INTRODUCTION Eukaryotes contain a highly conserved signaling pathway that becomes rapidly activated when adenosine triphosphate (ATP) levels decrease, as happens during conditions of nutrient shortage or mitochondrial dysfunction. The adenosine monophosphate (AMP)–activated protein kinase (AMPK) is activated within minutes of energetic stress and phosphorylates a limited number of substrates to biochemically rewire metabolism from an anabolic state to a catabolic state to restore metabolic homeostasis. AMPK also promotes prolonged metabolic adaptation through transcriptional changes, decreasing biosynthetic genes while increasing expression of genes promoting lysosomal and mitochondrial biogenesis. The transcription factor EB (TFEB) is a well-appreciated effector of AMPK-dependent signals, but many of the molecular details of how AMPK controls these processes remain unknown. RATIONALE The requirement of AMPK and its specific downstream targets that control aspects of the transcriptional adaptation of metabolism remain largely undefined. We performed time courses examining gene expression changes after various mitochondrial stresses in wild-type (WT) or AMPK knockout cells. We hypothesized that a previously described interacting protein of AMPK, folliculin-interacting protein 1 (FNIP1), may be involved in how AMPK promotes increases in gene expression after metabolic stress. FNIP1 forms a complex with the protein folliculin (FLCN), together acting as a guanosine triphosphate (GTP)–activating protein (GAP) for RagC. The FNIP1-FLCN complex has emerged as an amino acid sensor to the mechanistic target of rapamycin complex 1 (mTORC1), involved in how amino acids control TFEB activation. We therefore examined whether AMPK may regulate FNIP1 to dominantly control TFEB independently of amino acids. RESULTS AMPK was found to govern expression of a core set of genes after various mitochondrial stresses. Hallmark features of this response were activation of TFEB and increases in the transcription of genes specifying lysosomal and mitochondrial biogenesis. AMPK directly phosphorylated five conserved serine residues in FNIP1, suppressing the function of the FLCN-FNIP1 GAP complex, which resulted in dissociation of RagC and mTOR from the lysosome, promoting nuclear translocation of TFEB even in the presence of amino acids. FNIP1 phosphorylation was required for AMPK to activate TFEB and for subsequent increases in peroxisome proliferation–activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) mRNAs. Cells in which the five serines in FNIP1 were mutated to alanine were unable to increase lysosomal and mitochondrial gene expression programs after treatment with mitochondrial poisons or AMPK activators despite the presence and normal regulation of all other substrates of AMPK. By contrast, neither AMPK nor its control of FNIP1 were needed for activation of TFEB after amino acid withdrawal, illustrating the specificity to energy-limited conditions. CONCLUSION Our data establish FNIP1 as the long-sought substrate of AMPK that controls TFEB translocation to the nucleus, defining AMPK phosphorylation of FNIP1 as a singular event required for increased lysosomal and mitochondrial gene expression programs after metabolic stresses. This study also illuminates the larger biological question of how mitochondrial damage triggers a temporal response of repair and replacement of damaged mitochondria: Within early hours, AMPK-FNIP1–activated TFEB induces a wave of lysosome and autophagy genes to promote degradation of damaged mitochondria, and a few hours later, TFEB–up-regulated PGC1⍺ and ERR⍺ promote expression of a second wave of genes specifying mitochondrial biogenesis. These insights open therapeutic avenues for several common diseases associated with mitochondrial dysfunction, ranging from neurodegeneration to type 2 diabetes to cancer. Mitochondrial damage activates AMPK to phosphorylate FNIP1, stimulating TFEB translocation to the nucleus and sequential waves of lysosomal and mitochondrial biogenesis. After mitochondrial damage, activated AMPK phosphorylates FNIP1 (1), causing inhibition of FLCN-FNIP1 GAP activity (2). This leads to accumulation of RagC in its GTP-bound form, causing dissociation of RagC, mTORC1, and TFEB from the lysosome (3). TFEB is therefore not phosphorylated and translocates to the nucleus, inducing transcription of lysosomal or autophagy genes, with parallel increases in NT-PGC1α mRNA (4), which, in concert with ERRα (5), subsequently induces mitochondrial biogenesis (6). CCCP, carbonyl cyanide m-chlorophenylhydrazone; CLEAR, coordinated lysosomal expression and regulation; GDP, guanosine diphosphate; P, phosphorylation. [Figure created using BioRender]
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Hyperosmolality induces nuclear translocation of osmotic stress transcription factor 1 in Oreochromis mossambicus cells
Euryhaline fish tolerate a wide range of environmental salinity by employing molecular mechanisms for coping with the associated osmotic stress. We have previously shown that osmotic stress transcription factor 1 (OSTF1) is part of these mechanisms. OSTF1 is transiently and rapidly upregulated in gill epithelial cells of tilapia (Oreochromis mossambicus) exposed to hyperosmolality. Hyperosmotic induction of OSTF1 in tilapia gills was reproduced in the tilapia OmB cell neuroepithelial cell line. OSTF1 shares the signature sequence of the TSC-22 family suggesting that it is a transcriptional repressor. If, in fact, OSTF1 is a transcription factor, we hypothesize that it will localize to the nucleus during hyperosmotic stress. Using standard cloning procedures, OSTF1 was tagged with enhanced green fluorescent protein (EGFP) at either the C- or N-terminus. Using fluorescent microscopy we show that the fusion proteins are retained in the cytosol under iso-osmotic conditions. To evaluate potential nuclear translocation of OSTF1 during hyperosmotic stress, we subjected OmB cells expressing the OSTF1:EGFP fusion protein to hyperosmotic media and imaged at time intervals from 5 minutes to 4 hours using a Leica Dmi8 microscope with automated scanning stage. At four hours and 650 mOsmol/kg, subcellular localization quantified by LASX image analysis (Leica) demonstrated that OSTF1:EGFP was mostly localized to the nucleus. This result supports our hypothesis that OSTF1 is indeed an osmotically inducible transcription factor. Current work evaluates influence of specific OSTF1 domains on nuclear localization.
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- Award ID(s):
- 1656371
- NSF-PAR ID:
- 10227854
- Date Published:
- Journal Name:
- SICB Virtual Annual Meeting 2021
- Volume:
- P15
- Issue:
- 4
- Page Range / eLocation ID:
- 1
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1096/fasebj.2020.34.s1.07028
