TFEB degradation is regulated by an IKK/β-TrCP2 phosphorylation-ubiquitination cascade

Abstract

Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and cellular clearance pathways. TFEB activity is tightly controlled by multiple post-translational mechanisms, but the exact molecular mechanism controlling its stability has remained elusive. Here, we identify the IκB kinase (IKK) complex as a key regulator of TFEB protein stability through a phosphorylation–ubiquitination cascade. A high-content kinase inhibitor screen reveals that IKK inhibition increases TFEB protein levels, and genetic ablation of IKK components increases TFEB stability, upregulates lysosomal genes, and enhances lysosomal biogenesis and degradative capacity. Mechanistically, we show that IKK phosphorylates TFEB on a cluster of serine residues (⁴²³SPFPSLS⁴²⁹), generating a phosphodegron recognized by the E3 ligase β-TrCP2, which in turn targets TFEB for proteasomal degradation via ubiquitination of adjacent lysine residues (K430 and K431). Mutation of either the phosphosites or the ubiquitination sites stabilizes TFEB without impairing its ability to translocate to the nucleus, activate target gene expression, or promote tau clearance in a cell model of tauopathy. These findings establish IKK–β-TrCP2 as a core regulatory axis controlling TFEB protein turnover and levels and reveal a mechanistically distinct layer of TFEB regulation that may be leveraged to enhance lysosomal function in disease contexts.

Inflammatory signaling profoundly influences cellular metabolism and stress responses. Here, the authors show that the IKK complex directly controls TFEB stability through β-TrCP2–dependent proteasomal degradation, mechanistically linking inflammation to lysosomal regulation.

Introduction

Transcription factor EB (TFEB) is a master regulator of the autophagy-lysosomal pathway (ALP)¹. Active TFEB promotes the transcription of lysosomal and autophagy genes^1–3 and participates in the induction of ALP-dependent processes such as lysosome-mediated clearance¹, lysosomal proteostasis⁴, and lysosomal exocytosis⁵. TFEB also contributes to other cellular programs, including the ER stress response^4,6 and mitochondrial biogenesis^7,8. TFEB plays a pivotal role in lysosome-to-nucleus communication by integrating inputs from mechanistic target of rapamycin complex 1 (mTORC1)^9–13 and other key metabolic regulators, including protein kinase B (PKB/AKT)^14,15 and AMP-activated protein kinase (AMPK)¹⁶, all of which have specific roles at the lysosome^17–19. Importantly, these and other kinases phosphorylate TFEB on distinct sets of residues^{9–16,20–23}, indicating that TFEB is controlled by multiple independent regulatory layers that likely execute different branches of the cellular adaptive response or serve additional functions. Studies in cultured cells have shown that, under standard growth conditions, TFEB is predominantly cytosolic and inactive. TFEB cytosolic retention is mediated by binding of YWHA/14-3-3 proteins to TFEB phosphorylated on Ser211^12,13. Ser211 is a direct target of active mTORC1^12,13, which therefore limits the ALP-enhancing functions of TFEB. Conversely, lysosomal storage, cell starvation, and other stressors promote TFEB nuclear translocation^{1,2,6,11–13,24}. In the nucleus, TFEB binds to the “coordinated lysosomal expression and regulation” (CLEAR) motif located within the promoter regions of genes participating in lysosomal pathways and promotes their transcription¹.

Active TFEB and ALP enhancement promote cellular clearance by facilitating the elimination of protein aggregates and lysosomal storage material. Building on this principle, we and others have shown that TFEB is both a component of pathogenic cascades and a candidate therapeutic target in neurodegenerative storage disorders and beyond^{1,4,5,14,25–30}. Multiple studies have demonstrated that exogenous TFEB expression reduces aggregated tau and β-amyloid in cell lines as well as in the brains of mouse models of Alzheimer’s disease^26,28,31, underscoring the significance of TFEB as a potential target for therapeutic intervention.

While best known for their roles in Nuclear Factor kappa B (NF-κB) signaling, components of the IκB kinase (IKK) complex have more recently emerged as NF-κB-independent regulators of processes as diverse as mTORC1 signaling^32–34, apoptosis^35,36, cell proliferation and differentiation^37,38, and epigenetic control of gene transcription^39–41, through direct phosphorylation of key players of these processes. The IKK complex is composed of three components: the partially redundant catalytic subunits IKKα (encoded by the CHUK gene) and IKKβ (IKBKB), and a scaffold/regulatory subunit IKKγ (IKBKG or NEMO)^42–45. IKK has been linked to the mTOR pathway through two converging mechanisms. IKK was previously shown to inhibit Tuberous Sclerosis Complex 1 (TSC1)³², a key negative regulator of mTORC1 signaling⁴⁶. Subsequent work demonstrated that IKK subunits can associate with and phosphorylate mTOR directly, promoting mTORC1 kinase activity^33,34. Thus, active IKK functions as a positive regulator of mTORC1 via at least two independent mechanisms. IKK can be activated by various stimuli, including TNFα signaling⁴⁴. For several of its targets, IKK-dependent phosphorylation generates a phosphodegron—a specific motif recognized by β-transducin repeat-containing proteins (β-TrCP), the F-box substrate adapters of SCF^β-TrCP E3 ubiquitin ligases^47–50. The mammalian members of the β-TrCP family, β-TrCP1 and β-TrCP2, share sequence similarity and biochemical properties, but may have distinct substrates⁴⁷. For example, β-TrCP2 has been characterized as the dominant paralog in regulating autophagy and cell growth through starvation-induced recognition and ubiquitination of β-TrCP1 and the mTORC1 inhibitors DEPTOR and REDD1⁵¹.

In this study, we show that TFEB protein levels under standard culture conditions are markedly regulated by a phosphorylation-ubiquitination cascade executed by IKK and β-TrCP2. We find that IKK phosphorylates TFEB at multiple residues, generating a phosphodegron (⁴²³SPFPSLS⁴²⁹) that is recognized by β-TrCP2, which in turn promotes degradative ubiquitination of TFEB at adjacent lysine residues (K430 and K431). Selective genetic disruption of this phosphorylation-ubiquitination cascade increases TFEB protein levels several fold but does not interfere with signaling pathways that regulate TFEB nuclear translocation or its capacity to enhance expression of its target genes and promote cellular clearance. This mechanism provides a molecular explanation for how TFEB is targeted for proteasomal degradation and introduces a regulatory axis that may represent a promising therapeutic target in diseases characterized by impaired cellular clearance.

Results

IKK regulates TFEB protein levels

To efficiently screen for kinases that modify TFEB levels post-translationally, we generated a TFEB-GFP fusion construct as a reporter that is independent of transcriptional regulation of gene levels and does not require incubation with antibodies. We first tested this construct by transient transfection in HeLa cells, using fluorescence microscopy to monitor both TFEB protein levels and its subcellular localization. Calculation of a TFEB nuclear translocation index (TFEB_NTI; see “Methods” for details) across hundreds of cells showed that TFEB nuclear localization levels were positively associated with TFEB protein levels (Supplementary Fig. 1a, b), presumably because high levels of TFEB protein saturate the systems that promote its cytosolic retention. Thus, we generated cells stably expressing TFEB-GFP (henceforth referred to as HeLa/TFEB-GFP) by selecting a non-clonal pool of cells infected with a TFEB-GFP lentivirus. Fluorescence microscopy analysis showed that TFEB was generally expressed at lower levels compared to transiently transfected cells, and that nuclear localization levels were independent of TFEB expression levels in HeLa/TFEB-GFP cells (Supplementary Fig. 1c, d), indicating the suitability of these cells for screening procedures.

To identify candidate kinases that regulate TFEB stability, we carried out a human kinome screen using a library of 436 established kinase inhibitors. For each inhibitor, we applied five different combinations of incubation time and drug concentration: cells were incubated with kinase inhibitors at either 1 or 3 µM for 1 h, or at 1, 3, or 10 µM for 6 h. We used automated fluorescence microscopy to acquire images of hundreds to thousands of cells per condition, which we then analyzed to assess TFEB-GFP signal levels in whole cells, cytosol, and nuclei. In each plate, Torin1 (a potent catalytic inhibitor of mTORC1) was used as a positive control for its ability to alter cytosolic and nuclear TFEB levels, while DMSO was used as a vehicle control. To assess the robustness of the pipeline, we integrated the microscopy data from the five conditions to perform unsupervised hierarchical clustering of the drugs based on TFEB_NTI (Supplementary Data 1). The analysis showed that the rapalogs (allosteric mTORC1 inhibitors) present in the library clustered with Torin1 (Fig. 1a, red arrow). The unsupervised hierarchical clustering procedure is effect size-independent and leverages all changes observed across the five data points to make associations amongst drugs that alter, even minimally, TFEB distribution in similar patterns. As rapalogs have been characterized as very weak modulators of TFEB nuclear translocation at best^11,12,52,53, yet still clustered with Torin1 (a potent activator of TFEB nuclear translocation), this result underscores the high sensitivity and robustness of the analytical pipeline.

Fig. 1. Suppression of IKK function increases TFEB protein levels and enhances lysosomal biogenesis and function.

a Hierarchical clustering of all tested drugs based on TFEB_NTI distribution curves. The red arrow indicates a cluster of drugs targeting mTORC1, while the green arrow indicates a cluster of drugs affecting TFEB subcellular distribution, but distinct from the mTORC1 cluster. Vehicle controls (DMSO) are indicated in the clustering. b Dot plot displaying TFEB-GFP intensity levels and the product of the distances from DMSO and Torin1 in the hierarchical clustering. TFEB-GFP intensity levels were measured after 6-hour incubation with a drug concentration of 10 µM. c Immunoblot analysis of protein extracts from WT MEFs and MEF lines bearing targeted deletion of the genes encoding IKKα, IKKβ, or IKKγ. IκBα is used as an indicator of IKK activity (n = 3 independent experiments). d Quantification of the immunoblots in (c), normalized to the corresponding GAPDH levels. p-values are calculated based on a two-sided student’s t test. e Relative Tcfeb mRNA levels determined by RT-qPCR in samples shown in panel (c). Results were normalized to Ppia. One-way ANOVA with Dunnett’s multiple comparisons test. f Representative images of immunostaining for LAMP1 (green) and DAPI (blue). Scale bar = 20 µm. g, h quantification of LAMP1-positive area (g) and LAMP1 integrated intensity (h). for WT and IKKα -/- MEFs, n = 32 cells; for IKKβ-/- MEFs, n = 33 cells; for IKKγ-/- MEFs, n = 35 cells. Welch’s one-way ANOVA with Dunnett’s T3 multiple comparisons test. i Representative images of DQ BSA fluorescence (green) and DAPI (blue). Scale bar = 20 µm. j Quantification of the DQ BSA integrated intensity. n = 30 cells each condition. Welch’s one-way ANOVA with Dunnett’s T3 multiple comparisons test. k–m HEK293T cells were transfected with scramble siRNA or siRNAs targeting CHUK (IKKα) (k), IKBKB (IKKβ) (l), or IKBKG (IKKγ) (m). After 48 h, transcriptional analysis was performed, and gene expression levels were normalized to the housekeeping gene, GAPDH (n = 3 independent experiments). Multiple two-sided t tests with Benjamini–Hochberg FDR correction. Results are presented as mean ± SEM. Source data are provided as a Source Data file.

The vast majority of the kinase inhibitors screened clustered with DMSO, indicating little to no effect on TFEB levels in the cytosol or nucleus. Interestingly, a small cluster of kinase inhibitors that increased TFEB nuclear signal (indicated by the green arrow in Fig. 1a) was distinct from the cluster of mTORC1 inhibitors, suggesting the presence of additional mechanisms contributing to changes in cytosolic and/or nuclear TFEB levels. A scatterplot of total TFEB-GFP intensity versus the product of the distances from Torin1 and vehicle in the hierarchical cluster highlighted a small group of kinases whose inhibition simultaneously increased both TFEB nuclear intensity and total TFEB intensity—IKK, Axl, JAK2, and CDK4/6 (Fig. 1b). CDK4/6 is a known kinase modifier of TFEB²³, and both Axl and JAK2 are general receptor tyrosine kinases that modulate the PI3K/Akt/mTOR pathway, among others^54,55. IKK has multiple strong connections to mTORC1 signaling^32–34 but has not been studied in the context of TFEB biology. Thus, we chose to focus on IKK for functional studies.

