taz suppresses nfat5 activity through tyrosine phosphorylation

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TAZ suppresses NFAT5 activity through tyrosine phosphorylation 1

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Eun Jung Janga,1,, Hana Jeonga,1, Ki Hwan Hanb, Hyug Moo Kwonb, Jeong-Ho Hongd and 3

Eun Sook Hwanga,* 4

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aCollege of Pharmacy and Division of Life and Pharmaceutical Sciences and Center for Cell 6

Signaling & Drug Discovery Research, Ewha Womans University, Seoul 120-750, Korea 7

bSchool of Medicine, Ewha Womans University, Seoul 158-709, Korea 8

cSchool of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science 9

and Technology, Ulsan 689-798, Korea 10

dSchool of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea 11

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1These authors equally contributed to this work. 13

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Short title: NFAT5 suppression by phosphorylated TAZ 15

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Total 28,128 characters (excluding Materials and Methods and References) 17

Abstract includes 178 words 18

Materials and Methods include 739 words 19

Introduction, Results, and Discussion include 2,546 words 20

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*To whom correspondence should be addressed: 23

Eun Sook Hwang, Ph.D. 24

College of Pharmacy and Division of Life and Pharmaceutical Sciences 25

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.00392-12 MCB Accepts, published online ahead of print on 8 October 2012

on February 11, 2018 by guest

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Ewha Womans University 26

52 Ewhayeodae-gil, Seodaemun-gu 27

Seoul 120-750, Korea 28

Tel: 82-2-3277-4369, Fax: 82-2-3277-3760 29

E-mail: [email protected]


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Abstract 31

Transcriptional co-activator with PDZ-binding motif (TAZ) physically interacts with a variety 32

of transcription factors and modulates their activities involved in cell proliferation and 33

mesenchymal stem cell differentiation. TAZ is highly expressed in the kidney and its 34

deficiency displays multiple renal cysts and urinary concentration defects; however, the 35

molecular functions of TAZ in renal cells remain largely unknown. Here, we examined the 36

effects of osmotic stress on TAZ expression and activity in renal cells. We found that 37

hyperosmotic stress selectively increased protein phosphorylation at tyrosine 316 of TAZ, 38

which was enhanced by c-Abl activation in response to hyperosmotic stress. Of interest, the 39

phosphorylated TAZ physically interacted with nuclear factor of activated T-cells 5 (NFAT5), 40

a major osmoregulatory transcription factor, and subsequently suppressed DNA binding and 41

transcriptional activity of NFAT5. Furthermore, TAZ deficiency elicited an increase in 42

NFAT5 activity in vitro and in vivo, which then reverted to basal levels following restoration 43

of wild-type TAZ, but not the mutant TAZ (Y316F). Collectively, we suggest that TAZ 44

modulates cellular responses to hyperosmotic stress through fine-tuning of NFAT5 activity 45

via tyrosine phosphorylation. 46

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Keywords: TAZ, hyperosmotic stress, c-Abl, NFAT5, tyrosine phosphorylation 48

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Introduction 51

Transcriptional co-activator with PDZ-binding motif (TAZ), also known as WW domain-52

containing transcription regulator 1 (WWTR1), was first identified as a 14-3-3-interacting 53

phosphoprotein (16). Phosphorylation of TAZ at serine 89 is required for its interaction with 54

14-3-3 and substantial retention in the cytoplasm (16). In addition to the 14-3-3 binding motif, 55

TAZ contains multiple functional domains, including a WW domain, a coiled-coil domain, 56

and a PDZ-binding motif, through which it modulates the activity of transcription factors 57

involved in stem cell differentiation (13). For example, TAZ interacts with runt-related 58

transcription factor 2 (RUNX2) and MyoD during osteoblast and myogenic differentiation 59

and promotes cell lineage-specific gene transcription (6, 13, 15). During adipogenic 60

differentiation, TAZ suppresses the activity of peroxisome proliferator-activated receptor 61

(PPAR)-γ, thus inhibiting adipogenic differentiation from mesenchymal stem cells (13). TAZ 62

also controls organ development, including thyroid, cardiac, and limb development, by 63

association with thyroid transcription factor-1 (TTF-1), paired box gene (PAX) 8, and PAX3 64

(7, 28-30). Furthermore, TAZ promotes cell proliferation through activation of TEAD 65

transcription factors, resulting in tumor cell growth and epithelial-mesenchymal transition (4, 66

5, 19, 22, 37). Much of our understanding of the physiological function of TAZ in vivo has 67

been derived from studies using TAZ knockout (KO) mice, which develop significant 68

pathogenic phenotypes, such as lung emphysema and multiple kidney cysts (14, 24). 69

