doi: 10.1128/MCB.00392-12.
Epub 2012 Oct 8.
TAZ suppresses NFAT5 activity through tyrosine phosphorylation
Affiliations
- PMID: 23045390
- PMCID: PMC3510524
- DOI: 10.1128/MCB.00392-12
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TAZ suppresses NFAT5 activity through tyrosine phosphorylation
Mol Cell Biol.
2012 Dec.
Abstract
Transcriptional coactivator with PDZ-binding motif (TAZ) physically interacts with a variety of transcription factors and modulates their activities involved in cell proliferation and mesenchymal stem cell differentiation. TAZ is highly expressed in the kidney, and a deficiency of this protein results in multiple renal cysts and urinary concentration defects; however, the molecular functions of TAZ in renal cells remain largely unknown. In this study, we examined the effects of osmotic stress on TAZ expression and activity in renal cells. We found that hyperosmotic stress selectively increased protein phosphorylation at tyrosine 316 of TAZ and that this was enhanced by c-Abl activation in response to hyperosmotic stress. Interestingly, phosphorylated TAZ physically interacted with nuclear factor of activated T cells 5 (NFAT5), a major osmoregulatory transcription factor, and subsequently suppressed DNA binding and transcriptional activity of NFAT5. Furthermore, TAZ deficiency elicited an increase in NFAT5 activity in vitro and in vivo, which then reverted to basal levels following restoration of wild-type TAZ but not mutant TAZ (Y316F). Collectively, the data suggest that TAZ modulates cellular responses to hyperosmotic stress through fine-tuning of NFAT5 activity via tyrosine phosphorylation.
Figures
Tyrosine phosphorylation of TAZ by hyperosmotic stress-induced c-Abl activation. (A) mIMCD-3 cells were exposed to 300 and 400 mosmol/kg NaCl for 4 h. Whole-cell extracts (WCE) were immunoprecipitated (IP) with anti-TAZ Ab, and immune complexes were immunoblotted (IB) with Abs against phosphotyrosine (4G10), phosphoserine TAZ (pS89), and TAZ. (B) Stable mIMCD-3 cells (mock and Flag-TAZ) were cultured under low-salt (150 mosmol/kg), normal (300 mosmol/kg), and hyperosmotic (400 mosmol/kg) conditions for 4 h and then incubated with Flag-M2 beads. TAZ immune complexes were analyzed by immunoblotting with 4G10 and anti-TAZ Abs. (C) Total proteins were extracted from mIMCD-3 cells for immunoblotting of phosphorylated Abl (pAbl), Abl, and actin. (D) Purified Flag-tagged TAZ was incubated with recombinant c-Abl kinase (rAbl) in the presence of radiolabeled ATP. The reaction mixture was then analyzed by SDS-PAGE and radiography. Separately, the TAZ level was assessed by immunoblotting. (E) 293T cells were transfected with Flag-TAZ, with (+) or without (−) the c-Abl expression vector. TAZ immune complexes were resolved by SDS-PAGE, followed by immunoblotting with 4G10. (F) mIMCD-3 cells were infected with viruses expressing pSRP-Abl (siAbl), and whole-cell extracts were used for TAZ immunoprecipitation, followed by immunoblotting with 4G10. (G) 293T cells were transfected with Flag-TAZ and c-Abl expression vectors and subsequently cultured in the presence of genistein (GNS; 50 μM) or STI-571 (STI; 10 μM) for 1 h. TAZ immune complexes were analyzed by immunoblotting. (H) mIMCD-3 cells were stimulated with hyperosmotic stress in the presence or absence of STI (10 μM). TAZ immune complexes were subsequently analyzed with 4G10 and anti-TAZ Abs.
c-Abl-induced phosphorylation of TAZ at tyrosine 316. (A) 293T cells were transfected with Flag-TAZ and c-Abl expression vectors. TAZ immune complexes were resolved by SDS-PAGE and immunoblotted with 4G10 and Abs against pY116, pY141, pY300, pY316 TAZ, and TAZ. (B) Parental mIMCD-3 cells were incubated with hyperosmolar medium for 4 h. Endogenous TAZ protein was immunoprecipitated and immunoblotted with pY316 TAZ or TAZ Ab. (C) Purified TAZ proteins were incubated with recombinant c-Abl kinase in the presence of radiolabeled ATP. The reaction mixture was analyzed by SDS-PAGE and radiography. (D) 293T cells were transfected with WT TAZ and Y316F TAZ, with or without the c-Abl expression vector. Immune complexes were resolved and immunoblotted with 4G10, pY316 TAZ, and TAZ Abs. (E) mIMCD-3 cells were stably transfected with the TAZ expression vector, and stable cells were selected in the presence of puromycin. The cells were incubated under hyperosmotic stress for 4 h, followed by immunoprecipitation and immunoblot analysis.