To test whether endogenous TFEB levels are regulated by IKK, we monitored TFEB protein levels in mouse embryonic fibroblast (MEF) lines in which the Chuk/IKKα, Ikbkb/IKKβ, or Nemo/IKKγ genes had been knocked out^56–58. Immunoblot analyses of total cell lysates showed that deletion of any IKK subunit resulted in increased TFEB protein levels (Fig. 1c, d), confirming IKK’s control over TFEB protein levels. As a control, we monitored IκBα (inhibitor of nuclear factor kappa B), a downstream target of IKK. Knockout of either IKKβ or IKKγ increased IκBα levels (Fig. 1c, d), indicating that IKK maintains basal activity under the test conditions, even in the absence of external stimuli. RT-qPCR analysis showed that TFEB mRNA levels were not increased by deletion of either IKK subunit (Fig. 1e), indicating that the effect of IKK on TFEB must be post-translational.

Cell fractionation followed by immunoblotting showed that deletion of IKK subunits did not alter TFEB subcellular distribution (Supplementary Fig. 2a, b), a result confirmed by siRNA-mediated silencing of all IKK subunits in HeLa/TFEB-GFP cells (Supplementary Fig. 2c, d). Additional experiments clarified that IKK-16, the IKK inhibitor used in the screen, also inhibits mTORC1 in HeLa/TFEB-GFP (Supplementary Fig. 2e), which could be an off-target effect. We verified that, when mTORC1 is rendered constitutively active by expression of modified RagB and RagD constructs, IKK-16-induced nuclear localization is indeed abolished, demonstrating that this effect is mediated by mTORC1 (Supplementary Fig. 2f, h). Conversely, constitutively active mTORC1 did not antagonize the observed increases in TFEB signal intensity (Supplementary Fig. 2f, g–i), and Torin1, differently from IKK-16, was unable to increase TFEB levels (Supplementary Fig. 2j), indicating that IKK-16–induced increases in TFEB levels are not mediated by mTORC1 inhibition. Taken together, these data demonstrate that IKK deletion or inhibition increases TFEB levels through a mechanism that is uncoupled from mTORC1-mediated control of TFEB nuclear translocation. We finally tested whether the IKK-16–induced increase in TFEB expression requires IKK. To this aim, we treated WT and IKK-knockout MEF lines with DMSO or IKK-16 and evaluated TFEB levels by immunoblotting. The results showed that IKK-16 increased TFEB levels in WT cells but failed to do so in any of the IKK-knockout lines (Supplementary Fig. 2k, l), demonstrating that the observed effect of IKK-16 on TFEB protein levels is mediated by IKK.

Next, we tested whether IKK deletion caused any changes in lysosomal biogenesis and function. Staining the MEF lines with an antibody against LAMP1 (lysosomal-associated membrane protein 1), a lysosomal marker, showed that knockout of any IKK subunit resulted in a significant expansion of lysosomal compartments, indicating increased lysosomal biogenesis (Fig. 1f–h). We also carried out an analysis of lysosomal proteolytic function using DQ BSA, a self-quenched albumin probe that fluoresces upon lysosomal degradation. Cells were loaded with DQ BSA for 16 h before fixation and imaging. Torin 1 was used as a positive control of activation of lysosomal proteolytic function. Microscopy experiments showed that the knockout MEF lines exhibited enhanced DQ BSA fluorescence compared to WT MEFs (Fig. 1i, j). Together, these data show that knockout of IKK expands both the lysosomal compartment and its degradative capacity. Acute suppression of IKK function in HEK293T cells (Fig. 1k–m) and HeLa cells (Supplementary Fig. 3a–c) through siRNA-mediated silencing of any IKK subunit resulted in transcriptional upregulation of TFEB target genes, consistent with a role for IKK in suppressing TFEB levels.

IKK stimulation by TNFα or IL-1β in MEF cells resulted in a rapid decrease in TFEB protein levels, which was not observed in any of the IKK gene knockout lines (Fig. 2a–f). We also tested lipopolysaccharide (LPS), an activator of inflammatory responses previously shown to reduce TFEB levels by unknown mechanisms^21,59. The results showed that LPS also requires IKK proteins to reduce TFEB protein levels (Fig. 2g–i). To assess whether chronic LPS-mediated suppression of TFEB impacts lysosomal biogenesis, we treated WT and IKK-knockout MEF lines with LPS for 72 h prior to fixation and imaging. Confocal imaging showed that prolonged LPS exposure led to a marked reduction of lysosomal compartments in WT cells, whereas no such reduction was observed in any IKK-knockout line (Fig. 2j, k). Collectively, these results demonstrate that IKK activity controls TFEB stability and the abundance of lysosomal compartments under both basal and stimulated conditions.

Fig. 2. Reduction of TFEB levels by TNFα, IL-1β, and LPS requires IKK.

a, d, g Immunoblot analysis of protein extracts from WT MEFs and MEF lines bearing targeted deletion of the genes encoding IKKα, IKKβ, or IKKγ upon treatment with TNFα (a), IL-1β (d), or LPS (g). IκBα is used as an indicator of IKK activity (a, n = 4 independent experiments; (d, g) n = 3 independent experiments). b, c Quantification of the immunoblots in (a). e, f Quantification of the immunoblots in (d). h, i Quantification of the immunoblots in (g), normalized to corresponding β-actin levels. p-values are calculated based on two-sided student’s t test. j Representative immunofluorescence images of LAMP1 (green) and DAPI (blue) staining in WT MEFs and MEF lines bearing targeted deletion of the genes encoding IKKα, IKKβ, or IKKγ, upon treatment with water or LPS (10 µg/ml) for 72 h. Scale bars, 20 µm. k Quantification of LAMP1 integrated intensity (left) and LAMP1-positive area (right) in (j). WT_water, n = 42; WT_LPS, n = 34; IKKα -/-_water, n = 48; IKKα -/-_LPS, n = 41; IKKβ-/-_water, n = 37; IKKβ-/-_LPS, n = 30; IKKγ-/-_water, n = 39; IKKγ-/-_LPS, n = 43. Statistical significance was assessed using Welch’s one-way ANOVA with Dunnett’s T3 multiple-comparisons test (two-sided). Data are presented as mean ± SEM. Source data are provided as a Source Data file.

IKK phosphorylates TFEB at multiple serine residues

IKKα and IKKβ are structurally similar, have partially redundant functions, and have been reported to show some degree of substate selectivity^45,60,61. We tested whether IKKα and IKKβ phosphorylate TFEB by performing phos-tag analyses. In phos-tag gels, the rate of protein migration depends on the number of phosphorylated residues, and the presence of multiple bands indicates multiple phosphorylation sites on the same protein⁶². 3xFlag-tagged TFEB was transiently expressed in HeLa cells, immunoprecipitated, and dephosphorylated in vitro using $\lambda$ phosphatase before being incubated with commercially available IKKα or IKKβ for phos-tag analysis. The results showed that both IKKα and IKKβ phosphorylated TFEB, with IKKβ displaying higher efficacy and generating multiple phosphorylated bands (Fig. 3a). As a control, in vitro phosphorylation of TFEB by Akt using the same procedure produced a single phosphorylated band, which was completely abolished by alanine substitution of serine 467, as expected¹⁴ (Supplementary Fig. 4a). Thus, the presence of multiple phosphorylated bands after incubation with IKKβ indicates that IKKβ mediates multiple phosphorylation events on TFEB. Of note, the reconstituted canonical IKK complex displayed higher phosphorylation efficiency than IKKβ alone (Fig. 3b). This result indicates that the IKK complex as a whole mediates TFEB regulation, consistent with the data from the knock-out lines and gene silencing experiments shown above.

Fig. 3. IKK phosphorylates TFEB.

a TFEB-3xFlag was immunoprecipitated from HeLa cell lysate, dephosphorylated by λ phosphatase, and subjected to an in vitro kinase assay with recombinant IKKβ or IKKα. The reaction was stopped by 2 x Laemmli buffer and analyzed using phos-tag analysis with a TFEB antibody. b TFEB-3 × Flag was subjected to an in vitro kinase assay with either recombinant IKKβ alone or a reconstituted IKK complex containing recombinant IKKα, IKKβ, and IKKγ. c Higher energy collisional dissociation (HCD) MS2 mass spectrum of TFEB’s tryptic peptide from precursor m/z 881.73, corresponding to the amino acid sequence 406AA-430AA (EDEGPPGYPEPLAPGHGSPFPSLSK). Spectra were acquired on a Fusion Lumos Orbitrap Mass Spectrometer and are displayed across m/z ranges of 100 to 600 m/z (upper panel), 600 to 1300 m/z (middle panel), and 1300 to 2000 m/z (lower panel). d Summary map of all MS2 fragment ions that identify the TFEB sequence 406AA-430AA and localize the phosphorylation to S427. e In vitro kinase assays. Immunoprecipitated WT TFEB-3 x Flag or TFEB S427A-3xFlag were incubated with recombinant IKKβ and analyzed as described in (a). f In vitro kinase assays. Immunoprecipitated WT TFEB-3 x Flag or TFEB S3A 3 x Flag or 3xFlag-tagged TFEB fragments were incubated with recombinant IKKβ and analyzed as described in (a). g In vitro kinase assays. Immunoprecipitated WT TFEB-3 x Flag or 3 x Flag-tagged TFEB mutants with specific amino acid substitutions were incubated with recombinant IKKβ and analyzed as described in (a). h Gas Phase MS2 sequencing of phosphorylated TFEB that occurs upon IKK activation by treatment with 20 ng/mL IL-1β for 15 minutes. Peptide 406AA-430AA exhibits clear evidence of monophosphorylation at the SPFPSLS motif. The MS2 ions best localize the phosphorylation to S423ph; however, S427ph and S429ph are also supported, likely representing a mixture. i Summary map of all MS2 fragment ions identifying the TFEB sequence 406AA–430AA and inference of phosphorylation at S423, S427, and S429. j Quantification of TFEB{S423ph} signal normalized to total signal (TFEB + TFEB{S423ph}) at 0 and 15 min. The abundance of TFEB{S423ph} is below the detection limit at 0 min. n = 3 independent experiments. Results are presented as mean ± SEM. a, b, e–g Representative images are shown from n = 3 independent experiments with similar results. p-value is calculated based on the one-tail Welch’s t test. Source data are provided as a Source Data file.

In vitro peptide screens to define IKKα/β substrate preferences, along with the identification of several bona fide substrates, have shown that IKKα and IKKβ do not have strict requirements for the amino acids surrounding their target serine, making prediction of candidate substrate sites difficult^61,63,64. To identify the TFEB residues phosphorylated by IKK, we performed experiments of mass spectrometry coupled with mutagenesis. For simplicity and owing to its ability to phosphorylate TFEB in vitro even in the absence of the other IKK subunits, IKKβ was used in these experiments. Mass-spectrometry analyses of intact TFEB showed abundant phosphorylation localized to serine 427, supported by multiple fragment ions, including fragments containing intact phosphoserine (Fig. 3c, d). Specifically, we detected an intact phosphorylated peptide spanning residues 405-430, with an m/z 881.7311 (-1.99 ppm error; expected mass, 881.732851 m/z) (Supplementary Fig. 4b). MS2 analysis (where precursor peptide ions are fragmented and analyzed to determine sequence and PTM localization) produced multiple ion types supporting phosphorylation at S427. In phos-tag analyses, alanine substitution of S427 altered the pattern of phospho-TFEB bands and reduced the phosphorylation efficiency of TFEB, resulting in higher levels of unphosphorylated TFEB-S427A compared to wild-type (WT) TFEB (Fig. 3e). To narrow down the additional sites targeted by IKK, we first tested S3—the only serine present within the N-terminal domain of TFEB—and found that alanine substitution did not affect the TFEB phosphorylation pattern (Fig. 3f). Therefore, we excluded S3 as a candidate site. We then separately analyzed the phosphorylation of TFEB fragments spanning residues 53–218 and 219–476 (Supplementary Fig. 4c). The results showed that TFEB (53-218) was not phosphorylated by IKKβ, while TFEB (219–476) exhibited multiple phosphorylation bands (Fig. 3f). These findings indicate that the additional IKK target sites are also located within the 219–476 region.

