysates and FLAG immunoprecipitates were subjected to immunoblotting. b MAP4K3 directly interacts with TFEB. WT HEK293A cells were transfected with an expression vector for either HDAC6-FLAG, WT-MAP4K3-FLAG, or kinase dead (KD)-MAP4K3-FLAG, and FLAG immunoprecipitates were incubated with recombinant TFEB generated by in vitro transcription and translation (IVTT TFEB). Cell lysates and TFEB immunoprecipitates were then subjected to immunoblotting. c MAP4K3 directly interacts with the N-terminal 37 amino acids of TFEB. WT HEK293A cells were transfected with an expression vector for either kinase dead (KD)-MAP4K3-FLAG, WT-MAP4K3-FLAG, or HDAC6-FLAG, and FLAG immunoprecipitates were incubated with recombinant GST-TFEB amino acids 1-37 or recombinant GST alone, before mixing with GST-containing beads. Cell lysates and the eluate obtained from GST-bound fractions were subjected to anti-FLAG and anti-GST immunoblotting, as indicated. d MAP4K3 phosphorylates TFEB at serine 3. WT HEK293A cells were transfected with WT-MAP4K3-FLAG or KD-MAP4K3-FLAG, and either TFEB-FLAG, TFEB-S3A-FLAG, or TFEB-S211A-FLAG, as indicated. FLAG immunoprecipitates were subjected to in vitro kinase reactions with γ-P³²-ATP, with Torin1 and the general kinase inhibitor FSBA included in the reaction mixture. Phosphopeptide mapping was performed after enzymatic digestion with thermolysin by spotting the resulting peptide mix onto cellulose thin layer chromatography plates, followed by 2D gel electrophoresis and chromatography, and finally autoradiography to visualize phospho-labeled peptides. Circles indicate location of phospho-S3-TFEB. Note the absence of phospho-S3-TFEB for TFEB-S3A and for kinase-dead (KD) MAP4K3. e MAP4K3 heavily phosphorylates TFEB on serines and threonines. WT HEK293A cells were transfected with WT-MAP4K3-FLAG or KD-MAP4K3-FLAG, and TFEB-FLAG, as indicated. FLAG immunoprecipitates were subjected to in vitro kinase reactions with γ-P³²-ATP, and phospho-amino acid mapping performed by matching the resultant spots on the autoradiograph with ninhydrin-stained standards. Orange circles indicate phospho-serine, and purple circles indicate phospho-threonine. All experiments were performed in triplicate

Fig. 6

Phosphorylation of TFEB at serine 3 is a key determinant of TFEB cellular regulation and autophagy function. a TFEB serine 3 phosphorylation is required for mTORC1 phosphorylation at serine 211. WT HEK293A and MAP4K3 k.o. cells were transfected with TFEB-FLAG, TFEB-S3A-FLAG, or TFEB-S3E-FLAG, and Torin1 treated. FLAG immunoprecipitates were immunoblotted and TFEB serine 211 phosphorylation as a fraction of total TFEB was quantified by densitometry. b MAP4K3 phosphorylation of TFEB is required for mTORC1 phosphorylation at serine 211. WT HEK293A and MAP4K3 k.o. cells were transfected with TFEB-FLAG and either no MAP4K3 (--), WT-MAP4K3, or KD-MAP4K3. FLAG immunoprecipitates were immunoblotted, and TFEB serine 211 phosphorylation as a fraction of total TFEB was quantified by densitometry. c TFEB serine 3 phosphorylation is required for interaction with 14-3-3. TFEB k.o. cells were transfected with no TFEB (--), inducible TFEB-WT-FLAG, or inducible TFEB-S3A-FLAG, and Torin1 treated, whereas all cells received doxycycline to induce TFEB-WT or TFEB-S3A expression. Cell lysates and TFEB immunoprecipitates were immunoblotted and immunoprecipitated 14-3-3 was quantified by densitometry. One-way ANOVA with post-hoc Tukey’s test; *P < 0.05, **P < 0.01. d TFEB serine 3 phosphorylation regulates TFEB nuclear localization. TFEB k.o. cells were transfected with inducible TFEB-WT-FLAG or TFEB-S3A-FLAG, and cultured in CM or amino-acid starved (– AA). Under amino acid deprivation, TFEB localizes to nucleus, regardless of serine 3 status; however, upon amino acid satiety, mutation of TFEB serine 3 to phospho-resistant alanine prevents retention of TFEB in the cytosol. Quantification of TFEB nuclear localization to right. n > 100 cells per condition. One-way ANOVA with post-hoc Tukey’s test; ***P < 0.001. e TFEB serine 3 phosphorylation regulates autophagy activation. TFEB k.o. cells were transfected with GFP-mCherry-LC3 and either inducible TFEB-WT-FLAG or TFEB-S3A-FLAG, and cultured in CM and doxycycline, as indicated. Note the red puncta indicative of autolysosomes in cells expressing TFEB-S3A. Autophagosome number per cell was determined by counting yellow puncta per GFP-mCherry-LC3-expressing cell, and autolysosome number per cell was determined by counting red puncta per GFP-mCherry-LC3-expressing cell. n > 50 cells per condition. One-way ANOVA with post-hoc Tukey’s test; *P 0< 0.05, **P < 0.01. All experiments performed in triplicate. Error bars = SEM. Scale bars = 20 μm