Physical interaction between phosphorylated TAZ and NFAT5. (A to C) 293T cells were transfected with c-Abl, Myc-tagged WT or mutated (Y143F or MT) NFAT5, and Flag-tagged WT or Y316F TAZ, as indicated above each panel. (A) NFAT5 immune complexes were precipitated by incubation using anti-Myc Ab and analyzed by SDS-PAGE and immunoblotting. (B) Cells were additionally treated with STI-571 (10 μM) for 1 h before harvest. TAZ immune complexes were analyzed by immunoblotting with Abs against Myc, pY316, and TAZ. (C) TAZ immune complexes were resolved by SDS-PAGE and subjected to immunoblotting. (D to F) mIMCD-3 cells were stimulated with different tonicities for 4 h. (D) Endogenous TAZ immune complexes were precipitated with TAZ Ab, followed by SDS-PAGE and immunoblotting with NFAT5 and pY316 Abs. (E) Separately, mIMCD-3 cells were cultured either in the presence or in the absence of STI, and TAZ immune complexes were immunoblotted with NFAT Ab. (F) Tonicity-stimulated mIMCD-3 cells were fixed and incubated with TAZ and NFAT5 Abs, followed by incubation with specific secondary Abs with PLA probes, using a Duolink in situ PLA kit. Samples were observed under a confocal microscope, and the number of spots per cell was measured. nd, not determined.
Suppression of NFAT5 activity by TAZ. (A) 293T cells were transfected with NFAT5 and different amounts of TAZ expression vector together with pTonE-luc. pCMVβgal was used as a control. NFAT5 and TAZ expression was determined by immunoblot analysis. Relative luciferase activity was calculated after normalization to β-galactosidase activity. (B) 293T cells were transfected with NFAT5 and TAZ (WT or Y316F) expression vectors together with pTonE-luc and pCMVβgal. Reporter activity was calculated from 3 independent experiments and expressed as the mean. (C) NFAT5 was overexpressed in 293T cells together with WT or Y316F TAZ. Whole-cell extracts were incubated with biotinylated double-stranded DNA containing the NFAT5/TonEBP-binding element for 1 h and subsequently incubated with streptavidin-agarose beads for an additional 2 h, followed by SDS-PAGE and immunoblotting. DNA-binding complexes were analyzed with NFAT5 and TAZ Abs. (D) mIMCD-3 or stable mIMCD-3 cells (mock [con], TAZ, and Y316F TAZ) were stimulated with either 150 or 400 mosmol/kg for 4 h. Whole-cell extracts were immunoprecipitated with NFAT5 Ab, and chromatin was analyzed by a PCR using specific primers for the NFAT5-binding element within the SMIT gene promoter. (E) mIMCD-3 cells (shcon and shTAZ) were cultured under 400 mosmol/kg and harvested for ChIP and real-time PCR. (F) WT and KO MEF cells were stimulated with 400 mosmol/kg for 4 h, and cells were analyzed by ChIP and quantitative PCR. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.
Inhibitory effects of TAZ on NFAT5-induced gene expression. (A and B) Stable TAZ transfectants (TAZ) and control cells (CON) were cultured under different tonicity conditions for 4 h. (A) Expression of TAZ and NFAT5 was determined by immunoblotting. (B) Total RNA was isolated from cells and subjected to reverse transcription and real-time PCR for measuring BGT1 and SMIT1 levels. Relative expression levels were calculated after normalization against β-actin levels. (C and D) TAZ knockdown (shTAZ) and control (shcon) cells were established and stimulated with normal and hyperosmolar conditions for 4 h. (C) Protein levels of TAZ, NFAT5, and actin were confirmed by immunoblotting. (D) Relative mRNA levels of BGT1 and SMIT were determined by real-time PCR. *, P < 0.05; **, P < 0.005.
Resuppression of NFAT5 activity by restored TAZ, but not Y316F TAZ, in TAZ KO cells. (A) TAZ expression in WT and KO MEF cells was determined by immunoblotting. (B) WT and KO MEF cells were transfected with reporter genes (pTonE-luc and pCMVβgal) and treated with different tonicities for 24 h. (C) Total RNAs were harvested from WT and KO MEF cells incubated under hyperosmolar conditions and used for measuring mRNA levels of BGT1 and SMIT. (D) TAZ KO MEF cells were transfected with WT or Y316F TAZ expression vector together with reporter gene vectors (pTonE-luc and pCMVβgal). Cells were then challenged with normal or hyperosmolar medium for 24 h. Reporter activities (relative light units [RLU]) in panels B and D were calculated after normalization with β-galactosidase activity and are expressed as means ± SEM for three independent experiments. (E) KO MEF cells were restored with WT and Y316F TAZ expression vectors and subsequently treated with hyperosmolar medium. Total RNA was used for reverse transcription and real-time PCR analysis. (F) Total RNAs were isolated from the kidneys of WT and KO mice (10 to 12 weeks old; n = 4), followed by reverse transcription and real-time PCR analysis. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.
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