To identify these sites, we generated a library of TFEB mutants containing single alanine substitutions for each of the 24 serine residues present within the 219–476 region, and evaluated the phosphorylation status of each mutant by in vitro kinase assay. The results showed that alanine substitution of S401 and S429 also altered the phosphorylation pattern of TFEB (Supplementary Fig. 4d). The S401A/S427A double mutant exhibited a significant reduction in phosphorylation efficiency compared to WT TFEB, although a small fraction of phosphorylated protein was still detected (Fig. 3g). Alanine substitution of four additional adjacent serine residues (S397, S399, S423, and S429), together with S401 and S427 (henceforth referred to as TFEB-6xA), completely abolished TFEB phosphorylation by IKKβ (Fig. 3g) or by the reconstituted IKK complex (Supplementary Fig. 4e).

In parallel, we generated non-clonal pools of HeLa cells stably expressing 3xFlag-tagged WT TFEB (henceforth referred to as HeLa/TFEB). We immunoprecipitated TFEB under basal conditions or after a 15 min stimulus with 20 ng/mL IL-1β and proceeded with mass spectrometry analysis. The results showed abundant monophosphorylation of TFEB at the SPFPSLS motif within the 405AA-431AA peptide after a 15 min stimulus with IL-1β (Fig. 3h-j). MSFragger⁶⁵ localized this phosphorylation event to S423 with high confidence. However, manual inspection revealed that while S423 may be the most likely single site of localization, phosphorylation at S427 or S429 is not excluded by the same evidence, and both sites also show weaker but independent supporting evidence, consistent with the mutational analyses described above. Under basal conditions, the signal for the phosphorylated 405–431 aa peptide was below the detection limit. Together, the data from TFEB phosphorylation assays in vitro and in intact cells indicate phosphorylation events at neighboring serines clustered in two small regions. Interestingly, these six sites form two similar modules, each approximately the size of a typical kinase interaction surface: S397/399/401 (FSHSLSF) and S423/427/429 (SPFPSLS). Thus, we used TFEB-6xA, which abolishes all IKK-mediated phosphorylation of TFEB, to carry out functional analyses of this pathway.

IKK phosphorylation-resistant TFEB displays increased expression levels and stability

To investigate the functional consequences of abolishing IKK-mediated TFEB phosphorylation on TFEB stability, we generated non-clonal pools of HeLa cells stably expressing 3 x Flag-tagged TFEB-6xA (henceforth referred to as HeLa/TFEB-6xA). Immunoblot analyses showed that HeLa/TFEB-6xA displayed substantially higher TFEB levels than HeLa/TFEB (Fig. 4a, b), despite no significant differences in RNA expression levels between the two constructs (Fig. 4c). To test whether this effect was due to differences in protein stability, we used cycloheximide to block protein synthesis and measured degradation rates for each TFEB construct. The results showed that WT TFEB levels rapidly declined following inhibition of protein synthesis, whereas TFEB-6xA levels remained stable throughout the monitored time frame (Fig. 4d, e). These data indicate that loss of IKK phosphorylation sites increases TFEB stability. We next examined the stability of WT and 6xA TFEB under TNFα-induced activation of IKK. WT TFEB levels were reduced by 30 min of TNFα treatment, while TFEB-6xA levels remained unchanged (Fig. 4f, g). Together, these findings demonstrate that abolishment of IKK target sites increases TFEB protein expression levels and stability, indicating that IKK phosphorylation of TFEB promotes TFEB degradation.

Fig. 4. Elimination of IKK-targeted sites stabilizes TFEB and the downstream E3 ligase β-TrCP2 regulates TFEB ubiquitination.

a Immunoblot analysis of HeLa/TFEB and HeLa/TFEB-6xA using the indicated antibodies (n  =  3 independent replicates for each group). b Quantification of the immunoblots in (a), normalized to corresponding GAPDH levels. c Relative TFEB-3xFlag mRNA levels determined by RT qPCR in samples shown in panel (a). The expression levels were normalized to the housekeeping gene, GAPDH. d HeLa/TFEB and HeLa/TFEB-6xA were treated with 100 μg/ml cycloheximide (CHX), and the cells were harvested at the indicated time points. Immunoblot analyses were performed using the indicated antibodies (n  =  3 independent replicates for each group). To facilitate quantification, the ratio between the concentrations of loaded proteins for WT TFEB and TFEB-6 x A is 5:1 to take into account that TFEB-6 x A’ signal intensity is several-fold greater than that of WT TFEB. e Quantification of the immunoblots in (d), normalized to corresponding β-actin levels. f HeLa/TFEB and HeLa/TFEB-6 x A were treated with 20 ng/ml TNFα, and the cells were harvested at the indicated time points. Immunoblot analyses were performed using the indicated antibodies (n  =  3 independent replicates for each group). g Quantification of the immunoblots in (f), normalized to corresponding GAPDH levels. h HEK293T cells were cotransfected with TFEB-3 x Flag, HA-ubiquitin, and one of the indicated E3 ligases (β-TrCP1, β-TrCP2, STUB1 or DCAF7). 24 h after transfection, TFEB proteins were immunoprecipitated, and the ubiquitination levels were checked using the HA antibody. i HEK293T cells were cotransfected with HA-ubiquitin and TFEB-3xFlag. 24 h after transfection, the cells were split and subjected to a second round of transfection with scramble or FBXW11 (β-TrCP2) siRNA. After an additional 28 h, the cells were incubated with 100 nM CFZ or DMSO for 20 h before harvest. TFEB proteins were then immunoprecipitated, and ubiquitination levels were assessed using an HA antibody. h, i Representative images are shown from n = 3 independent experiments with similar results. Results are presented as mean ± SEM. p-values are calculated based on two-sided student’s t test. Source data are provided as a Source Data file.

β-TrCP2 mediates TFEB ubiquitination and stability downstream of IKK phosphorylation

IKK-mediated phosphorylation primes certain protein substrates for degradative ubiquitination by generating a phosphodegron recognized by the E3 ubiquitin ligases β-TrCP1/2^48–50. To investigate whether β-TrCPs mediate TFEB ubiquitination, we assessed TFEB ubiquitination levels upon exogenous expression, in HEK293T cells, of β-TrCP1, β-TrCP2, or the E3 ubiquitin ligases STUB1 (STIP1 homology and U-Box containing protein 1) and DCAF7 (DDB1 and CUL4 associated factor 7), which had previously been associated with TFEB degradation^66,67. Coexpression with HA-tagged ubiquitin and TFEB-3 x Flag followed by TFEB immunoprecipitation and HA immunoblotting, showed that, β-TrCP2—but not β-TrCP1—promoted TFEB ubiquitination (Fig. 4h). Next, we silenced β-TrCP2 to test whether it mediates TFEB ubiquitination. Inhibition of proteasomal degradation using low concentrations of carfilzomib (CFZ) resulted in the accumulation of ubiquitinated TFEB, which was completely prevented by β-TrCP2 (FBXW11) silencing (Fig. 4i and Supplementary Fig. 5a). These data show that β-TrCP2 functions as a ‘default’ TFEB ubiquitin ligase and indicate that TFEB tagging for proteasomal degradation is a continuously occurring process in unstimulated, standard cell culture conditions. Due to the unavailability of suitable antibodies against endogenous β-TrCP2, silencing efficiency was evaluated by monitoring endogenous FBXW11 mRNA levels (Supplementary Fig. 5a) and the protein levels of an HA-β-TrCP2 construct transiently transfected into HEK293T cells (Supplementary Fig. 5b).

Silencing of β-TrCP2 in unstimulated HEK293T cells resulted in a significant increase in endogenous TFEB levels (Fig. 5a–c), similar to the silencing of IKK subunits (Supplementary Fig. 6a–c), indicating that IKK and β-TrCP2 participate in TFEB turnover in multiple cell types. In contrast to TFEB protein levels, TFEB mRNA levels remained unchanged upon β-TrCP2 or IKK subunit silencing (Fig. 5d and Supplementary Fig. 6d), confirming that the IKK–β-TrCP2 axis affects TFEB post-transcriptionally. In contrast, DCAF7 silencing did not affect endogenous TFEB levels under the same conditions (Supplementary Fig. 6e–g). Similarly, knockdown of β-TrCP2 in WT MEFs significantly increased endogenous TFEB levels, an effect that did not occur in any of the IKK-knockout lines (Fig. 5e–g). These results demonstrate that β-TrCP2–mediated regulation of TFEB occurs downstream of IKK and that the IKK–β-TrCP2 axis regulates TFEB turnover in multiple cell types.

Fig. 5. β-TrCP2 regulates TFEB protein levels and ubiquitination downstream of IKK.

a Immunoblot analysis of HEK293T cells transiently transfected with scramble or FBXW11 siRNA (n  =  3 independent experiments) using the indicated antibodies. b Quantification of the immunoblots in (a), normalized to corresponding β-actin levels. c Relative FBXW11 mRNA levels determined by RT-qPCR in samples shown in panel (a). d Relative TFEB mRNA levels determined by RT-qPCR in samples shown in panel (a). e Immunoblot analysis of WT MEFs and MEF lines bearing targeted deletion of the genes encoding IKKα, IKKβ, or IKKγ, transiently transfected with scramble or Fbxw11 siRNA (n  =  4 independent experiments) using the indicated antibodies. f Quantification of the immunoblots in (e), normalized to corresponding GAPDH levels. g Relative Fbxw11 mRNA levels determined by RT-qPCR in samples shown in panel (e) (n  =  3 technical replicates). h Immunoblot analysis of HeLa/TFEB and HeLa/TFEB-6xA transiently transfected with scramble or FBXW11 siRNA (n  =  3 independent experiments) using the indicated antibodies. To facilitate quantification, the ratio between the concentrations of loaded proteins for WT TFEB and TFEB-6 x A is 5:1 to take into account that TFEB-6 x A’ signal intensity is several-fold greater than that of WT TFEB. i Quantification of the immunoblots in (h), normalized to corresponding β-actin levels. j Relative FBXW11 mRNA levels determined by RT-qPCR in samples shown in panel (h). k HEK293T cells were cotransfected with HA-ubiquitin and TFEB-3 x Flag or TFEB 6xA-3 x Flag. 24 h after transfection, the cells were split and subjected to a second round of transfection with scramble or FBXW11 siRNA. After an additional 48 h, TFEB proteins (WT and 6xA) were immunoprecipitated, and ubiquitination levels were assessed using an HA antibody. Representative images are shown from n = 3 independent experiments with similar results. l HeLa/TFEB were transiently transfected with scramble or FBXW11 siRNA. 48 h later, the cells were treated with 20 ng/ml TNFα for 15 minutes. Immunoblot analyses were performed using the indicated antibodies (n  =  3 independent replicates for each group). m Quantification of the immunoblots in (l), normalized to corresponding β-actin levels. n HEK293T cells were transiently transfected with scramble or FBXW11 siRNA. 72 h later, the cells were treated with 250 nM IKK-16 or DMSO for 6 h. Immunoblot analyses were performed using the indicated antibodies (n  =  3 independent replicates for each group). o Quantification of the immunoblots in (n), normalized to corresponding β-actin levels. Results are presented as mean ± SEM. p-values are calculated based on two-sided student’s t test. Source data are provided as a Source Data file.

Next, we investigated mechanistically whether β-TrCP2-mediated modulation of TFEB levels requires the IKK-targeted phosphodegron by testing the effects of β-TrCP2 silencing on the IKK-resistant TFEB-6xA construct. To enable comparison with WT TFEB, we silenced β-TrCP2 in both HeLa/TFEB and HeLa/TFEB-6xA. The results showed that β-TrCP2 silencing nearly doubled WT TFEB levels in standard cell culture conditions, while having only a minimal effect on TFEB-6xA levels (Fig. 5h–j). We next tested whether IKK-mediated phosphorylation of TFEB is necessary for its ubiquitination by β-TrCP2. To this aim, we performed sequential transfections by first introducing either WT TFEB or TFEB-6xA constructs along with HA-tagged ubiquitin, and subsequently transfecting the cells with siRNAs targeting FBXW11 or scrambled controls. Immunoprecipitation of TFEB followed by HA immunoblotting showed that IKK-resistant TFEB-6xA exhibited markedly lower levels of ubiquitination compared to WT TFEB (Fig. 5k). As expected, β-TrCP2 silencing abated the ubiquitination of WT TFEB but had no effect on TFEB-6xA (Fig. 5k and Supplementary Fig. 5c). Remarkably, TFEB-6xA treated with scramble siRNAs displayed ubiquitination levels similar to those of WT TFEB upon β-TrCP2 silencing, indicating that removal of IKK-targeted phosphorylation sites from TFEB mimics a lack of engagement of β-TrCP2. Furthermore, β-TrCP2 silencing abrogated TNFα-induced degradation of TFEB (Fig. 5l, m) as well as IKK-16-induced increase in TFEB levels (Fig. 5n, o), further demonstrating that IKK regulates TFEB degradation in a β-TrCP2–dependent manner.

