| 1999 |
NFAT5 contains an NFAT-like Rel homology domain, binds DNA sequences similar to NFAT1-4 target sites, but lacks the majority of Fos/Jun contact residues and does not bind cooperatively with Fos and Jun to DNA. Unlike NFAT1-4, NFAT5 is constitutively nuclear and its nuclear localization is not regulated by calcineurin-mediated dephosphorylation. |
DNA binding assays, co-transfection/reporter assays, immunofluorescence localization, sequence analysis of contact residues |
Proceedings of the National Academy of Sciences of the United States of America |
High |
10377394
|
| 2002 |
Crystal structure of TonEBP/NFAT5 Rel homology domain bound to DNA reveals that TonEBP adopts an NF-κB-like homodimeric structure and completely encircles its TonE DNA target, providing increased kinetic stability of the DNA complex. |
X-ray crystallography, electrophoretic mobility shift assay (EMSA), biochemical DNA-binding kinetics |
Nature structural biology |
High |
11780147
|
| 2002 |
Dimerization of TonEBP via the C-terminus of the Rel homology domain (CRHD) is required for DNA binding and proper phosphorylation (especially under hypertonic conditions); deletion of the CRHD abolishes DNA binding and eliminates dominant-negative activity. |
Biochemical dimerization assays, deletion mutagenesis, DNA binding assays, phosphorylation analysis |
Biochemical and biophysical research communications |
High |
12074571
|
| 2002 |
The C-terminal transactivation domain (TAD; 983 C-terminal amino acids) of TonEBP/OREBP is osmotically regulated at the post-translational level, with activity decreasing >80% at low osmolality and increasing 8-fold at high NaCl, dependent on tonicity-driven phosphorylation. Tyrosine kinase inhibitor herbimycin and CK2 inhibitor DRB reduce TAD activity, implicating these kinases. |
GAL4-TAD fusion reporter assay in HepG2 cells, pharmacological kinase inhibition |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
11792870
|
| 2002 |
p38 MAPK and Fyn tyrosine kinase are both required for maximal hypertonic activation of OREBP/TonEBP; the transactivation domain is the target of p38- and Fyn-mediated activation. Combined inhibition of p38 in Fyn-deficient cells almost completely abolishes hypertonic ORE reporter induction. |
Dominant-negative mutants, pharmacological inhibition (SB203580), Fyn-deficient cells, ORE reporter assay |
The Journal of biological chemistry |
High |
12359721
|
| 2004 |
ATM kinase, activated by high NaCl-induced DNA damage via autophosphorylation at Ser-1981, contributes to TonEBP/OREBP activation. TonEBP/OREBP physically associates with ATM (reciprocal co-IP), ATM phosphorylates TonEBP at consensus sites (Ser-1197, Ser-1247, Ser-1367), and mutation of these sites reduces TonEBP transcriptional activity. |
Co-immunoprecipitation, site-directed mutagenesis (S1197A, S1247A, S1367A), ATM-deficient (AT) cells, wortmannin inhibition, EMSA supershift |
Proceedings of the National Academy of Sciences of the United States of America |
High |
15173573
|
| 2005 |
High NaCl stabilizes TonEBP/OREBP mRNA (via its 5'-UTR, not the 3'-UTR), which accounts for the increase in mRNA abundance without requiring increased transcription. |
Actinomycin D chase assay, luciferase reporters containing 5'- and 3'-UTR fragments |
American journal of physiology. Renal physiology |
Medium |
15900024
|
| 2005 |
ATM kinase also contributes to high NaCl-induced nuclear translocation of TonEBP/OREBP; nuclear localization of TonEBP and its N-terminal Rel homology domain is reduced in ATM-deficient (AT) cells and restored by reconstitution with functional ATM. |
Western blot of nuclear/cytoplasmic fractions, ATM-deficient cells, wortmannin inhibition, genetic reconstitution |
American journal of physiology. Renal physiology |
Medium |
15840767
|
| 2005 |