Although the pathogenic mechanisms at play in the TAZ KO mice largely remain to be 70

explored, TAZ is known to suppress the expression and activity of the transmembrane protein 71

polycystin-2 (PC2) in the kidney (8, 33). Increased PC2 expression in TAZ KO mice may be 72

involved in the development of polycystic kidney disease (33). In addition, TAZ interacts 73

with the transcription factor Glis-3 and enhances its transcriptional activity. It is evident that 74

deficiency of either TAZ or Glis-3 in mice leads to abnormal cilia formation and polycystic 75


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kidneys (17). Collectively, these studies suggest that TAZ plays a critical role in normal 76

kidney development and function through different mechanisms. 77

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Since hyperosmolar medullary interstitial fluid is essential for urinary concentration in the 79

kidney, renal medullary cells are normally exposed to extracellular hyperosmotic stress, 80

which can cause cell shrinkage, DNA damage, and cell apoptosis. To avoid hyperosmotic 81

stress-induced damage, renal medullary cells exhibit rapid adjustment via a complex network 82

of osmoprotective molecules, including kinases, heat shock proteins, p53, and osmolyte-83

accumulating genes (2). The osmolyte-accumulating genes include the betaine-GABA 84

transporter (BGT1) (35) and the sodium/myo-inositol cotransporter (SMIT) (34, 36). 85

Induction of these genes results in organic osmolyte accumulation, which prevents acute 86

hyperosmotic stress-induced water loss and cell shrinkage. Expression of BGT1 and SMIT is 87

transcriptionally controlled by the nuclear factor of activated T-cells 5 (NFAT5)/tonicity-88

responsive enhancer-binding protein (TonEBP), hereafter referred to as NFAT5 (10, 26, 27). 89

NFAT5 deficiency blocks the expression of osmolyte-accumulating genes in the kidney 90

medulla and produces a severe urinary concentration defect in mice, suggesting a critical role 91

for NFAT5 in the hyperosmotic stress-signaling pathway (21). However, recent studies 92

indicate that NFAT5 is activated by various tonicity-independent mechanisms and exhibits 93

tissue-specific functions, such as cell migration, vascular remodeling, and carcinoma invasion 94

(12). Although the hyperosmolarity-dependent and -independent activation and functions of 95

NFAT5 have been extensively characterized in numerous cell types, mechanisms underlying 96

the suppression of NFAT5 activity remain unclear. 97

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In this study, we investigated the effects of hyperosmotic stress on TAZ activity and its 99

relationship with NFAT5 function in renal medullary cells. Of interest, TAZ underwent 100


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tyrosine phosphorylation in response to hyperosmotic stress and specifically associated with 101

NFAT5, suppressing its DNA-binding activity and transcriptional activity. Our results suggest 102

that TAZ functions as a modulator of NFAT activity in response to hyperosmotic stress. 103

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Materials and Methods 105

106

Reagents 107

Antibodies (Abs) against phosphorylated serine 89 of TAZ (pS89), TAZ, Oct1, β-actin, 108

phospho-Abl (pAbl), phosphotyrosine (pY, 4G10), c-Abl and NFAT5 were purchased from 109

Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), Cell Signaling Technology (Boston, MA), 110

Upstate Biotechnology Inc. (Lake Placid, NY), and Abcam Inc. (Cambridge, MA). Flag Ab 111

and Flag-M2 agarose beads were from Sigma-Aldrich (St Louis, CA). Recombinant c-Abl 112

kinase and imatinib (STI-571) were obtained from Calbiochem (San Diego, CA) and 113

Novartis Pharmaceuticals (Basel, Switzerland), respectively. Abs recognizing phospho-114

tyrosine (pY118, pY141, pY300, and pY316) were generated by AbFrontier Inc. (Seoul, 115

Republic of Korea) 116

117

Animals 118

Wild type (WT) C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, MN). 119

WT and TAZ knockout (KO) mice were maintained under specific pathogen-free conditions. 120

All animal experiments were performed in accordance with the IACUC guidelines and were 121

approved by IACUC at Ewha Womans University (ELAGC-09-1017 and IACUC 2010-13-3). 122

123

Cell culture 124

Cos7 and HEK293T cells were obtained from American Type Culture Collection (ATCC, 125

Manassas, VA) and cultured in Dulbecco's modified Eagle medium (DMEM, Invitrogen, 126

Carlsbad, CA). Cells were transiently transfected with calcium phosphate transfection 127

methods. Tonicity-responsive immortalized mouse inner medullary collecting duct-3 cells 128

(mIMCD-3, ATCC) and mouse embryonic fibroblasts (MEF) of WT and TAZ KO mice were 129


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maintained in DMEM supplemented with 10% heat inactivated FBS (HyClone, Logan, UT). 130