Collectively, these findings demonstrate that β-TrCP2 ubiquitinates TFEB downstream of IKK phosphorylation in both unstimulated and stimulated cells, presumably to maintain tight control of TFEB levels.

TFEB ubiquitination and degradation is mediated by phosphodegron-adjacent lysines

β-TrCP proteins interact with their substrates via a phosphodegron that contains two phosphorylated serine residues separated by three or four amino acids [pSxxx(x)pS]⁶⁸. The canonical phosphodegron targeted by β-TrCP has a DpSGxx(x)pS consensus sequence⁶⁸, but β-TrCP also interacts with non-canonical phosphodegrons featuring different amino acids around the two phosphoserines^69–71. Lysine residues that undergo ubiquitination are typically located in proximity to their cognate phosphodegrons^72–74. Although TFEB does not contain the canonical β-TrCP phosphodegron, serines 423 and 427 are separated by three amino acids (⁴²³SPFPS⁴²⁷) and lie adjacent to two consecutive lysines, K430 and K431 (Fig. 6a). Mass-spec analysis confirmed ubiquitination at K430 and K431 in cells expressing TFEB-3×Flag and ubiquitin (Supplementary Fig. 7a–e). To test whether these lysines are the sites of β-TrCP-mediated ubiquitination, we generated HeLa cells stably expressing a TFEB mutant in which both lysines were replaced by arginine residues (HeLa/TFEB-KKRR). Notably, compared with HeLa/TFEB, HeLa/TFEB-KKRR showed markedly elevated TFEB expression (Supplementary Fig. 8a, b), despite no differences in mRNA levels between the two transgenes (Supplementary Fig. 8c). Thus, similar to the IKK-resistant TFEB-6xA construct, the increase in TFEB-KKRR protein levels must result from a post-translational mechanism.

Fig. 6. β-TrCP2 targets lysine residues adjacent to the IKK phosphodegron.

a Consecutive lysine residues K430 and K431 are adjacent to the TFEB phosphodegron ⁴²³SPFPSLS⁴²⁹. b Immunoblot analysis of HeLa/TFEB and HeLa/TFEB-KKRR transiently transfected with scramble or FBXW11 siRNA (n  =  3 independent experiments) using the indicated antibodies. c Quantification of the immunoblots in (b), normalized to corresponding GAPDH levels. d Relative FBXW11 mRNA levels determined by RT-qPCR in samples shown in panel (b). The expression levels were normalized to the housekeeping gene, HPRT1. e Immunoblot analysis of HeLa/TFEB and HeLa/TFEB-KKRR transiently transfected with myc-β-TrCP2 (n  =  3 independent experiments) using the indicated antibodies. To facilitate quantification, the ratio between the concentrations of loaded proteins for WT TFEB and TFEB-KKRR is 5:1 to take into account that TFEB-KKRR’ signal intensity is several-fold greater than that of WT TFEB. f Quantification of the immunoblots in (e), normalized to corresponding GAPDH levels. g HEK293T cells were cotransfected with HA-ubiquitin and TFEB-3xFlag or TFEB-KKRR-3xFlag. 24 h after transfection, the cells were split and subjected to a second round of transfection with scramble or FBXW11 siRNA. After an additional 48 hours, TFEB proteins (WT and KKRR) were immunoprecipitated, and ubiquitination levels were assessed using an HA antibody. h Relative FBXW11 mRNA levels determined by RT-qPCR in samples shown in panel (g). The expression levels were normalized to HPRT1 (n  =  3 technical replicates). i HeLa cells were cotransfected with HA-ubiquitin, myc-β-TrCP2 or pcDNA, and TFEB-3 x Flag or TFEB-KKRR-3 x Flag. 24 h after transfection, TFEB proteins were immunoprecipitated, and the ubiquitination levels were checked using the HA antibody. Representative images are shown from n = 3 independent experiments with similar results. Results are presented as mean ± SEM. p-values are calculated based on two-sided student’s t test. Source data are provided as a Source Data file.

A cycloheximide chase assay demonstrated that mutation of the phosphodegron-adjacent lysines increased TFEB protein stability (Supplementary Fig. 8d), again mimicking the effect of mutating the serines within TFEB’s phosphodegron (see Fig. 4d). A comparison of protein levels from WT TFEB, TFEB-6xA, TFEB-KKRR, and a phospho-mimetic TFEB-6xE mutant showed that 6xA and KKRR protein levels were similar to each other, whereas the levels of the phospho-mimetic mutant were comparable to those of WT TFEB—albeit slightly increased, presumably because the glutamic acid residues do not fully mimic phosphorylation (Supplementary Fig. 8e). IKK inhibition increased the levels of WT TFEB but had no effect on any of the mutants, consistent with the conclusion that IKK acts through the identified phosphorylation and ubiquitination sites (Supplementary Fig. 8e, f).

Notably, TFEB-KKRR was insensitive to β-TrCP2 silencing (Fig. 6b–d), mimicking the behavior of TFEB-6xA. Overexpression of β-TrCP2 decreased TFEB protein levels, and this effect was abolished by arginine substitution of the two lysines (Fig. 6e, f). In contrast, overexpression of DCAF7 had no effect on the protein levels of either construct (Supplementary Fig. 9a, b). A ubiquitination assay showed that arginine substitution also reduced TFEB ubiquitination to levels comparable to those observed for WT TFEB upon β-TrCP2 silencing (Fig. 6g, h). Furthermore, neither silencing nor overexpression of β-TrCP2 altered the ubiquitination levels of TFEB-KKRR (Fig. 6g–i), confirming that β-TrCP2-mediated ubiquitination of TFEB requires these phosphodegron-adjacent lysines. In comparison, arginine substitution of the two lysines did not affect DCAF7-induced changes in TFEB ubiquitination levels (Supplementary Fig. 9c). Of note, both TFEB-6xA and TFEB-KKRR exhibited minimal ubiquitination upon proteasome blockade (Supplementary Fig. 10), indicating that the IKK–β-TrCP2-mediated phosphorylation-ubiquitination cascade is the primary pathway regulating TFEB proteasomal degradation.

To directly link IKK-mediated phosphorylation of TFEB to regulation of its stability via the phosphodegron-adjacent lysines, we monitored the protein levels of WT TFEB and TFEB-KKRR following siRNA-m ediated silencing of IKK. Immunoblotting showed WT TFEB levels increased upon silencing of IKKγ and IKKβ in unstimulated cells, whereas TFEB-KKRR levels remained unchanged (Fig. 7a, b and Supplementary Fig. 8g, h). IKK activation by IL-1β induced a rapid decrease in WT TFEB protein levels, an effect that was not observed for either TFEB-6xA or TFEB-KKRR (Fig. 7c, d). Furthermore, the IL-1β–induced acute decrease in TFEB levels was abolished by proteasomal inhibition, indicating that IL-1β promotes TFEB degradation through the proteasome (Fig. 7e, f). Similarly, LPS treatment reduced WT TFEB levels but had no effect on TFEB-6xA or TFEB-KKRR (Fig. 7g, h). It is worth noting that LPS treatment also decreased IκBα levels (Fig. 7i), confirming IKK activation. These results indicate that IL-1β and LPS-induced degradation of TFEB require the IKK-targeted phosphodegron.

Fig. 7. Elimination of IKK or β-TrCP2 target sites stabilizes TFEB and renders it resistant to IKK.

a Immunoblot analysis of HeLa/TFEB and HeLa/TFEB-KKRR transiently transfected with scramble or IKBKG siRNA (n  =  3 independent experiments) using the indicated antibodies. b Quantification of the immunoblots in (a), normalized to corresponding GAPDH levels. c HeLa/TFEB, HeLa/TFEB-KKRR and HeLa/TFEB-6xA were treated with 20 ng/ml IL-1β, and the cells were harvested at the indicated time points. Immunoblot analyses were performed using the indicated antibodies (n  =  4 independent replicates for each group). a, c to facilitate quantification, the ratio between the concentrations of loaded proteins for WT TFEB and TFEB mutants is 5:1 to take into account that TFEB mutants’ signal intensity is several-fold greater than that of WT TFEB. d Quantification of the immunoblots in (c), normalized to corresponding GAPDH levels. e HeLa/TFEB were treated with CFZ or DMSO. 7 hours later, the cells were treated with 20 ng/ml IL-1β for 30 min. Immunoblot analyses were performed using the indicated antibodies (n  =  3 independent replicates for each group). f Quantification of the immunoblots in (e), normalized to corresponding GAPDH levels. g MEF/TFEB, MEF/TFEB-6xA and MEF/TFEB-KKRR were treated with LPS at the specified concentrations for 48 h, and the cells were harvested for immunoblot analyses using the indicated antibodies (n  =  3 independent experiments). h, i Quantification of the immunoblots in (g), normalized to corresponding GAPDH levels. Results are presented as mean ± SEM. p-values are calculated based on two-sided student’s t test. Source data are provided as a Source Data file.

Together, these results show that TFEB levels are regulated by a phosphorylation-ubiquitination cascade mediated by IKK and β -TrCP2 under both basal and IKK-activating conditions.

Inactivation of TFEB degron does not interfere with TFEB nuclear translocation and activation of clearance pathways

Next, we investigated whether degron-dependent modifications of TFEB affect its nuclear translocation or its ability to modulate clearance pathways. To this aim, we first tested whether TFEB-6xA and TFEB-KKRR remain responsive to starvation and mTORC1 inhibition, both of which are known to promote TFEB nuclear translocation. Confocal microscopy of HeLa/TFEB, HeLa/TFEB-6xA, and HeLa/TFEB-KKRR cells showed that inactivation of either component of the TFEB degron did not alter its responsiveness to starvation or mTORC1 inhibition (Fig. 8a, b)—a result confirmed by immunoblotting following subcellular fractionation (Fig. 8c).

Fig. 8. Phosphorylation and ubiquitination of TFEB degron do not interfere with TFEB nuclear translocation and transcriptional capabilities.

a HeLa/TFEB, HeLa/TFEB-6xA and HeLa/TFEB-KKRR were treated with DMSO, Torin1, or starved for amino acids (HBSS) for 4 hours prior to immunofluorescent labeling of TFEB with Flag antibody. Representative images are shown. Scale bar: 30 μm. b Quantification of TFEB_NTI of cells under the above-mentioned three conditions in (a). n = 30 cells per condition. One-way ANOVA with Tukey’s multiple comparisons test. c HeLa/TFEB, HeLa/TFEB-6xA and HeLa/TFEB-KKRR treated as in (a) were fractionated into cytosol and nuclei. Immunoblot analyses of each fraction was performed with the indicated antibodies. Representative images are shown from n = 3 independent experiments with similar results. d HEK293T cells were transfected with 3xFlag tagged WT TFEB, TFEB-6 x A or TFEB-KKRR. 24 hours after transfection, immunoblot analyses were performed with the indicated antibodies (n  =  3 independent experiments) (upper); quantification of TFEB-6xA and KKRR levels, respectively, relative to WT TFEB levels (lower). Two-sided student’s t test. e Expression analysis of cells in (d). Gene expression levels were normalized to the housekeeping gene, GAPDH. MCOLN1, ATP6V1H, GNS and LAMP1 (n = 3 per group); SCPEP1, NEU1, CTSA and PIP4P1 (pcDNA, n = 3; WT/6xA/KKRR, n = 4). Multiple two-sided t tests with Benjamini–Hochberg FDR correction. f HEK293T cells were transfected with 3 x Flag tagged WT TFEB, TFEB-6 x A or TFEB-KKRR, together with V5 tagged full length human tau with P301L mutation. 48 h after transfection, immunoblot analyses were performed with the indicated antibodies (n  =  6 independent replicates for each group). g Quantification of total tau, phospho-tau and TFEB levels, respectively. One-way ANOVA with Tukey’s multiple comparisons test. Results are presented as mean ± SEM. Source data are provided as a Source Data file.