High NaCl increases reactive oxygen species (specifically superoxide from mitochondrial complexes I and III), which contribute to TonEBP/OREBP activation by increasing its transactivating activity but not nuclear translocation. |
Pharmacological inhibitors (rotenone, myxothiazol, NAC, MnTBAP), ORE/TonE reporter assay, BGT1 mRNA measurement |
American journal of physiology. Renal physiology |
Medium |
15769933 16303854
|
| 2006 |
TonEBP is physically associated in vivo with RNA helicase A (RHA), which binds the E'F loop of the DNA-binding domain. Overexpression of RHA inhibits TonEBP activity; elevated tonicity decreases the TonEBP-RHA interaction, suggesting RHA dissociation is a mechanism for TonEBP activation. |
Co-immunoprecipitation, domain mapping, overexpression, reporter assays |
The Biochemical journal |
Medium |
16173919
|
| 2006 |
TonEBP/OREBP nuclear export under isotonic conditions is mediated by a CRM1-dependent leucine-rich nuclear export sequence (NES) in the N-terminus; hypotonicity-induced nuclear export additionally requires a distinct auxiliary export domain. CRM1 physically interacts with TonEBP, demonstrated by bimolecular fluorescence complementation. |
Immunocytochemistry, GFP fusions, site-directed mutagenesis of NES, bimolecular fluorescence complementation assay, leptomycin B treatment |
The Journal of biological chemistry |
High |
16782704
|
| 2006 |
Proteomic analysis identified 14 proteins physically associated with TonEBP/OREBP in nuclei under high NaCl conditions, including DNA-PK (catalytic subunit and Ku86), RNA helicases (RHA, nucleolar RNA helicase II/Gu, DEAD-box p72), several hnRNPs and snRNPs, Hsp90β, Hsc70, and PARP-1. PARP-1 reduces TonEBP transcriptional and transactivating activities, while Hsp90 enhances them and sustains TonEBP protein abundance. |
Stable expression of N-terminal TonEBP fragment, immunoprecipitation, mass spectrometry, Western blot confirmation, EMSA, reporter assays |
American journal of physiology. Renal physiology |
High |
17148781
|
| 2008 |
p38α and p38δ isoforms have opposing effects on TonEBP/OREBP activity: p38α increases TonEBP activity while p38δ decreases it. Overexpression of MKP-1 inhibits both p38 isoforms but has no net effect on TonEBP/OREBP activity due to these opposing actions. |
siRNA knockdown of p38 isoforms, overexpression of dominant-negative MKP-1, TonEBP reporter assay |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
18367666
|
| 2009 |
Phospholipase C-γ1 (PLC-γ1) contributes to TonEBP/OREBP activation by high NaCl. High NaCl induces phosphorylation of TonEBP at Y143; phospho-Y143 creates a binding site for PLC-γ1 (reciprocal co-IP, abolished by Y143A mutation); PLC-γ1 promotes TonEBP transactivating activity and nuclear localization, and is present in the TonEBP-DNA complex. |
PLC-γ1 null cells, siRNA knockdown, co-immunoprecipitation, Y143A mutagenesis, nuclear localization assay, reporter assay, EMSA |
Proceedings of the National Academy of Sciences of the United States of America |
High |
20080774
|
| 2009 |
Inducible nucleosome depletion occurs at OREBP/TonEBP binding sites (OREs) in response to hypertonic stress. Initial nucleosome loss is OREBP-independent but is potentiated by OREBP; OREBP-dependent histone hyperacetylation spans the 5' upstream sequences and exons of the aldose reductase gene, but nucleosome loss is not regulated by acetylation status. |
Chromatin immunoprecipitation (ChIP), nucleosome occupancy assays, aldose reductase promoter analysis |
PloS one |
Medium |
20041176
|
| 2010 |
SHP-1 protein tyrosine phosphatase dephosphorylates TonEBP/OREBP at Y143 both in vivo and in vitro, reducing TonEBP nuclear localization and transactivating activity. SHP-1 co-immunoprecipitates with TonEBP. High NaCl inhibits SHP-1 by increasing its phosphorylation at Ser591, which reduces SHP-1 phosphatase activity and nuclear localization, thereby enabling TonEBP activation. |