Cells were exposed to low salt, normal, or hyperosmolar medium (150, 300, or 400 131

mOsm/Kg H2O final osmolarity) for 2–4 h with NaCl. For hyperosmotic stimulation, cells 132

were exposed to low salt (150 mOsm/kg) conditions for 2 h and subsequently cultured under 133

normal or hyperosmolar medium for 4 h. 134

135

In situ proximity ligation assay (PLA) 136

Renal medullary mIMCD-3 cells were plated onto a slide glass and incubated under normal 137

and hyperosmotic medium for 4 h. Cells were fixed and incubated with mouse anti-TAZ Ab 138

(1:100, Abcam) and rabbit anti-NFAT5 Ab (27). In situ PLA was performed according to the 139

manufacturer’s description (DUO-link, Olink Bioscience, Uppsala, Sweden). Fluorescence 140

was then examined under confocal fluorescence microscopy (LSM510 META, Carl Zeiss Inc., 141

Germany). 142

143

Reporter gene assays 144

293T cells were plated onto a 6-well plate with number of 5×105 cells/well and transiently 145

transfected with NFAT5 and TAZ expression vector and NFAT5/TonEBP-responsive 146

element–linked luciferase (pTonE-luc) as well as pCMVβgal as a transfection control. 147

Luciferase activity was assayed using the Bright-Glo luciferase assay kit (Promega, Madison, 148

WI). Relative luciferase units were calculated after normalization with β-galactosidase 149

activity (TROPIX, Bedford, MA). 150

151

Real-time PCR analysis 152

Total RNA was isolated from cells using TRIzol (Gibco-BRL, Invitrogen) and used for 153

reverse transcription for cDNA synthesis (Invitrogen). Real-time PCR reactions were 154


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performed with SYBR Green pre-mix buffer and an ABI-Prism 7300 sequence detector 155

(Perkin-Elmer Applied Biosystems, Foster City, CA). Relative expression levels were 156

determined after normalization to the Ct values for β-actin. Gene-specific primers were as 157

follows: BGT1, 5′-ctgggagagacgggttttgggtattacatc-3′, 5′-ggaccccaggtcgtggat-3′; SMIT, 5′-158

ccgggcgctctatgacctggg-3′, 5′-caaacagagaggcaccaatcg-3′; and β-actin, 5′-159

agagggaaatcgtgcgtgac-3′, 5′-caatagtgatgacctggccgt-3′. 160

161

DNA pull-down assay 162

Cells were lysed with HKMG buffer (10 mM Hepes, pH 7.9, 100 mM KCl, 5 mM MgCl2, 163

10% glycerol, 0.1% NP-40, and 1 mM DTT) and incubated with biotinylated double stranded 164

DNA and subsequently with streptavidin agarose beads. Precipitates were washed with 165

HKMG buffer three times and applied onto SDS-PAGE for immunoblot assay. DNA 166

sequence containing NFAT5/TonEBP-binding element is as follow: 5′-167

cttggtggaaaattaccgctggt-3′ and 5′-aaccagcggtccttttccaccaa-3′. 168

169

Chromatin immunoprecipitation (ChIP) and quantitative real time-PCR 170

Cells were formaldehyde cross-linked in a 1% solution for 10 min as described previously 171

(15). Chromatin extracts were prepared and incubated with anti-NFAT5 Ab at 4°C overnight, 172

followed by incubation with protein A/G-agarose beads (Pierce, Rockford, IL, USA). 173

Immune complexes were sequentially washed and then heated to reverse the formaldehyde 174

cross-linking of the protein and DNA complexes. The DNA fragments were purified using the 175

QIAquick PCR Purification kit (QIAGEN, Hilden, Germany) and then used for PCR. Primers 176

were as follows: SMIT-ChIP-F 5′-ttgcggcttggtgcagcagtc-3′, SMIT-ChIP-R 5′-177

tcgctcggcacgcacccgctc-3′. 178

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Establishment of stable cell lines by retroviral transduction 181

Viral-producing phoenix cells were cultured in DMEM supplemented with 10% FBS and 182

transfected with the TAZ-expressing retroviral vector using the calcium phosphate 183

transfection method. Viral supernatants were harvested and added to mIMCD-3 or MEF cells 184

in the presence of polybrene (4 μg/ml, Sigma-Aldrich) for 24 h. Stable cells were established 185

by puromycin resistance (Sigma-Aldrich). 186

187

Statistical analysis 188

All data are expressed as the mean ± SEM. The statistical significance of the experimental 189

results was calculated by unpaired Student’s t-test. Values under 0.05 were considered 190

statistically significant. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.191


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Results 192

Hyperosmotic stress induces tyrosine phosphorylation of TAZ through c-Abl activation 193

In order to examine the effects of osmotic stress on TAZ expression and activity, mouse renal 194

medullary cells, mIMCD-3 were cultured under normal (300 mOsm/kg) and hyperosmotic 195