Next, we tested the ability of the three constructs to promote transcription of TFEB target genes. To allow a fair comparison, we did not use the stable cell lines, as they express TFEB proteins at markedly different levels despite similar mRNA levels, as shown above. We noticed that transiently expressed WT TFEB, TFEB-6xA, and TFEB-KKRR exhibited similar protein levels when standard amounts of plasmids were transfected (Fig. 8d), whereas the protein levels of the IKK- and β-TrCP2-insensitive TFEB mutants were far greater than those of WT TFEB when lower amounts of plasmid were used (Supplementary Fig. 11a, b). This difference likely reflects saturation of IKK/β-TrCP2 or their associated machineries under conditions of high protein expression, as standard transient transfection results in higher TFEB levels than those achieved in stable clones (see Supplementary Fig. 1). Thus, to compare the transcriptional responses of the three TFEB constructs, we transfected standard amounts of plasmids. RT-qPCR analysis of RNA from transiently transfected cells showed that all three constructs similarly enhanced the transcription of TFEB target genes, with TFEB-KKRR exhibiting a slightly stronger effect (Fig. 8d, e).

We have previously shown that exogenous TFEB expression enhances tau fibril uptake and reduces tau spreading in cell lines and mouse models of Alzheimer’s disease^28,31. To assess the potential of phosphorylation/ubiquitination-resistant TFEB constructs to attenuate tauopathy, we cotransfected HEK293T cells with a V5-tagged tau-P301L construct along with WT TFEB or either TFEB mutant construct. Quantification of total tau and phospho-tau (PHF1) levels under comparable expression levels of the three TFEB constructs showed similar overall efficiencies in promoting the degradation of both total tau and phospho-tau, with the two TFEB mutants being slightly more effective at reducing total tau levels (Fig. 8f, g). Taken together, these results indicate that the TFEB modifications involved in the phosphorylation-ubiquitination cascade are functionally distinct from those that control TFEB nuclear translocation, transcriptional activity, and clearance function.

Discussion

Over the past decade, the lysosome has gained increased recognition as a central signaling hub that modulates cell metabolism by processing information regarding the cell’s nutritional status and its endogenous or exogenous stressors⁷⁵, substantially enriching the historical view of the lysosome as the primary site of cellular catabolism^76–80. TFEB is arguably a key player in this emerging signaling role and the prime effector of lysosome-to-nucleus communication^29,81–83. TFEB activity is directly controlled by mTORC1 and other kinases that sit at major signaling nodes in cell metabolism and the cell cycle^{9–16,20–23,84,85}. Owing to the observed disruption of lysosomal homeostasis in the pathogenesis of late-onset neurodegenerative diseases, TFEB has been tested as a tool to counteract disease progression by modulating cellular clearance^29,30,86,87. Exogenous expression of TFEB in animal models of disease has indeed demonstrated TFEB’s ability to ameliorate disease phenotypes relevant to Alzheimer’s disease^26,28,31,88 and Parkinson’s disease^89–91, among others. Given that a pharmacologically viable route to enhance the activity of endogenous TFEB has yet to be established, defining the key mechanisms that regulate TFEB levels and function is critically important to lay the foundation for the clinical translation of TFEB-mediated enhancement of cellular clearance.

In this study, we demonstrate that TFEB is targeted for degradation through a phosphorylation-ubiquitination cascade mediated by the IKK–β-TrCP2 axis (Fig. 9). Specifically, we identify an IKK-generated phosphodegron recognized by β-TrCP2, which ubiquitinates adjacent lysine residues (K430 and K431), leading to TFEB proteasomal degradation. This study provides the molecular definition of a specific phosphorylation-dependent degron that directly controls TFEB stability. These findings uncover that TFEB undergoes proteasomal degradation in both unstimulated and stimulated conditions, with a mechanism that is distinct from previously characterized pathways governing TFEB’s nuclear translocation and transcriptional activity^92,93. Mass spectrometry analyses have revealed that IKK targets a serine-enriched peptide (⁴²³SPFPSLS⁴²⁹), with S423 and S427 being the most abundantly phosphorylated residues according to parallel analyses in cell and in vitro. It is worth mentioning that these residues lie within a very large ( ~ 6 kDa), highly acidic (pI ~ 4) peptide fragment that remains refractory to proteomic methods. Additional mutagenesis and functional assays have indicated that a second, nearby serine-enriched peptide (³⁹⁷SHSLS⁴⁰¹) is also targeted by IKK. Remarkably, simple genetic abolishment of the modifications mediated by IKK and β-TrCP2 increases TFEB protein levels by several fold, even in the absence of any external stimuli, indicating that TFEB degradation is a continuously occurring process. Our data also show a complete functional separation between TFEB stability (regulated by IKK–β-TrCP2) and its nuclear localization (regulated by mTORC1). Pharmacological inhibition of mTORC1 with Torin1 induces TFEB nuclear translocation without increasing TFEB protein levels, whereas phosphodegron-inactivated TFEB accumulates to high levels but remains fully sensitive to mTORC1-dependent nuclear exclusion. This dual regulatory architecture is expected to allow precise, context-specific modulation of TFEB function to respond to cellular needs dictated by metabolic or inflammatory conditions.

Fig. 9. Schematic of the proposed phosphorylation/ubiquitination/degradation mechanism.

IKK phosphorylates TFEB, triggering β-TrCP2-mediated ubiquitination and leading to TFEB degradation by the proteasome.

Our data show that genetic inhibition of IKK activity enhances transcription of TFEB target genes and expands the lysosomal compartment and its degradative capacity. The finding that IKK regulates lysosomal biogenesis and function through controlling TFEB abundance significantly widens the functional repertoire of the IKK complex and underscores its broader role in linking inflammatory signaling with metabolic control. While IKK is traditionally recognized for its canonical role in NF-κB activation, emerging evidence indicates that various stimuli, including inflammatory signals and lysosomal damage, recruit inflammatory signaling molecules—including IKK—to the lysosomal surface to modulate NF-κB and mTORC1-dependent signaling^94–97. This supports the notion that the lysosome may serve as a physical platform for both metabolic and inflammatory signal integration. This spatial proximity could facilitate coordinated regulation of TFEB and mTORC1 by IKK-dependent phosphorylation and lysosome-associated signaling. IKKβ has indeed been reported to activate mTORC1 in response to TNFα stimulation through two converging mechanisms: direct phosphorylation of mTOR itself, and phosphorylation of TSC1, a negative regulator of mTORC1^32–34. IKK therefore emerges as a bidirectional regulator of lysosomal function and inflammation, with the IKK–β-TrCP2–TFEB cascade mirroring the classical IKK–β-TrCP–IκBα pathway in which phosphorylated IκBα is recognized by β-TrCP and degraded, liberating NF-κB dimers to enter the nucleus⁹⁸. Our data show that genetic knock-out or knock-down of IKK subunits results in a robust increase in TFEB protein levels even in the absence of external stimuli, indicating a fundamental role for IKK in maintaining basal TFEB protein abundance. Since active IKK enhances the function of mTORC1, which in turn phosphorylates TFEB, promoting its cytosolic sequestration, an integrated model of IKK regulation of TFEB emerges in which IKK can exert a dual inhibitory effect on TFEB function when fully active—driving its proteasomal degradation through β-TrCP2-mediated ubiquitination and promoting its cytosolic retention through mTORC1. Interestingly, prior work has shown that neuron-specific deletion of IKKβ in APP/PS1 or tau transgenic mice reduces amyloid plaque deposition and phosphorylated tau levels, respectively⁹⁹, effects recapitulated by AAV-mediated expression of TFEB in similar models by other studies¹⁰⁰. Neuronal deletion of IKKβ resulted in upregulation of the autophagy flux, indicating that active IKK signaling normally limits autophagy⁹⁹. Thus, the finding that IKK primes TFEB for degradation may offer insight into how IKKβ modulates autophagy and cellular clearance, indicating that direct regulation of TFEB by IKK could play a significant role in these processes.

An interesting question is why a key effector of inflammatory pathways, such as IKK, has a direct role in suppressing TFEB. This may relate to the temporal regulation of autophagy during inflammation. Findings across multiple systems (including macrophages, microglia, and epithelial cells) have indeed shown that the role of autophagy in inflammation changes over time, with an interplay between autophagy and immune signaling ensuring a robust initial inflammatory response followed by resolution to restore tissue homeostasis¹⁰¹. In the early stages of inflammation, autophagy is often actively suppressed. Autophagy typically restrains inflammation by eliminating pro-inflammatory substrates—it limits NLRP3 inflammasome activation and sequesters and degrades pro-cytokines, reducing their maturation and secretion^102,103. Notably, IKKβ itself is targeted for selective autophagic degradation via p62, a process that suppresses NF-κB activation and inflammatory cytokine production¹⁰⁴. Thus, autophagy generally acts as a brake on inflammation. Conversely, blocking autophagy or autophagic targeting of IKKβ amplifies inflammatory signaling^102–104. Indeed, an early autophagic block has been observed in various models of acute inflammatory responses, where mTORC1 activation is associated with decreased autophagy markers, sustained NF-κB activity, and increased cytokine production^105–107. Importantly, this early autophagic suppression can also be exploited by pathogens: for instance, Salmonella stimulates mTORC1 to acutely suppress autophagy and aid bacterial survival within macrophages¹⁰⁸. As inflammation progresses towards resolution, the balance shifts and autophagy is upregulated to promote recovery. Changes in the cellular environment, including energy stress and damage signals, lead to the activation of AMPK and other stress-responsive kinases that antagonize mTOR, thereby lifting autophagic suppression¹⁰¹. This delayed activation of autophagy facilitates clearance of inflammasome complexes, curbing further cytokine secretion¹⁰⁹. The resolution phase of inflammation is also driven by mediators that include lipids capable of triggering autophagy in immune cells to clear debris (such as apoptotic cells) and restore tissue homeostasis¹¹⁰.

TFEB occupies an ideal position in this interplay between the two systems. TFEB is known to exert context-dependent inflammatory roles¹¹¹. It promotes the expression of pro-inflammatory cytokines in macrophages upon stimulation^59,112–114, but it can also suppress IKK activity, restraining NF-κB activation¹¹⁵, and induce expression of PPAR-α and PGC-1α, which exert anti-inflammatory effects by promoting mitochondrial biogenesis and reducing reactive oxygen species production^11,116. IKK-driven TFEB degradation might therefore remove a brake on IKK itself, promoting the inflammatory response in its early phase. This negative feedback loop between IKK and TFEB, therefore, provides an essential mechanistic link to the interplay between inflammation and autophagy pathways by connecting their two key effectors.

Our findings also broaden the scope of β-TrCP2 function, a factor that serves as an integrator of inflammatory, metabolic, and catabolic programs. In addition to TFEB, β-TrCP2 regulates the turnover of other critical proteins involved in lysosome function and autophagy, including DEPTOR and REDD1⁵¹. β-TrCP2 is also a key regulator of SREBP, a master transcription factor for lipid homeostasis¹¹⁷. Given that both TFEB and SREBP respond to nutrient availability and drive major transcriptional programs governing metabolic adaptation, the identification of β-TrCP2 as a common regulator places it at the intersection of clearance, lipid metabolism, and inflammation. TFEB does not appear to be a substrate of the β-TrCP2 paralog β-TrCP1, possibly due to the predominantly nuclear localization of β-TrCP1^118,119, which may preclude interaction with cytosolic phosphodegron-bearing TFEB. It is interesting to note that the TFEB phosphodegron identified here is conserved across vertebrates, but is not present in other members of the microphthalmia/transcription factor E (MiT/TFE) family, such as TFE3 or MITF¹²⁰. This feature distinguishes this phosphodegron from other MiT/TFE signaling modules^{12–14,16,23} and may open opportunities for TFEB-specific therapeutic regulation without interfering with related transcription factors that have roles in melanocyte biology, immune regulation, or oncogenesis.