Genome-wide phosphatase siRNA screen, siRNA knockdown, overexpression, in vitro phosphatase assay, co-immunoprecipitation, nuclear fractionation |
Proceedings of the National Academy of Sciences of the United States of America |
High |
20351292
|
| 2010 |
c-Abl kinase is responsible for high NaCl-induced phosphorylation of TonEBP/OREBP at Y143 in cell culture and in rat renal inner medulla in vivo. c-Abl associates with TonEBP (co-immunoprecipitation), phosphorylates TonEBP-Y143 in vitro, and its inhibition (imatinib, siRNA, kinase-dead mutant) drastically reduces TonEBP nuclear localization and transactivating activity. High NaCl-induced ATM activation depends on c-Abl activity. |
Co-immunoprecipitation, in vitro kinase assay, imatinib treatment, siRNA, kinase-dead construct, nuclear fractionation, in vivo renal inner medulla analysis |
FASEB journal : official publication of the Federation of American Societies for Experimental Biology |
High |
20585028
|
| 2010 |
TonEBP interacts with NF-κB p65 in a tonicity-dependent manner and enhances NF-κB activity at κB elements of NF-κB-responsive genes. This interaction is dependent on Akt signaling. TonEBP affects neither IκBα degradation nor p65 nuclear translocation but acts as a cofactor enhancing NF-κB transcriptional output. |
Co-immunoprecipitation, reporter assays, kinase inhibition, knockdown in renal epithelial cells and macrophages |
Molecular biology of the cell |
Medium |
20685965
|
| 2011 |
CDK5, activated by high NaCl, directly phosphorylates TonEBP/OREBP at Thr135 (identified by mass spectrometry), contributing to its rapid nuclear localization. Inhibition of CDK5 reduces early (≤4 h) but not late nuclear TonEBP accumulation and transcriptional activity. S155A mutation increases nuclear localization while S134A/T135A mutations decrease it. |
Mass spectrometry phospho-site mapping, site-directed mutagenesis, CDK5 inhibition, nuclear fractionation, reporter assay, in vivo rat renal medulla phosphorylation analysis |
Molecular biology of the cell |
High |
21209322
|
| 2011 |
Rac1 and its scaffold OSM (osmosensing scaffold for MEKK3) support TonEBP/OREBP transcriptional/transactivating activity via PLC-γ1, not via p38 MAPK. siRNA knockdown of Rac1 or OSM reduces TonEBP activity; this effect is abolished in PLC-γ1-null cells and restored by PLC-γ1 reconstitution. |
siRNA knockdown, PLC-γ1 null cells, reconstitution, reporter assay, phosphorylation analysis |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
21712438
|
| 2013 |
GSK-3β inhibits TonEBP/NFAT5 by suppressing its transactivating activity (not nuclear localization or protein abundance). High NaCl activates PKA, PI3K, and AKT1 which phosphorylate GSK-3β at Ser9, inhibiting GSK-3β and thereby relieving its suppression of TonEBP. |
siRNA knockdown, GSK-3β null MEFs, S9A GSK-3β mutant transfection, overexpression of PKA/AKT/p38, reporter assay |
American journal of physiology. Renal physiology |
Medium |
23324178
|
| 2013 |
IKKβ regulates NFAT5 expression in thymocytes; NFAT5 in turn promotes survival of TCRαβ thymocytes and the β-selection checkpoint transition in an osmostress-independent manner by regulating pro-survival factors A1 and Bcl2 and attenuating the p53/Noxa axis. |
NFAT5-deficient mice, T cell-specific NFAT5 knockout, IKKβ manipulation, apoptosis assays, gene expression analysis |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
24043824
|
| 2014 |
PKC-α contributes to high NaCl-dependent NFAT5 activation through ERK1/2 (not through SHP-1-S591). PKC-α knockout reduces NFAT5 protein and target gene expression in the inner medulla, and PKC-α knockdown attenuates NFAT5 transcriptional and transactivating activity; combined knockdown of PKC-α and ERK2 is not additive, indicating a common pathway. |