(400 mOsm/kg) conditions. Hyperosmotic stimulation of mIMCD-3 cells had no effect on 196

either expression of TAZ or phosphorylation of TAZ at serine 89, compared to that under 197

normal conditions. However, phosphorylation of TAZ on tyrosine residues was strikingly 198

elevated under hyperosmotic stress (4G10) (Fig. 1A). This observation was confirmed by the 199

finding that tyrosine phosphorylation of ectopically expressed TAZ proteins was selectively 200

enhanced by hyperosmolarity (Fig. 1B). Interestingly, endogenous c-Abl tyrosine kinase was 201

activated by hyperosmotic stimulation, as demonstrated by the increased detection of 202

phosphorylated, but not total c-Abl, in response to hyperosmotic stress (Fig. 1C). To 203

determine whether c-Abl was an upstream tyrosine kinase for TAZ, we performed an in vitro 204

kinase assay using recombinant c-Abl kinase. Immunoprecipitated TAZ protein was directly 205

phosphorylated by c-Abl in a cell-free system (Fig. 1D). Furthermore, co-expression of c-Abl 206

with TAZ in 293T cells enhanced the tyrosine phosphorylation of TAZ (Fig. 1E). In contrast, 207

Abl knockdown abolished TAZ phosphorylation induced by hyperosmotic stimulation (Fig. 208

1F). Furthermore, the c-Abl-induced tyrosine phosphorylation was inhibited by genistein, a 209

non-selective tyrosine kinase inhibitor and by STI-571, a c-Abl kinase-specific inhibitor (Fig. 210

1G). Hyperosmotic stress also promoted tyrosine phosphorylation of the endogenous TAZ 211

protein along with c-Abl activation, but failed to induce tyrosine phosphorylation of TAZ in 212

the presence of STI-571 (Fig. 1H). 213

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Tyrosine 316 of TAZ is the c-Abl phosphorylation site 215

We next attempted to identify the c-Abl phosphorylation sites in TAZ. Since TAZ contains 4 216


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tyrosine residues (Y118, Y141, Y300, and Y316), we first generated TAZ-specific 217

phosphotyrosine Abs (pY118, pY141, pY300, and pY316) using synthetic TAZ peptides 218

containing the appropriate phosphotyrosine. The c-Abl-induced tyrosine phosphorylation of 219

TAZ was prominently detected by 4G10 and by immunoblotting with the pY316 TAZ Ab, but 220

not with other Abs (Fig. 2A). We also confirmed that hyperosmotic stress enhanced the 221

tyrosine phosphorylation of endogenous TAZ selectively on Y316 (Fig. 2B). We next 222

constructed the tyrosine mutants of TAZ (Y118F, Y141F, Y300F, and Y316F) and performed 223

in vitro Abl kinase assay using purified TAZ proteins. TAZ proteins including wild-type (WT), 224

Y118F, Y141F, and Y300F were phosphorylated by c-Abl but Abl phosphorylation was not 225

appeared in Y316F mutant (Fig. 2C). Overexpression of c-Abl with WT and Y316F TAZ also 226

increased the phosphorylation of WT TAZ but failed to phosphorylate the Y316F mutant, as 227

demonstrated by immunoblotting with 4G10 and pY316 TAZ Abs (Fig. 2D). In addition, 228

stable mIMCD-3 cells expressing Flag-tagged WT TAZ and its tyrosine mutants were 229

established and exposed to hyperosmotic stress. Hyperosmotic stimulation induced tyrosine 230

phosphorylation in WT, Y118F, Y141F, and Y300F TAZ proteins, but this hyperosmotic 231

stress-induced phosphorylation was impaired in the Y316F mutant (Fig. 2E), indicating that 232

tyrosine 316 of TAZ is the c-Abl phosphorylation site. 233

234

TAZ physically interacts with NFAT5 via hyperosmotic stress-induced phosphorylation 235

To identify the functions of phosphorylated TAZ in the hyperosmotic stress-mediated 236

signaling pathway, we examined the relationship between TAZ and NFAT5, a key regulator 237

of the hyperosmotic stress-signaling pathway (3, 27). For determining the interaction between 238

TAZ and NFAT5, we transiently expressed Flag-TAZ and NFAT5 together with c-Abl in 239

293T cells. Interestingly, co-immunoprecipitation and immunoblotting analysis demonstrated 240

the physical interaction between TAZ and NFAT5 (Fig. 3A). However, Y316F mutant TAZ 241


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was incapable of interacting with NFAT5 (Fig. 3A). In addition, treatment of cells with STI-242

571 reduced the physical interaction between TAZ and NFAT5 and diminished the tyrosine 243

phosphorylation of TAZ (Fig. 3B), indicating a tyrosine phosphorylation-dependent 244

interaction of TAZ with NFAT5. As NFAT5 is also phosphorylated at tyrosine 143 by c-Abl 245