In summary, this study provides the molecular characterization of a phosphorylation-ubiquitination cascade that targets TFEB for proteasomal degradation. This mechanism operates independently of TFEB nuclear translocation and does not intersect with the control of its transcriptional activity. The involvement of IKK and β-TrCP2 as the executioners of this cascade reveals a crucial regulatory mechanism that integrates lysosomal biogenesis, inflammatory signaling, and cellular metabolism. These findings further expand the scope of molecular signaling at the lysosome, supporting its role as an integrative platform to coordinate inflammatory responses and autophagy. By defining a distinct and pharmacologically accessible layer of TFEB regulation, our findings offer critical insight into TFEB and IKK biology and lay the groundwork for therapeutic strategies aimed at modulating their function. This has particular relevance for neurodegenerative diseases, where impaired autophagy and lysosomal dysfunction contribute to pathological protein accumulation. TFEB’s role in promoting clearance of aggregated proteins, including pathogenic tau and amyloid-beta, indeed positions it as a prime therapeutic target. Our demonstration that selective suppression of the IKK–β-TrCP2 axis markedly enhances TFEB stability, lysosomal biogenesis, and tau clearance suggests therapeutic avenues that could simultaneously dampen neuroinflammation and enhance proteostasis. As pharmacological inhibitors of IKK are available¹²¹, these findings identify a therapeutic avenue to modulate TFEB in diseases characterized by abnormal lysosomal function and accumulation of undegraded material.

Methods

Eukaryotic cell lines

HeLa cells (female; human cervical carcinoma; ATCC, CCL-2) and HEK293T cells (female; human embryonic kidney; ATCC, CRL-3216) were purchased from ATCC. Wildtype, IKKα^−/−, IKKβ^−/− and IKKγ^–/–MEFs were kindly provided by Dr. Michael Karin’s laboratory (University of California at San Diego), the sex of these MEF lines was not determined. HeLa/TFEB-GFP, HeLa/TFEB, HeLa/TFEB-6xA and HeLa/TFEB-KKRR stable cell lines were generated from parental HeLa cells using the corresponding expression constructs following standard stable cell line generation protocols. MEF/TFEB, MEF/TFEB-6xA and MEF/TFEB-KKRR stable cell lines were generated analogously from parental WT MEFs.

Cell culture

HeLa and HEK were cultured in DMEM (Cat# 10013CV, Corning™), supplemented with 10% inactivated FBS (Cat# 16140071, Gibco), 2 mM L-glutamine (Cat# 25030081, Life Technologies), and Penicillin/streptomycin (100 μ/mL) (Cat# 15140122, Life Technologies). MEFs were cultured in DMEM supplemented with 15% inactivated FBS, 2 mM L-glutamine and Penicillin/streptomycin (100 μ/mL). All cells were maintained at 37 °C and 5% CO2.

Lentivirus infection and stable cell lines

HEK293T Cells were plated at a density of 3.5 × 10⁶ per 60 mm diameter dish 18 h prior to transfection. HEK293T packaging cells were cotransfected with pLenti expression construct, packaging vector psPAX2 (Addgene#12260) and envelope vector pMD2.G (Addgene #12259) using Lipofectamine™ 3000 Transfection Reagent (Cat# L3000001, Thermo Fisher Scientific). The lentiviruses were harvested 52 hr after transfection. Virus particles were purified by centrifugation at 2000 × g for 10 min, followed by filtration through a 0.22 μm pore size syringe filter. For viral transduction, HeLa cells and MEFs with a C57BL/6 background (Dr. Mark Sands’ laboratory, Washington University in St. Louis) were seeded at 50–60% confluence and treated overnight with the medium containing freshly harvested lentivirus in the presence of 8 μg/mL polybrene (Cat# TR-1003-G, MilliporeSigma). Selection of stable clones was carried out using puromycin (Cat# A1113802, Thermo Fisher Scientific).

Antibodies

Primary antibodies: Antibodies to TFEB (E5P9M) (Cat# 83010S) (1:1000), Flag (D6W5B) (Cat# 14793) (1:1000), β-actin (Cat# 3700S) (1:20,000), HA (C29F4) (Cat# 3724S) (1:1000), α-tubulin (Cat# 2144S) (1:2000), Histone H3 (Cat #9715S) (1:30,000), IKKγ Kinase (DA10-12) (Cat# 2695 T) (1:1000), Phospho-S6 Ribosomal Protein (Ser240/244) (Cat# 2215S) (1:10,000), S6 Ribosomal Protein (5G10) (Cat# 2217S) (1:2000), 4E-BP1 (53H11) Rabbit Monoclonal Antibody (Cat #9644) (1:1000) and phospho-4E-BP1 (Ser65) Antibody (Cat #9451S) (1:1000) were from Cell Signaling Technology; antibodies to IKKβ Kinase (Cat# ab124957) (1:5000), IKKα Kinase (Cat# ab32041) (1:5000), IKKγ Kinase [EPR16629] (Cat# ab178872) (1:5000) and WDR68 (Cat# ab138490) (1:5000) were from Abcam; antibodies to GAPDH (Cat# 60004-1-Ig) (1:150,000) and IκBα (Cat# 10268-1-AP) (1:2000) were from Proteintech; antibodies to Flag (produced in mouse) (Cat# F1804) (1:5000), Flag (produced in Rabbit) (Cat# F7425) (1:2000) and c-Myc (Cat# C3956) (1:1000) were from Sigma-Aldrich; antibodies to TrCP1 (C-6) (Cat# sc-390629) (1:1000), STUB1 (Cat# sc-133066) (1:1000) and Lamp1 (Cat# sc-19992) (1:1000) were from Santa Cruz; antibodies to V5 (Cat# MCA1360GA) (1:1000) was from Bio-Rad; antibodies to PHF1 antibody (1:1000) was a gift from Dr. Peter Davies (Feinstein Institute for Medical Research).

Secondary antibodies: Goat anti-mouse HRP-conjugated antibody (Cat# 1705047) (1:2000) and Goat anti-rabbit HRP-conjugated antibody (Cat# 1705046) (1:2000) were from Biorad, IRDye® 680RD Goat anti-Mouse IgG Secondary Antibody (Cat #926-68070) (1:10000) and IRDye® 800CW Goat anti- Rabbit IgG Secondary Antibody (Cat #926-32211) (1:10000) were from LI-COR Biosciences, Goat anti-mouse antibody, Alexa Fluor™ 555 (Cat# A-21422) (1:400) and Goat anti-mouse antibody Alexa Fluor™ 488 (Cat# A-11001) (1:400) were from Invitrogen.

Reagents

Torin 1 (Cat# 424710) was from Tocris; DMSO (Cat# D8418), Tumor Necrosis Factor-α (TNFα) (Cat# H8916), cycloheximide (Cat# 01810-1 G) and lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Cat# L4391-1MG) were from Sigma-Aldrich; carfilzomib (Cat# 17554) was from Cayman Chemical; Protease Inhibitor Cocktail (Cat# P3100-010) and Phosphatase Inhibitor Cocktail (Cat# P3200-010) were from Gendepot. Opti-MEM (Cat# 31985070) and HBSS (Cat# 14025092) were from Gibco. Recombinant mouse IL-1β/IL-1F2 protein (Cat# 401-ML-005/CF), recombinant mouse TNFα protein (Cat# 410-MT-010), recombinant human TNFα protein (Cat# 210-TA-005) and recombinant human IL-1β/IL-1F2 protein (Cat# 201-LB-005/CF) were from R&D systems. DQ Green BSA (Cat #D12050) was from Life Technologies.

Primers

RT–qPCR primers were obtained from published studies or designed using the PrimerQuest™ Tool (Integrated DNA Technologies, IDT) based on reference gene sequences. Primer specificity was verified using NCBI Primer-BLAST. Primers for In-Fusion cloning were designed using the In-Fusion Cloning Primer Design Tool (Takara Bio), and mutagenesis primers were designed using NEBaseChanger (New England Biolabs). All oligonucleotides were synthesized by MilliporeSigma. Sequences of all primers are provided in Supplementary Data 2.

Plasmids

CMV-TFEB-3 × Flag: Human TFEB was expressed from a pcDNA3.1(+)-based mammalian expression vector containing a C-terminal 3 × Flag tag under control of the CMV promoter. The vector confers ampicillin resistance for bacterial selection and neomycin resistance for mammalian selection. pCDH-EF1-TFEB-GFP: A third-generation self-inactivating lentiviral transfer vector containing the EF-1α promoter (with intron A) was used to express human TFEB as a C-terminal Myc–EGFP fusion. CMV-TFEB (aa53–218)-3 × Flag, CMV-TFEB (aa219–476)-3 × Flag, Lenti-TFEB-3  ×Flag, β-TrCP2, DCAF7, and STUB1 were synthesized by Twist Bioscience. TFEB_6 x A-3 × Flag and TFEB_6xE-3 × Flag were synthesized by Twist Bioscience and cloned into a pcDNA3.1-based expression vector using In-Fusion® Snap Assembly Master Mix (Cat# 638948, Takara Bio). Myc-β-TrCP2, Lenti-TFEB-6xA-3 × Flag, and Lenti-TFEB-KKRR-3 × Flag were generated using In-Fusion® Snap Assembly Master Mix (Cat# 638948, Takara Bio). Other TFEB mutants were generated from the CMV-WT-TFEB-3 × Flag construct using the Q5® Site-Directed Mutagenesis Kit (Cat# E0554, New England Biolabs) according to the manufacturer’s instructions. All plasmids were verified by Sanger sequencing prior to use. Detailed information on all plasmids is provided in Supplementary Data 2.

Human kinome screen

All drug treatment, washing, fixing and Hoechst staining steps were performed using the SAMI-integrated automated screening system (Beckman Colter). HeLa/TFEB-GFP were seeded at a density of 7000 cells per well in 96-well plates using a Multidrop and incubated at 37 °C in a humidified 5% CO₂ incubator for 12 h prior to treatment. On each plate, four wells were seeded with parental HeLa cells for imaging background controls, eight wells of cells were treated with 0.1% DMSO as drug vehicle controls, and four wells of cells were treated with 250 nM Torin1 as nuclear translocation positive controls. For the remaining wells, each well was treated with one of the 436 drugs within the L1200 library from SelleckChem. Each drug was applied under five different conditions: 10 μM for 6 hours, 3 μM for 6 h, 1 μM for 6 h 10 μM for 1 hour, and 3 μM for 1 hour. After treatment, cells were fixed with 4% paraformaldehyde at room temperature for 20 min and incubated with 2 μM Hoechst 33342 overnight at 4 °C. The cells were washed with PBS, and the plates were sealed. Imaging was performed using the IN Cell 2000 Analyzer (GE Healthcare Life Sciences) with FITC (for TFEB-GFP) and DAPI (for nuclei) channels at 20X magnification. Sixteen fields were captured for each well. The acquired images were analyzed with the IN Cell 1000 Workstation analyzer software. DAPI channel images were used to segment the nuclei and identify individual cells for TFEB-GFP intensity measurements. Intensity analysis was conducted by measuring the average intensity of the whole cell. Drugs that yielded intensities greater than the average intensity for all 436 drugs plus four times the standard deviation were considered as outliers and were excluded from further analysis. The Multi-Target Analysis Module was used to measure the average nuclear intensity (N) and the average intensity within a 2 µm collar (C) surrounding the nucleus, as the cytoplasmic sampling region.

Kinome data analysis

TFEB nuclear translocation index (NT index) was calculated as N/(N + C), which ranges from 0 to 1, and was divided into 100 equal intervals. The frequencies of cells falling into each interval were calculated, resulting in 100 data points for each drug, which constitute the distribution curve for each drug and are referred to as the frequency data points. To assess the similarity of the 436 distribution curves to the control distribution curves of DMSO and Torin1, a heatmap was generated using Python. The similarity was quantified using the formula: D_DMSO * D_Torin1, where D_DMSO represents the similarity to the DMSO control curves, and DTorin1 represents the similarity to the Torin1 control curves. In addition, intensity analysis was conducted by measuring the average intensity of the whole cell. To cluster components based on the frequency data points, several steps were taken to ensure proper normalization and clustering. Initially, min-max normalization was applied to each drug’s frequency data points. This step scaled the values to the range of 0 to 1. Subsequently, we applied quantile normalization to the control components’ data (DMSO and Torin1), which rescaled the frequency data points to a unified scale among all controls and mitigated variations in measurement scales. Following this, frequency data points of all other drugs were adjusted. This adjustment was performed based on the average impact of the quantile normalization on the control data for the associated plates. We then combined the data from different conditions into a single data table for ease of analysis.