Fig. 7

MAP4K3 exhibits lysosomal localization. a HEK293A cells were transfected with FLAG-tagged MAP4K3 for 16 h, then cultured in complete media (CM) or subjected to amino acid starvation (– AA) for 1 h. Cells were subjected to subcellular organelle fractionation via sucrose gradient density ultracentrifugation. The whole homogenate and isolated lysosomal fractions (P1 or P2) were collected for immunoblotting analysis, as indicated. Densitometry of MAP4K3 and Lamp1 was performed on the immunoblot of the P1 lysosomal fraction to quantify MAP4K3 in the lysosomal fraction, normalized to Lamp1. The relative ratio of MAP4K3 in the P1 fraction for CM-cultured HEK293A cells and amino-acid starved HEK293A cells is given below their respective lanes, with MAP4K3 in CM-cultured HEK293A cells arbitrarily set to 1. b HEK293A cells were transfected with MAP4K3-mNeonGreen and maintained under conditions of amino acid satiety for at least 60 min, before being switched to media lacking amino acids for 60 min, after which cells were fixed and immunostained for Lamp2. Note numerous puncta (arrows indicate representative examples) revealing colocalization of MAP4K3 with Lamp2. Scale bar = 10 μm. c HEK293A cells were transfected with MAP4K3-mNeonGreen and maintained under conditions of amino acid satiety for at least 60 min, before being switched to media lacking amino acids. Note the marked increase in MAP4K3 localization to cytosolic puncta upon amino acid starvation. Scale bar = 10 μm (see Supplementary Movie 1 for live cell imaging). d HEK293A cells were transfected with MAP4K3-mNeonGreen and starved of amino acids for 60 min, before being switched to amino acid-replete media. Note the MAP4K3 disassociation from cytosolic puncta to diffuse cytosolic localization upon supplying amino acids. Scale bar = 10 μm. e HEK293A cells were transfected with MAP4K3-mNeonGreen, treated with Lysotracker Red, and starved of amino acids for 60 min, before being switched to amino acid-replete media. Note the prominent MAP4K3 colocalization with Lysotracker Red in cytosolic puncta during amino acid starvation, then upon amino acid supplementation, and MAP4K3 movement from cytosolic puncta and Lysotracker Red colocalization to a more diffuse cytosolic localization as well. Scale bar = 10 μm (see Supplementary Movie 2 for live-cell imaging). All experiments were performed in triplicate

Fig. 8

MAP4K3 preferentially localizes to lysosomes upon amino acid depletion and interacts with Rag GTPases. a HEK293A cells were starved of amino acids for 60 min and then switched to amino acid-replete media for 15 min, after which cells were fixed and immunostained for endogenous MAP4K3 and Lamp2. It is noteworthy that extensive MAP4K3–Lamp2 colocalization upon amino acid starvation diminishes with resupply of amino acids. Scale bar = 10 μm. b Quantification of MAP4K3–Lamp2 colocalization in a. We counted the number of colocalized puncta in 10 cells per field for 3 fields per condition, performed in triplicate, and determined the mean puncta count per condition. *P < 0.05; two-tailed t-test. c MAP4K3 physically interacts with RagC. WT HEK293A cells were transfected with an expression vector for Hey1-FLAG, WT-MAP4K3-FLAG, or kinase dead (KD)-MAP4K3-FLAG as indicated, and cell lysates and RagC immunoprecipitates were subjected to immunoblotting. d MAP4K3 interaction with RagA is dependent on amino acid status. WT HEK293A cells cultured in complete media (CM) or subjected to amino acid starvation (– AA) were transfected with an expression vector for either HDAC6-FLAG or WT-MAP4K3-FLAG as indicated, and cell lysates and RagA immunoprecipitates were subjected to immunoblotting. All experiments were performed in triplicate

Fig. 9

Model for MAP4K3 regulation of TFEB activation-dependent autophagy. Right: when amino acids are abundant, MAP4K3 phosphorylates TFEB on serine 3 in the cytosol. TFEB serine 3 phosphorylation enables the Rag GTPases to recruit TFEB, which may still be in complex with MAP4K3, to the surface of the lysosome via the interaction of Rag GTPases with the Ragulator complex. Recruitment of TFEB to the lysosomal surface facilitates mTORC1 interaction with TFEB and mTORC1 phosphorylation of TFEB on serine 211. Upon serine 211 phosphorylation, TFEB is released from the lysosome to the cytosol, where 14-3-3 binds to TFEB and retains inactive TFEB sequestered in the cytosol. Left: when amino acids are scarce, MAP4K3 localizes to the lysosome and TFEB is thus no longer phosphorylated, permitting TFEB to translocate into the nucleus and activate the expression of genes that promote autophagy-lysosome pathway function