(11), we tested whether tyrosine phosphorylation of NFAT5 was also essential for its 246

interaction with TAZ. The interaction of NFAT5 with TAZ was sustained in Y143F mutant 247

NFAT5, as indicated by the comparable presence of WT and mutant NFAT5 in TAZ immune 248

complexes (Fig. 3C). Most importantly, analysis of endogenous TAZ immune complexes 249

verified that TAZ interacted with NFAT5 under normal conditions and more strongly under 250

hyperosmotic stress in a tyrosine phosphorylation-dependent manner (Fig. 3D). However, 251

treatment of cells with STI-571 attenuated the interaction between TAZ and NFAT5 under 252

hypertonic conditions (Fig. 3E). Furthermore, the in situ proximity ligation assay (PLA) 253

system visualized the individual TAZ-NFAT5 interaction in the nucleus of mIMCD-3 cells 254

cultured in normal or hyperosmolar medium, but not in low salt (150 mOsm/kg) medium (Fig. 255

3F). 256

257

TAZ suppresses DNA-binding and transcriptional activities of NFAT5 258

Next, we determined the effects of the TAZ-NFAT5 interaction on NFAT5 activity. 259

Transcriptional activity of NFAT5 was assessed with a NFAT5/TonEBP-responsive luciferase 260

reporter gene (pTonE-luc) in the presence or absence of TAZ. Ectopic expression of NFAT5 261

increased the luciferase activity of pTonE-luc, but co-expression of TAZ significantly 262

suppressed this activity in a dose-dependent manner (Fig. 4A). Furthermore, the NFAT5-263

induced reporter activity was inhibited by overexpression of WT or Y118F TAZ, whereas 264

Y316F TAZ did not suppress NFAT5 activity (Fig. 4B), which was consistent with the 265

inability of Y316F to interact with NFAT5. The TAZ-mediated suppression of NFAT5 266


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transcriptional activity led us to examine the DNA-binding activity of NFAT5 in the presence 267

of TAZ. A DNA-pull-down assay revealed that NFAT5 directly bound to a DNA sequence 268

containing the NFAT5/TonEBP-binding element, and this DNA-NFAT5 association was 269

markedly inhibited by overexpression of WT TAZ, but not the Y316F mutant (Fig. 4C). More 270

significantly, ChIP assay demonstrated that the NFAT5 bound to SMIT promoter region in 271

response to hyperosmolarity. The NFAT5-DNA association was suppressed by the 272

overexpression of WT TAZ, but not by Y316F TAZ (Fig. 4D). More significantly, NFAT5 273

binding to SMIT promoter was augmented in TAZ-deficient cells, as evidenced by ChIP and 274

real time-PCR analysis (Fig. 4E and F). These results indicate that the TAZ-NFAT5 275

interaction suppresses the transcriptional activity of NFAT5 by inhibiting the DNA-binding 276

activity of NFAT5. 277

278

Expression of BGT1 and SMIT is negatively regulated by TAZ expression 279

Since TAZ suppressed DNA binding and transcriptional activity of NFAT5, we investigated 280

the effects of TAZ overexpression on the expression of endogenous NFAT5 target genes, such 281

as BGT1 and SMIT. TAZ protein level was increased in TAZ stable cells compared to control 282

mIMCD-3 cells, whereas NFAT5 expression was comparable between control and TAZ stable 283

cells and unchanged by tonicity challenges (Fig. 5A). Of interest, the relative expression of 284

BGT1 and SMIT was drastically increased by hyperosmotic stimulation in control cells but 285

this increase was significantly attenuated by the overexpression of TAZ (Fig. 5B). Conversely, 286

we measured the levels of BGT1 and SMIT in TAZ knockdown cells (shTAZ) (Fig. 5C). 287

Hyperosmotic stress consistently increased the expression of BGT1 and SMIT in control 288

(shcon) cells. However, the level of BGT1 and SMIT was substantially elevated by a 289

reduction in TAZ expression under both normal and hyperosmolar conditions (Fig. 5D). 290

These results suggest that TAZ expression level may be important for fine-tuning NFAT5 291


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activity induced by hyperosmotic stimulation. 292

293

Restoration of TAZ, but not Y316F, in TAZ-deficient cells leads to suppression of the 294

enhanced NFAT5 activity 295

We wanted to confirm that the increase in NFAT5 activity caused by the lack of TAZ could be 296

directly reversed by TAZ restoration. For the re-introduction of TAZ or the Y316F mutant, 297

MEF cells were isolated from WT and KO mice and cellular responses were examined under 298

different osmotic stresses. TAZ expression in the WT and KO MEF cells was verified by 299

immunoblot analysis (Fig. 6A). MEF cells were then transfected with the pTonE-luc reporter 300

gene and challenged with media of different osmolarity. Hyperosmotic stimulation increased 301

the reporter activity in WT MEF cells, but had a much greater effect on the TAZ KO cells 302