PKC-α knockout mice, siRNA knockdown, ERK1/2 phosphorylation analysis, reporter assay, in vivo renal medulla analysis |
American journal of physiology. Renal physiology |
Medium |
25391900
|
| 2016 |
TonEBP is required for LPS-induced NF-κB enhanceosome activity in macrophages by recruiting the co-activator p300 to NF-κB; TonEBP molecules incapable of recruiting p300 do not stimulate NF-κB. Myeloid-specific TonEBP deletion reduces inflammation and sepsis severity. The natural compound cerulenin disrupts this TonEBP/NF-κB/p300 enhanceosome without affecting NF-κB activation itself. |
Myeloid-specific conditional KO mice, p300-recruitment-deficient TonEBP mutants, reporter assays, co-immunoprecipitation, cerulenin treatment, sepsis model |
Scientific reports |
High |
27118681
|
| 2019 |
TonEBP acts as an epigenetic suppressor of white adipose tissue beiging by recruiting DNMT1 DNA methylase to the β3-adrenoreceptor gene promoter, causing promoter methylation and transcriptional silencing. Adipocyte-specific TonEBP deletion or haploinsufficiency prevents high-fat diet-induced obesity and metabolic defects. |
Adipocyte-specific and haploinsufficient mouse models, ChIP, DNMT1 recruitment assay, promoter methylation analysis, ex vivo and cultured adipocyte reporter assays |
Nature communications |
High |
31387996
|
| 2019 |
NFAT5 is selectively required for CD8+ T cell exhaustion in the tumor microenvironment but not during chronic viral infection. NFAT5 transcriptional activity in the tumor context requires hyperosmolarity. NFAT5 overexpression reduces tumor control; deletion reduces TOX and PD-1 expression and increases cytokine production in precursor exhausted PD-1+TCF1+TIM-3- CD8+ T cells. |
NFAT5 conditional KO mice, NFAT5 overexpression in CD8+ T cells, tumor implantation and LCMV clone 13 infection models, flow cytometry, cytokine assays |
Nature immunology |
High |
37709986
|
| 2021 |
TonEBP recognizes R-loops (RNA-DNA hybrids) generated by DNA damaging agents (UV, CPT), binds R-loops via both 3D collision and 1D diffusion along DNA in vitro, and recruits METTL3 to R-loops through its Rel homology domain to promote m6A RNA methylation. TonEBP also recruits RNaseH1 to R-loops through a METTL3 interaction. TonEBP or METTL3 depletion increases R-loop accumulation and reduces cell survival under genotoxic stress. |
Single-molecule imaging, biochemical R-loop binding assays, co-immunoprecipitation, METTL3 recruitment assay, domain mapping (RHD), RNaseH1 recruitment, siRNA knockdown, cell survival assays |
Nucleic acids research |
High |
33313823
|
| 2021 |
PARP1-mediated PARylation of TonEBP is required for its recruitment to sites of R-loop-associated DNA damage induced by camptothecin. TonEBP interacts with PARP1, and loss of TonEBP increases R-loop accumulation and DNA damage and promotes cell death under CPT treatment. |
Co-immunoprecipitation, PARylation assays, R-loop detection, siRNA knockdown, cell death assays |
DNA repair |
Medium |
34049076
|
| 2023 |
EGFR activation induces phosphorylated EZH2 (Ser21) binding to NFAT5 and triggers NFAT5 methylation at K668. This methylation prevents NFAT5 interaction with E3 ligase TRAF6 in the cytoplasm, blocking TRAF6-mediated K63-linked ubiquitination and lysosomal degradation, resulting in NFAT5 protein stabilization, nuclear accumulation, and activation. Methylated NFAT5 upregulates MGMT expression, mediating temozolomide resistance in glioblastoma. |
Co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis (K668), reporter assays, orthotopic xenograft and PDX models, MGMT expression analysis |
Nature communications |
High |
37429858
|
| 2016 |
TonEBP suppresses HO-1 (heme oxygenase-1) expression in M1 macrophages by blocking Nrf2 recruitment to the HO-1 promoter, thereby promoting M1 polarization. TonEBP suppresses IL-10 expression and M2 macrophage phenotype by enhancing chromatin accessibility and Sp1 recruitment to the IL-10 promoter when TonEBP is knocked down. |