To identify the relationships between drugs, we computed cosine similarities (distances). These values were utilized in hierarchical clustering, where we implemented single-linkage clustering. In this methodology, the distance between clusters is defined as the shortest distance between any two points in the clusters. In the resulting matrix, each entry signifies the correlation value between the adjusted frequency data points of the two corresponding drugs. Moreover, we calculated the distances between each drug and the mean frequency data points of the corresponding Torin1 and DMSO values. These distances were computed using the correlation values that were previously calculated for each drug in relation to all other drugs.

Transient transfection

Cells were seeded in six-well plates at 70% confluence 12 h prior to transfection. Transfection was performed using Jetprime Transfection Reagent (Cat# 101000027, Polyplus) according to the manufacturer’s protocols. Cells were harvested after 24–48 h.

RNA interference and quantitative PCR

HEK293T cells were seeded in six-well plates at 30% confluence 12 h prior to transfection. Cells were transfected with a total of 20 nM siRNAs using the Lipofectamine RNAiMAX transfection reagent (Cat# 13778075, Invitrogen) according to the manufacturer’s protocols. To enhance gene silencing efficiency, two individual siRNAs were used for one gene target. Cells were harvested 48 h or 72 h after transfection. Silencer® Select Negative Control #1 siRNA (Cat# 4390843) and gene specific siRNAs for FBXW11 (β-TrCP2) (Cat# 4392420, assay ID: s23487 and s23485), IKBKB (IKKβ) (Cat# 4392420, assay ID: s223926 and s7263), CHUK (IKKα) (Cat# 4390824, assay ID: s3077 and s3078), IKBKG (IKKγ) (Cat# 4390824, assay ID: s16186 and Cat # 4392420, assay ID: s533388), WDR68 (DCAF7) (Cat# 4427037, assay ID: s19985 and s19986) and fbxw11 (β-Trcp2) (Cat# 4390771, assay ID: s98104) were obtained from Thermo Fisher Scientific. For expression studies, RNA was extracted from cells using the NucleoSpin® RNA kit (Cat# 740955.50, Takara) according to the manufacturer’s instructions. The concentration and quality of the extracted RNA were assessed using a Nanodrop ONE^c (Thermo Scientific) spectrophotometer. For cDNA synthesis, 1 µg of RNA was used per sample using the QuantiTect Reverse Transcription Kit (Cat# 205311, Qiagen). The quantitative RT-qPCR reactions were carried out using the PowerUp™ SYBR™ Green Master Mix (Cat# A25742, Applied Biosystems) and the QuantStudio-3 real-time PCR systems with the following conditions: 95 °C, 2 min; (95 °C, 15 s; 55 °C, 15 s; 72 °C, 60 s) x 40. Primer sequences are listed in Supplementary Data 2. Relative RNA levels were normalized against an internal control (GAPDH or HPRT1) and were calculated as 2^−ΔΔCT.

Cell lysis and immunoblotting

Following completion of the treatment, cells were washed once with cold PBS and lysed using either NP-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-Cl pH 8.0) or RIPA buffer (150 mM NaCl, 1% Triton-X100, 50 mM Tris-Cl pH 8.0, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with 1X protease and phosphatase inhibitors. The total lysates were kept at 4 °C with agitation for 1 h followed by centrifugation at 16,000 × g for 15 min at 4 °C to remove debris. The protein concentration was measured using the Pierce™ BCA Protein Assay Kit (Cat# 23225, Thermo Scientific), with Pierce™ Bovine Serum Albumin Standard, 2 mg/mL (Cat# 23210, Thermo Scientific) as the standard. Protein samples were prepared by adding Laemmli sample buffer (Cat# 1610737EDU, biorad) and incubating at 95 °C for 5 min. The samples were then stored at − 80 °C until use. Denatured samples were separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Cat# 1620177, Biorad). The membranes were incubated in blocking buffer (5%, w/v, dried skimmed milk in Tris-buffered saline, pH 7.4, and 0.1% Tween 20, TBST), followed by overnight incubation with the indicated primary antibodies, appropriately diluted in the blocking buffer. Subsequently, the membranes were washed three times with TBST and incubated with the appropriate secondary antibodies, either Goat anti-mouse HRP-conjugated antibody (Cat# 1705047, Biorad) or Goat anti-rabbit HRP-conjugated antibody (Cat# 1705046, Biorad) or IRDye® 680RD Goat anti-Mouse IgG Secondary Antibody (926-68070, 1:10000, LI-COR Biosciences) or IRDye® 800CW Goat anti- Rabbit IgG Secondary Antibody (926-32211, 1:10000, LI-COR Biosciences), diluted in blocking buffer, for 1 h at room temperature. After washing, the membranes were detected using SuperSignal™ West Dura Extended Duration Substrate reagent (Cat# A38554, ThermoFisher Scientific). Images were taken with ImageQuant LAS 4000 (GE Healthcare) and quantified by Fiji analysis software. Uncropped scans of all blots are provided in Source data file.

Immunoprecipitation

Protein extraction from cultured HEK293T and HeLa cells was performed with either RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM

NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS) or NP-40 lysis buffer. Centrifugation was performed at 16,000 × g for 15 min at 4 °C to remove cell debris. For lysates in RIPA buffer, the volume of the lysate was roughly measured, and powdered SDS was added in proportion to the volume to achieve a final concentration of 1% SDS. The lysate was then boiled at 95 °C for 5 minutes to dissociate any non-covalently bound proteins. Subsequently, RIPA buffer without SDS was added to reduce the SDS concentration back to 0.1%. The lysates were then pre-cleared using dynabeads (Cat# 10003D, Invitrogen) conjugated with normal mouse IgG (Cat# 12-371, Sigma Aldrich). The pre-cleared lysates were then incubated with dynabeads conjugated to the indicated primary antibodies, rotating at 4 °C for 2 h Subsequently, the beads were washed four times with 500 μL of RIPA lysis buffer and eluted in 2x Laemmli buffer at 95 °C for 5 min.

Immunofluorescence

HeLa cells were seeded at 50–60% confluence on 24-well glass bottom plates (Cat# P24-1.5H-N, Cellvis) and subjected to either transfection or treatment. Cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, followed by permeabilization in 0.1% Triton X-100 in PBS for 10 min at room temperature with agitation. Next, cell samples were incubated in a blocking buffer consisting of 5% (w/v) Bovine Serum Albumin and 0.1% saposin. Overnight incubation with Flag antibodies (diluted 1:500 in the blocking buffer) was carried out. Subsequently, the cell samples were washed three times with PBS and incubated with the appropriate secondary antibodies: Goat anti-mouse antibody, Alexa Fluor™ 555 (Cat# A-21422, Invitrogen) or Goat anti-mouse antibody, Alexa Fluor™ 488 (Cat# A-11001, Invitrogen), both diluted 1:400 in blocking buffer, for 1 h at room temperature. Following this, the cell samples were incubated with 300 nM DAPI (Cat# 28718-90-3, Roche) in PBS for 15 min and washed three times with PBS. A Zeiss cell discoverer 7 microscope and accompanying Zen software were used to acquire confocal images.

In vitro kinase assay and phos-tag analysis

3xFlag-tagged WT and mutant TFEB were transiently transfected and immunoprecipitated from HeLa cell lysates using Flag antibody-conjugated beads. The immunoprecipitated TFEB proteins were first dephosphorylated by λ phosphatase (Cat# P9614, Sigma-Aldrich). The dephosphorylation master mix is composed of 1x λ Protein Phosphatase Buffer (Cat# L9288), 1 x MnCl2 (Cat# M0564), 1 x protease inhibitor (Cat# P3100-010) and about 16 units per μl λ phosphatase. Resuspend 25 μl of beads conjugated with immunoprecipitated TFEB in 50 μl of dephosphorylation master mix and incubate the reaction in a 30 °C water bath for 40 min, pipetting the beads up and down every 10 min to resuspend them. Remove the master mix and wash the beads with lysis buffer. Elute the dephosphorylated TFEB using 200 mM glycine-HCl, pH 2.6. Immediately neutralize the elute with 1 μL 1 M Tris, pH 10.6. Subsequently, the dephosphorylated TFEB molecules were subjected to a kinase assay using 300 nM IKKα (Cat# ab102103, Abcam) or IKKβ (Cat# 81066, Active Motif) or Akt (Cat# 1775-KS-010, R&D Systems) in the presence of 100 μM ATP, 2 mM MgCl2, 1 mM DTT, 100 μM Na3VO4, and 500 μM EDTA. The reaction mixtures were incubated for either 60 or 120 min at 25 °C, and the reaction was stopped by adding 2X Laemmli sample buffer, followed by boiling for 5 min.

Phos-tag analysis was performed according to the manufacturer’s instructions (Cat# F4002, APExBIO). Briefly, the resolving gel solution is composed of 10% (w/v) acrylamide, 30 μM Phosbind Acrylamide (PB-A), 0.1 mM MnCl2, 0.375 M Tris/HCl solution (pH 8.8), 0.1% (w/v) SDS solution, 0.1% TEMED (tetramethylethylenediamine), and 0.05% (w/v) ammonium persulfate solution. The stacking gel follows the standard composition of regular SDS-PAGE gels. Subsequently, the samples were separated by Phos-tag SDS-PAGE and transferred onto PVDF membranes for immunoblotting analysis.

Sample preparation and LC-MS analysis for phosphorylation detection

For phosphorylated TFEB from the in vitro IKK kinase phosphorylation: Phosphorylated TFEB protein was prepared as described in the “in vitro kinase assay” section. Three aliquots of in vitro phosphorylated TFEB was used as n = 3 replicates for this analysis. To remove MS-incompatible reagents, TFEB was isolated by acetone precipitation. Dry TFEB was reconstituted in 50 mM ammonium bicarbonate buffer, pH 7, reduced using dithiothreitol (Cat#20291, Pierce), alkylated using iodoacetamide (Cat#A3221, Sigma-Alrich), and digested using sequencing grade trypsin (Cat#V5113, Promega). TFEB tryptic peptides were dried and reconstituted in LC-MS mobile phase A (2% acetonitrile (Cat# 047138.M6, Thermo Scientific), 98% MilliQ purified water, 0.1% formic acid (Cat# 85178, Pierce)) for LC-MS analysis. We used a Thermo Fisher Ultimate RSLC3000 nanoLC system equipped with a micro silica column packed with 20 cm of ReproSil-Pur C18 resin (120 Å, C18-AQ,1.9 μmresin; Cat# r119.aq, Dr. Maisch) and an Orbitrap Fusion Lumos Tribrid Mass Spectrometer. Our acquisition method includes an untargeted acquisition in parallel with a targeted acquisition for the tryptic phosphopeptides 406AA-430AA and 406AA-431AA. This approach allows a general analysis of TFEB tryptic peptides and targeted analysis of select candidate phosphopeptides for further fragmentation that ultimately enables the rigorous localization of the phosphorylation sites. The untargeted acquisition settings include Orbitrap as the detector, top 15 data dependent MS2, charge state selection 2–6, 60 K and 30 K was set as the Orbitrap resolution for MS1 and MS2 respectively, 250–2000 m/z scan range, 30% RF lens, 0.8 m/z isolation window, stepped HCD at 28%, 30%, 34% collision energy for fragmentation, and profile as acquired data type. The targeted acquisition involves MS2 acquisition, and the settings include a targeted precursor list of m/z 881.73 and m/z 924.43, Orbitrap as the detector, 30 K Orbitrap resolution, scan range is 110–2000 m/z, 30% RF lens, stepped HCD fragmentation with 28, 30, and 32% collision energy. These fragmentation parameters are optimized to minimize the loss of phosphate for the targeted phosphopeptides. We used our in-house software (TDMS 7.04) to process the LC-MS data using UniProt database P19484 for TFEB^122,123. The data shows a clear presence of the intact phosphorylated peptide at 406AA-430AA with m/z 881.7311 (-1.99 ppm error, expected mass of 881.732851 m/z). The subsequent MS2 data exhibit multiple ion types supporting phosphorylation localization at S427. We also manually validated the MS2 fragment ions independently using Qualbrowser (Thermo Scientific Excalibur 4.3) to ensure confident phosphorylation site localization. No statistical tests were applied to the mass spectrometry data, as phosphorylation site localization was determined based on qualitative MS2 fragment ion evidence.