(Fig. 6B). Consistently, BGT1 mRNA level was much greater in hyperosmolarity-stimulated 303

KO cells than in WT cell (Fig. 6C). Next, the increased pTonE-luc activity in TAZ KO cells 304

reverted to basal levels upon restoration of WT TAZ. However, introduction of Y316F TAZ 305

was much less effective in inhibiting reporter activity (Fig. 6D). The endogenous mRNA 306

level of SMIT was accordingly regulated by the restoration of WT or the Y316F mutant (Fig. 307

6E). Lastly, we attempted to measure the in vivo effect of TAZ deficiency on expression 308

levels of NFAT5-mediated osmoregulatory genes because TAZ suppressed NFAT5 activity in 309

vitro. Despite severe morphological defects in the kidneys of TAZ KO mice, expression of 310

SMIT and BGT1 was significantly increased in the renal medulla of TAZ KO kidney 311

compared to that in WT kidney (Fig. 6F). 312

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Discussion 314

Our results demonstrate that TAZ undergoes tyrosine phosphorylation at tyrosine 316 by c-315

Abl kinase upon hyperosmotic stimulation and then specifically interacts with and suppresses 316

the transcriptional activity of NFAT5. While ectopic TAZ overexpression suppresses the 317

transcription of NFAT5 target genes, such as SMIT and BGT1, TAZ deficiency increases the 318

expression of SMIT and BGT1 in vitro and in vivo. Collectively, our data are the first to 319

demonstrate that TAZ functions as a suppressor of NFAT5 activity in response to 320

hyperosmotic stress. 321

322

Interestingly, TAZ selectively associates with and modulates activity of NFAT5 in a tyrosine 323

phosphorylation-dependent manner. Hyperosmotic stress activates multiple mitogen-activated 324

kinases (1), protein kinase A (9), and tyrosine kinases, such as syk and yes (25, 31), which 325

then modulate the expression of genes required for osmotic regulation. TAZ is also 326

phosphorylated by Ser/Thr protein kinases at serine residues (19, 20), but its S89 327

phosphorylation was not affected by hyperosmotic stimulation. Recently, c-Abl was 328

identified as the kinase responsible for the hyperosmotic stress signaling via NFAT5 329

phosphorylation (11). Gallazzaini et al. explored that c-Abl was activated within 1 h upon 330

500 mOsm/kg stimulation and the activated c-Abl phosphorylated NFAT5 on tyrosine 143 331

and stimulated its nuclear localization and transcriptional activity (11). Our results 332

demonstrate that TAZ, like NFAT5, is a novel substrate for c-Abl in kidney cells stimulated 333

with hyperosmolarity for 4 h. However, the phosphorylated TAZ subsequently suppresses 334

NFAT5 activity through a phosphorylation-dependent physical interaction. Interestingly, c-335

Abl-induced tyrosine phosphorylation of TAZ is a prerequisite for suppressing NFAT5 336

activity, whereas c-Abl-mediated NFAT5 phosphorylation is not essential for its interaction 337

with TAZ. These findings suggest that c-Abl may have dual roles in timely regulation of renal 338


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tonicity by accelerating and decelerating NFAT5 activity through sequential phosphorylation 339

of NFAT5 and TAZ. The detailed mechanisms and in vivo implications of c-Abl-mediated 340

NFAT5 and TAZ regulation remain to be uncovered. In addition, a few punctate spots of 341

TAZ-NFAT5 complex were visualized in the nucleus, indicating the fine-tuning of NFAT5 342

activity by a phosphorylated TAZ. 343

344

Our results suggest that TAZ deficiency may cause the constitutive activation of NFAT5 in 345

renal medullary cells and thus lead to the cellular resistance to hyperosmotic stress-induced 346

damage. However, TAZ KO mice die shortly after birth due to developmental defects in 347

multiple organs, a few viable TAZ KO mice display multicystic kidneys with polyuria (24). 348

Despite abnormal renal structure or function, the renal medullas of TAZ KO mice express 349

significantly higher levels of BGT1 and SMIT than those of WT mice. Most importantly, 350

however, the in vivo importance of TAZ-mediated NFAT5 suppression should be verified 351

from viable mice with kidney-specific knockout of TAZ exposed to hyperosmotic stress. On 352

the other hand, NFAT5 have been extensively characterized to have hyperosmolarity-353

independent and tissue-specific functions in a variety of cell types (12, 18, 32). NFAT5 KO 354

mice are embryonic lethal due to impaired cardiac development and function and display 355

renal atrophy (21, 23); thus, it notes that NFAT5 is essential for heart and kidney 356

development. Therefore, TAZ-mediated NFAT5 regulation may also be important in 357

regulating heart and kidney development, but its detailed molecular functions and 358

mechanisms are poorly understood. Together, our results suggest that TAZ functions in fine-359

tuning NFAT5 activity through tyrosine phosphorylation and contributes to cellular 360

adaptation to hyperosmotic stress. 361

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Acknowledgements 363

This work was supported by the Ewha Global Top5 Grant 2011 of Ewha Womans University, 364

