Fig 2: MiR‐142‐3p blocked NSCLC development in vitro by depleting MAP4K3. In H1299 and A549 cells transfected with miR‐142‐3p mimic or miR‐142‐3p mimic+pc‐MAP4K3, (a and b) cell proliferation was assessed by CCK‐8 assay and colony formation assay. (c and d) cell migration and cell invasion were assessed by transwell assay. (e) The angiogenic ability was investigated by Matrigel tube formation assay. (f and g) Cell cycle distribution was determined by flow cytometry assay. (h) Cell apoptosis was investigated by flow cytometry assay. (i and j) The expression of PCNA and Bax was detected by western blot. *p < 0.05

Fig 3: 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 4: 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 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