For phosphorated TFEB from the IL-1β treatment

Three cell plates were used in each IL-1β treatment timepoint as n = 3 replicates for this analysis. After IL-1β treatment, cultured HeLa/TFEB cells were harvested, washed in PBS, resuspended in modified RIPA buffer, incubated on Thermomixer for 15 minutes at 37 °C at 600 rpm, sonicated using BioRuptor Pico on high power for 7 cycles (30 s on, 30 s off), and centrifuged at 10,000 × g for 10 min at 4 °C to pellet cell membrane debris. The supernatant cell lysate was then transferred to a low-bind tube for Flag-tag immunoprecipitation. For a 15 cm plate of IL-1β-treated cells, 50 µL of Anti-DYKDDDDK Mag Agarose bead (Cat# A36798, Pierce) was used. The beads were washed using 500 µl modified RIPA 3 times, and the washed buffer was discarded each time with the aid of a DynaMag rack. The beads were incubated in cell lysate on a rotator for 20 min at room temperature. Conjugated beads were collected, and the supernatant was discarded. Beads were washed with 500 µl PBS twice and with 500 µl milliQ water once. Flag-tagged TFEB was eluted from the beads using 100 µl Glycine-HCl, pH 3.0 on the thermomixer at 1400 rpm, 25 °C for 5 min. DynaMag rack was used to retain the beads, while the elute was collected to a low bind tube. This was repeated once more, and all elutes were combined. The final elution was neutralized with 30 µl 1 M Tris buffer, pH 8.0. This eluate was reduced using dithiothreitol (Cat# 20291, Pierce), alkylated using iodoacetic acid (Cat#43964, Alfa Aesar), and digested using sequencing-grade trypsin (Cat# V5113, Promega). TFEB tryptic peptides were dried and reconstituted in LC-MS mobile phase A (2% acetonitrile (Cat# 047138.M6, Thermo Scientific), 98% MilliQ purified water, 0.1% formic acid (Cat# 85178, Pierce)) for LC-MS analysis. The LC-MS method used here was the same to the method described above. Briefly, we used a Thermo Fisher Ultimate RSLC3000 nanoLC system equipped with a micro silica column packed with 20 cm of ReproSil-Pur C18 resin (120 Å, C18-AQ,1.9-μmresin; Cat# r119.aq, Dr. Maisch) and an Orbitrap Fusion Lumos Tribrid Mass Spectrometer. Our acquisition method includes an untargeted acquisition in parallel with a targeted acquisition for the tryptic phosphopeptides 406AA-430AA and 406AA-431AA. The untargeted acquisition settings include Orbitrap as the detector, top 15 data dependent MS2, charge state selection 2-6, 60 K and 30 K was set as the Orbitrap resolution for MS1 and MS2 respectively, 250–2000 m/z scan range, 30% RF lens, 0.8 m/z isolation window, stepped HCD at 28%, 30%, 34% collision energy for fragmentation, and profile as acquired data type. The targeted acquisition involves MS2 acquisition, and the settings include a targeted precursor list of m/z 881.73 and m/z 924.43, Orbitrap as the detector, 30 K Orbitrap resolution, scan range is 110–2000 m/z, 30% RF lens, stepped HCD fragmentation with 28%, 30%, and 32% collision energy. MS Fragger v.21.1 was used for data search against the reviewed Uniprot human proteome database (UP000005640) with preset LFQ-phospho workflow, strict-trypsin as protease at K, R residue, 3 allowed miscleavage, mass shifts for: variable oxidation modification at M residue 15.9949 Da, variable acylation modification at N terminal 42.0106 Da, variable phosphorylation modification at S, T, Y residue 79.96633 Da, fix modification at C (cysteine) from iodoacetic acid 58.005 Da. Other parameters that are not mentioned here used default values set by the workflow. MS1 quantification data was analyze and plotted using GraphPad Prism v.9.5.1, and the statistical analysis used one-tail Welch’s t test to obtain a p-value of 0.0008. Phosphorylation site localization was further evaluated by manual inspection of MS2 fragment ions.

Sample preparation and LC-MS analysis for ubiquitination detection

The samples were digested using a modified filter-aided sample preparation method¹²⁴. The beads were washed four times with 1 ml of 50 mM cold ammonium bicarbonate buffer, pH 8.0, and samples were eluted and reduced with SDS buffer (4% (wt/vol) SDS, 100 mM Tris-HCl, pH 8.0) containing 100 mM DTT, and then the samples were boiled at 95 °C for 10 min. The reduced samples were mixed with 200 µl of 100 mM Tris-HCL buffer, pH 8.5, containing 8 M urea (UA buffer) and transferred onto the top chamber of a 30,000 MWCO filtration unit (Cat# MRCF0R030, Millipore) and spun in a microcentrifuge at 14,000 x g for 10 min. An additional 200 µl of UA buffer was added to the top chamber of the filter unit, and the filter was spun at 14,000 x g for 15 to 20 min in a microcentrifuge (Eppendorf 5424). The flow through was discarded, and the proteins were alkylated by adding 100 µl of UA buffer containing 50 mM iodoacetamide (Cat# A39271, Pierce) to the top chamber of the filtration unit and gyrating at 550 rpm in the dark at room temperature for 30 min using a thermomixer (Thermomixer R, Eppendorf). The filter was spun at 14,000 x g for 15 min and the flow through was discarded. Unreacted iodoacetamide was washed away from the filter with two sequential additions of 200 µl UA buffer and centrifugation at 14,000 x g for 15 to 20 min after each addition. The urea buffer was exchanged into 50 mM ammonium bicarbonate buffer, pH 8 (digestion buffer), two sequential additions of 200 µl digestion buffer with centrifugation after each addition to the top chamber was performed. The top filter units were transferred to a new collection tube, and 0.2 µg of Asp-N (Cat# 9053, Pierce) was added, and samples were digested at 37 °C overnight. The next day, 0.1 µg of sequencing-grade trypsin (Cat# V5113, Promega) was added, and samples were digested for two hours at 37 °C. The filters were spun at 14,000 x g for 15 min to collect the peptides in the flow-through. The filter was washed with 50 µl of 100 mM ammonium bicarbonate buffer, and the wash was collected with the peptides. In preparation for desalting, peptides were acidified to 1% (vol/vol) TFA final concentration. The peptides were desalted using two micro-tips sequentially. (C4, BIOMEKNT3C04, and C18, BIOMEKNT3C18) (Glygen) on a Beckman robot (Biomek NX), as previously described¹²⁵. The peptides were eluted with 60% (vol/vol) acetonitrile in 0.1% TFA (vol/vol) and dried in a Speed-Vac (Thermo Scientific, Model No. Savant DNA 120 concentrator). The peptides were dissolved in 20 µl of 1% (vol/vol) acetonitrile in water. An aliquot (10%) was removed for quantification using the Pierce Quantitative Fluorometric Peptide Assay kit (Cat# 23290, Thermo Scientific). The remaining peptides were transferred to autosampler vials (Cat# 200046, Sun-Sri), dried and stored at − 80 °C for LC-MS analysis.

The peptides were analyzed using a nano-Elute chromatograph (Bruker Daltonics) coupled online to a hybrid trapped ion mobility-quadrupole time of flight mass spectrometer (timsTOF Pro2, Bruker Daltonics with a modified nano-electrospray source (CaptiveSpray, Bruker Daltonics). The mass spectrometer was operated in dda-PASEF mode¹²⁶. The samples in 0.1% (vol/vol) aqueous formic acid (FA) were loaded (2 µl) onto a 75 µm i.d. × 25 cm Aurora Series column with CSI emitter (Ionopticks) on a Bruker nano-ELUTE (Bruker Daltonics). The column temperature was set to 50 °C. The column was equilibrated using constant pressure (800 bar) with 8 column volumes of solvent A (0.1% (vol/vol) aqueous FA). Sample loading was performed at constant pressure (800 bar) at a volume of 1 x sample pick-up volume plus 2 µl. The peptides were eluted using the one column separation mode with a flow rate of 300 nL/min and using solvents A and B (0.1% (vol/vol) FA/MeCN): solvent A containing 2%B increased to 17% B over 60 min, to 25% B over 30 min, to 37% B over 10 min, to 80% B over 10 min and constant 80% B for 10 min.

The MS1 and MS2 spectra were recorded from m/z 100 to 1700. Suitable precursor ions for PASEF-MS/MS were selected in real time from TIMS-MS survey scans by a PASEF scheduling algorithm¹²⁶. A polygon filter was applied to the m/z and ion mobility plane to select features most likely representing peptide precursors rather than singly charged background ions. The quadrupole isolation width was set to 2 Th for m/z < 700 and 3 Th for m/z > 700, and the collision energy was ramped stepwise as a function of increasing ion mobility: 52 eV for 0–19% of the ramp time; 47 eV from 19–38%; 42 eV from 38–57%; 37 eV from 57–76%; and 32 eV for the remainder. The TIMS elution voltage was calibrated linearly using the Agilent ESI-L Tuning Mix (m/z 622, 922, 1222).

Data from the mass spectrometer were converted to peak lists using DataAnalysis (version 6.1, Bruker Daltonics). The MS2 spectra with charges + 2, + 3 and + 4 were analyzed using Mascot software¹²⁷ (Matrix Science, London, UK; version 2.8.2). Mascot was set up to search against a UniProt (ver July 2019) database of human proteins (20,667 entries), assuming the digestion enzyme was AspN with a maximum of 4 missed cleavages allowed. The searches were performed with a fragment ion mass tolerance of 50 ppm and a parent ion tolerance of 25 ppm. Carbamidomethylation of cysteine was specified in Mascot as a fixed modification. Deamidation of asparagine, deamidation of glutamine, formation of pyro-glutamic acid from N-terminal glutamine, acetylation of protein N-terminus, oxidation of methionine, and diglycine modification of lysine were specified as variable modifications. Peptides and proteins were filtered at 1% false-discovery rate (FDR) by searching against a reversed protein sequence database. Scaffold (version Scaffold_5.3.0, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 54.0% probability to achieve an FDR less than 1.0% by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii, Al et al Anal. Chem 2003;75(17):4646-58).

Subcellular fractionation

Cells were cultured to 70% confluence in a 6-well plate. Subcellular fractionation was performed using the Subcellular Protein Fractionation Kit for Cultured Cells (Cat# 78840, Thermo Scientific) following the manufacturer’s protocols. Each fraction was subsequently subjected to immunoblotting analyses.

Data analysis

Data are presented as mean ± SEM. unless otherwise stated. Statistical comparisons between two groups were performed using two-tailed unpaired Student’s t tests. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparisons test, one-way ANOVA followed by Dunnett’s multiple-comparisons test, or Welch’s one-way ANOVA followed by Dunnett’s T3 multiple-comparisons test, as indicated. In analyses involving multiple pairwise comparisons, multiple two-sided t-tests with Benjamini–Hochberg false discovery rate (FDR) correction were applied. Where specified, one-tailed Welch’s t tests were used. A p-value  < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

Y.X. and M.S. conceived the project and designed the experiments. M.N.Y. and N.L.Y. designed mass-spectrometry experiments. Y.X. and M.X.G.I designed high-throughput screening experiments. Y.X., J.S., W.X., M.N.Y., K.F.P., M.X.G.I., B.G. and Q.W. performed experiments and analyzed the data under the supervision of H.Z., N.L.Y. and M.S. A.J. performed bioinformatic analyses. Y.X. and M.S. wrote the manuscript with input from all authors.

Peer review

Peer review information

Nature Communications thanks Claus Scheidereit and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

Raw mass spectrometry data have been deposited in the MassIVE publicly available repository under accession code: Accession: MSV000093106 (TFEB pS427 detection) ftp://massive-ftp.ucsd.edu/v06/MSV000093106/ Accession: MSV000095363 (TFEB phospho-peptide analysis following IL-1β treatment) ftp://massive-ftp.ucsd.edu/v08/MSV000095363/ Accession: MSV000096549 (TFEB ubiquitination analysis) ftp://massive-ftp.ucsd.edu/v07/MSV000096549/ All other data generated or analyzed in this study are included in the article and its Supplementary Information and the Source data file. Source data are provided in this paper.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-026-71001-1.