R&D program (A080908) of Korea Health Industry Development Institute and partly by a 365

National Research Foundation grant funded by the Ministry of Education and Science 366

Technology of Korea (2012-0000952). 367

368

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502

503

504


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Figure legends 505

506

Figure 1. Tyrosine phosphorylation of TAZ by hyperosmotic stress-induced c-Abl activation. 507

(A) mIMCD-3 cells were exposed to 300 and 400 mOsm/kg NaCl for 4 h. Whole cell extracts 508

(WCE) were immunoprecipitated with anti-TAZ Ab, and immune complexes were 509

immunoblotted with Abs against phosphotyrosine (4G10), phosphoserine TAZ (pS89), and 510

TAZ. (B) Stable mIMCD-3 cells (mock and Flag-TAZ) were cultured under low salt (150 511

mOsm/kg), normal (300 mOsm/kg), and hyperosmotic (400 mOsm/kg) conditions for 4 h and 512

incubated with Flag-M2 beads. TAZ immune complexes were analyzed by immunoblotting 513

with 4G10 and anti-TAZ Ab. (C) Total proteins were extracted from mIMCD-3 cells for 514

immunoblotting of phosphorylated Abl (pAbl), Abl, and actin. (D) Purified Flag-tagged TAZ 515

was incubated with recombinant c-Abl kinase (rAbl) in the presence of radiolabeled ATP. The 516

reaction mixture was then analyzed by SDS-PAGE and radiography. Separately, TAZ level 517

was assessed by immunoblotting. (E) 293T cells were transfected with Flag-TAZ with (+) or 518

without (–) the c-Abl expression vector. TAZ immune complexes were resolved by SDS-519

PAGE, followed by immunoblotting of 4G10. (F) mIMCD-3 cells were infected with viruses 520

expressing pSRP-Abl (siAbl) and whole cell extracts were used for TAZ immunoprecipitation, 521

followed by immunoblotting of 4G10. (G) 293T cells were transfected with Flag-TAZ and c-522

Abl expression vectors and subsequently cultured in the presence of genistein (GNS; 50 μM) 523

or STI-571 (STI; 10 μM) for 1 h. TAZ immune complexes were analyzed by immunoblotting. 524

(H) mIMCD-3 cells were stimulated with hyperosmotic stress in the presence or absence of 525

STI (10 μM). TAZ immune complexes were subsequently analyzed with 4G10 and anti-TAZ 526

Abs. 527

528

Figure 2. c-Abl-induced phosphorylation of TAZ at tyrosine 316. (A) 293T cells were 529


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transfected with Flag-TAZ and c-Abl expression vectors. TAZ immune complexes were 530

resolved by SDS-PAGE and immunoblotted with 4G10 and Abs against pY116, pY141, 531

pY300, pY316 TAZ, or TAZ. (B) Parental mIMCD-3 cells were incubated with hyperosmolar 532

medium for 4 h. Endogenous TAZ protein was immunoprecipitated and immunoblotted with 533

pY316 TAZ or TAZ Ab. (C) Purified TAZ proteins were incubated with the recombinant c-534

Abl kinase in the presence of radiolabeled ATP. Reaction mixture was analyzed by SDS-535

PAGE and radiography. (D) 293T cells were transfected with WT TAZ and Y316F TAZ with 536

or without the c-Abl expression vector. Immune complexes were resolved and immunoblotted 537

with 4G10, pY316 TAZ, and TAZ Abs. (E) mIMCD3 cells were stably transfected with the 538

TAZ expression vector and stable cells were selected in the presence of puromycin. The cells 539

were incubated under hyperosmotic stress for 4 h, followed by immunoprecipitation and 540

immunoblot analysis. 541

542

Figure 3. Physical interaction between phosphorylated TAZ and NFAT5. (A–C) 293T cells 543

were transfected with c-Abl, Myc-tagged WT or mutated (Y143F; MT) NFAT5 and Flag-544

tagged TAZ WT or Y316F, as indicated above each panel. NFAT5 immune complexes were 545

precipitated by incubation using anti-Myc Ab and analyzed by SDS-PAGE and 546

immunoblotting (A). Cells were additionally treated with STI-571 (10 μM) for 1 h before 547

harvest. TAZ immune complexes were analyzed by immunoblotting with Abs against Myc, 548

pY316, and TAZ (B). TAZ immune complexes were resolved by SDS-PAGE and subjected to 549

immunoblotting (C). (D–F) mIMCD-3 cells were stimulated with different tonicities for 4 h. 550