ChIP, siRNA knockdown, reporter assays, neutralizing antibodies, macrophage polarization assays |
Frontiers in immunology / Scientific reports |
Medium |
27160066 31057560
|
| 2020 |
In microglia, TonEBP acts as a transcriptional cofactor for both NF-κB and AP-1; LPS induces assembly of a TonEBP/NF-κB/AP-1/p300 complex. Microglial-specific TonEBP deletion blocks LPS-induced pro-inflammatory cytokine expression, microglial activation, and subsequent neuronal cell death and memory loss. Cerulenin disrupts this complex. |
shRNA knockdown, myeloid-specific conditional KO mice, reporter assays, co-immunoprecipitation, LPS challenge, behavioral memory tests |
Journal of neuroinflammation |
Medium |
33292328
|
| 2010 |
NFAT5 controls the expression of CD24 in T cells in response to hypertonicity; NFAT5 binds to the Cd24 promoter in response to hyperosmotic stress, facilitating local chromatin derepression and increased CD24 mRNA and protein expression, which is required to sustain T cell expansion under osmostress. |
ChIP, chromatin accessibility assay, NFAT5 KO and T cell-specific KO mice, in vitro hypernatremia, flow cytometry |
Journal of immunology |
Medium |
21037089
|
| 2015 |
TonEBP binds the PPARγ2 promoter and blocks the epigenetic transition required for its activation, suppressing adipogenesis and insulin signaling. TonEBP reduction permits PPARγ2 epigenetic remodeling, enhanced adipogenesis, and improved insulin response. |
ChIP, epigenetic chromatin analysis, reporter assays, knockdown/overexpression, adipogenesis functional assays |
Scientific reports |
Medium |
26042523
|
| 2016 |
NFAT5 binds a consensus sequence TGGAAGCGTTC in the promoter of Cacna1c (L-type calcium channel α1C) and activates its transcription. siRNA knockdown of Nfat5 suppresses Cacna1c expression and decreases L-type calcium current in neonatal cardiomyocytes; morpholino-mediated knockdown in zebrafish abolishes cacna1c expression and results in non-contractile ventricle, rescued by overexpression of either cacna1c or nfat5. |
Promoter binding/reporter assay, siRNA knockdown, electrophysiology (L-type Ca2+ current), zebrafish morpholino knockdown, rescue experiments |
Journal of molecular medicine |
High |
27368804
|
| 2024 |
Aurora kinase B (AURKB) phosphorylates NFAT5, enhancing its protein stability and nuclear translocation, which directly controls AQP4 (aquaporin-4) expression in astrocytes. The AURKB-NFAT5-AQP4 pathway regulates astrocyte swelling and contributes to neuropathic pain in the spinal dorsal horn. |
Phosphorylation assays, protein stability assays, nuclear translocation assays, AQP4 promoter reporter, spinal cord neuropathic pain model, selective astrocyte vs. microglia analysis |
Advanced science |
Medium |
38195869
|
| 2022 |
NLRC3 inhibits NF-κB p65 binding to the NFAT5 promoter by decreasing NF-κB activation and p300 co-activator activity through sequestration of mTOR and p300 in the NLRC3-mTOR-p300 complex, reducing NFAT5-dependent glycolytic gene expression in immunosuppressed macrophages. |
Myeloid-specific NLRC3 deletion, co-immunoprecipitation of NLRC3-mTOR-p300 complex, ChIP for p65 at NFAT5 promoter, glycolysis assays |
Molecular therapy |
Medium |
36068919
|
| 2024 |
Serum amyloid A (SAA) activates NFAT5 expression and activity in macrophages via the TLR2/4-JNK signaling pathway. The SAA-TLR2/4-NFAT5 axis promotes macrophage migration and chemotaxis in an IL-6- and CCL2-dependent manner. Genetic ablation of NFAT5 or TLR2/4 rescues SAA-induced arthritis pathology in mice. |
In vitro macrophage stimulation with SAA, TLR2/4 and NFAT5 genetic KO mice, JNK pathway inhibition, migration/chemotaxis assays, intraarticular SAA injection model |
The Journal of clinical investigation |
Medium |
38426494
|