Endogenous TAZ immune complexes were precipitated with TAZ Ab, followed by SDS-551

PAGE and immunoblotting with NFAT5 and pY316 Abs (D). Separately, mIMCD-3 cells 552

were cultured either in the presence or in the absence of STI and TAZ immune complexes 553

were immunoblotted with NFAT Ab (E). Tonicity-stimulated mIMCD3 cells were fixed and 554


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incubated with TAZ and NFAT5 Abs, followed by incubation with specific secondary Abs 555

with PLA probes using the Duolink in situ PLA kit. Samples were observed under a confocal 556

microscope and the number of spots per cell was measured (F). 557

558

Figure 4. Suppression of NFAT5 activity by TAZ. (A) 293T cells were transfected with 559

NFAT5 and different amounts of TAZ expression vector together with the pTonE-luc. 560

pCMVβgal was used as a control. NFAT5 and TAZ expression were determined by 561

immunoblot analysis. Relative luciferase activity was calculated after normalization to β-562

galactosidase activity. (B) 293T cells were transfected with NFAT5 and TAZ expression 563

vectors (WT or Y316F) together with pTonE-luc and pCMVβgal. Reporter activity was 564

calculated from 3 independent experiments and are expressed as mean. (C) NFAT5 was 565

overexpressed in 293T cells together with WT or Y316F TAZ. Whole cell extracts were 566

incubated with biotinylated double-stranded DNA containing the NFAT5/TonEBP-binding 567

element for 1 h and subsequently with streptavidin-agarose beads for an additional 2 h, 568

followed by SDS-PAGE and immunoblotting. DNA-binding complexes were analyzed with 569

NFAT5 and TAZ Abs. (D) mIMCD3 or stable mIMCD-3 cells (mock, TAZ, and Y316F) were 570

stimulated with either 150 or 400 mOsm/kg for 4 h. Whole cell extracts were 571

immunoprecipitated with NFAT5 Ab and chromatins were analyzed by PCR using specific 572

primers for NFAT5-binding element within SMIT gene promoter. (E) mIMCD3 cells (shcon 573

and shTAZ) were cultured under 400 mOsm/kg and harvested for ChIP and real time-PCR. 574

(F) WT and KO MEF cells were stimulated with 400 mOsm/kg for 4 h and cells were 575

analyzed by ChIP and quantitative PCR. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. 576

577

Figure 5. Inhibitory effects of TAZ on NFAT5-induced gene expression. (A, B) TAZ stable 578

transfectants (TAZ) and control (CON) cells were cultured under different tonicity conditions 579


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for 4 h. Expression of TAZ and NFAT5 was determined by immunoblotting (A). Total RNA 580

was isolated from cells and subjected to reverse transcription and real-time PCR for 581

measuring BGT1 and SMIT1 levels. Relative expression levels were calculated after 582

normalization against β-actin levels (B). (C, D) TAZ knockdown (shTAZ) and control (shcon) 583

cells were established and stimulated with normal and hyperosmolar conditions for 4 h. 584

Protein levels of TAZ, NFAT5, and actin were confirmed by immunoblotting (C). Relative 585

mRNA levels of BGT1 and SMIT were determined by real time-PCR (D). *, P < 0.05; **, P < 586

0.005. 587

588

Figure 6. Re-suppression of NFAT5 activity by restored TAZ, but not Y316F, in TAZ KO 589

cells. (A) TAZ expression was determined in WT and KO MEF cells by immunoblotting. (B) 590

WT and KO MEF cells were transfected with reporter genes (pTonE-luc and pCMVβgal) and 591

treated with different tonicity for 24 h. (C) Total RNA was harvested from WT and KO MEF 592

cells incubated under hyperosmolar conditions and used for measuring mRNA level of BGT1 593

and SMIT. (D) TAZ KO MEF cells were transfected with WT or Y316F TAZ expression 594

vector together with reporter genes (pTonE-luc and pCMVβgal). Cells were then challenged 595

with normal or hyperosmolar medium for 24 h. Reporter activity (RLU) in panels B and D 596

was calculated after normalization with β-galactosidase activity and are expressed as mean ± 597

SEM of three independent experiments. (E) KO MEF cells were restored with WT and 598

Y316F TAZ expression vector and subsequently treated with hyperosmolar medium. Total 599

RNA was used for reverse transcription and real time-PCR analysis. (F) Total RNA was 600

isolated from the kidneys of WT and KO mice (10–12 weeks, n = 4), followed by reverse 601

transcription and real time-PCR analysis. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. 602


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taz suppresses nfat5 activity through tyrosine phosphorylation

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