Nuclear Factor of Activated T3 Is a Negative Regulator of Ras-JNK1/2-AP-1–Induced Cell Transformation

Research Article

Ke Yao,1 Yong-Yeon Cho,1 H. Robert Bergen III,2 Benjamin J. Madden,2 Bu Young Choi,1 Wei-Ya Ma,1 Ann M. Bode,1 and Zigang Dong1

1Hormel Institute, University of Minnesota, Austin, Minnesota and 2Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, Minnesota

Abstract and the activation signal is transmitted to downstream substrate(s) such as c-Jun (11). JNK1 and JNK2 are well known for the activation The c-jun-NH2-kinases (JNK) play a critical role in tumor 63 73 promoter–induced cell transformation and apoptosis. Here, and phosphorylation of c-Jun at Ser and Ser . However, other we showed that the nuclear factor of activated T3 (NFAT3) is downstream target proteins include Elk-1 (12), c-Myc (13), p53 (14), phosphorylated by JNK1 or JNK2 at Ser213 and Ser217, which are and NFATc2 (15), as well as several members of the apoptosis- located in the conserved SP motif. The transactivation domain related family of proteins, including Bcl-2, Bcl-XL, Bim, and Bad of NFAT3 is found between amino acids (aa) 113 and 260 and (16–18). These functions of JNKs have been primarily attributed to includes the phosphorylation targets of JNK1 and JNK2. the fact that JNKs activate different substrates based on the specific À À NFAT3 transactivation activity was suppressed in JNK1 / or stimulus or cell type. À À JNK2 / mouse embryonic fibroblast (MEF) cells compared Although the nuclear factor of activated T cell (NFAT) family of transcription factors has been primarily identified in immune cells, with wild-type MEF cells. Moreover, a 3xNFAT-luc reporter recent studies indicated that NFAT is functionally active in several gene assay indicated that NFAT3 transcriptional activity was other non-immune cell types, including vascular endothelial cells, increased in a dose-dependent manner by JNK1 or JNK2. 213 217 embryonic exon cells, and 3T3-L1 fibroblasts (19–22). Four different Double mutations at Ser and Ser suppressed NFAT3 isotypes of NFATs, including NFAT1 (1a, 1b, and 1c), NFAT2 (2a and transactivation activity; and SP600125, a JNK inhibitor, sup- 2b), NFAT3, and NFAT4 (4x, 4a, 4b, and 4c) were shown to have pressed NFAT3-induced 3xNFAT-luciferase activity. Knock- differential tissue distribution (23). These findings suggested that down of JNK1 or JNK2 suppressed foci formation in NIH3T3 distinct NFAT isotypes play different roles in diverse tissues under cells. Importantly, ectopic expression of NFAT3 inhibited AP-1 various physiologic conditions (24). Calcineurin, a Ca2+/calmodu- activity and suppressed foci formation. Furthermore, knock- lin-dependent protein phosphatase that is a downstream target of down of NFAT3 enhanced Ras-JNK1 or JNK2-induced foci intracellular Ca2+ signaling, is a well-known effector of the NFAT formation in NIH3T3 cells. Taken together, these results family of transcription factors (NFAT1–4; ref. 23). Classically, provided direct evidence for the anti-oncogenic potential of calcineurin dephosphorylates NFAT1–4, allowing NFAT to translo- the NFAT3 transcription factor. [Cancer Res 2007;67(18):8725–35] cate to the nucleus, bind to consensus DNA sites, and control gene transcription (24). Upon cessation of the Ca2+ signal, NFAT proteins Introduction are re-phosphorylated by kinases such as GSK-3 (25), resulting in The JNKs signal transduction pathway has been shown to play the translocation of NFAT to the cytoplasm (24). However, recent an important role in coordinating various cellular responses studies indicated that the Ras signaling pathway positively such as apoptosis (1), proliferation (2), and neoplastic transforma- regulates NFAT3 activity (26) by forming an activation complex tion (3). Mice that are deficient in both jnk1 and jnk2 exhibit to regulate PPARg2 promoter activity, which leads to adipocyte embryonic death at E10.5 due to enhanced apoptosis in the differentiation (27). In addition, RSK2-mediated phosphorylation of hindbrain (4) and forebrain regions (4, 5), which clearly suggests NFAT3 regulates NFAT3 activity and induces muscle cell differen- that JNK1 and JNK2 are involved in cell survival during develop- tiation (28). Furthermore, the NFAT3 protein forms a complex with ment. Furthermore, specific antisense oligonucleotides against CBP to activate transcription machinery (29), and NFATc1 binds JNKs inhibited tumor cell growth (6) and jnk2-deficient mice with AP-1 to enhance its transcriptional activity (30). Activation of displayed significant suppression of skin papilloma development NFATc1 was also reported to induce cell transformation (22). On induced by 12-O-tetradecanoylphorbol-13-acetate (TPA; ref. 7). the other hand, NFATc2 was shown to repress cyclin-dependent These types of observations may have been due to an enhanced kinase 4 (CDK4), resulting in cell cycle arrest at G0-G1 (20, 31). In apoptosis in jnk-deficient cells and mice (8). addition, when the lymphomagenic virus SL3-3 was infected in When cells are stimulated by environmental stress, cytokines, or NFAT4-deficient mice, T-cell lymphoma developed faster and with toxins (9), JNK phosphorylation is increased through MKK4/7(10), higher frequency compared with wild-type mice (20). These reports strongly indicate that the oncogenic or anti-oncogenic activities of the NFAT proteins are dependent on the isotype and specific physiologic condition. However, the role of NFAT3 in the tumori- Note: Supplementary data for this article are available at Cancer Research Online genesis is not yet understood. (http://cancerres.aacrjournals.org/). K. Yao and Y-Y. Cho contributed equally to this work. In this study, we showed that NFAT3 is a strong binding partner Requests for reprints: Zigang Dong, Hormel Institute, University of Minnesota, of JNK1 and JNK2. The phosphorylation of NFAT3 at 213 and 217 801 16th Avenue NE, Austin, MN 55912. Phone: 507-437-9600; Fax: 507-437-9606; by JNK1/2 induced NFAT3 transactivation activity. Importantly, E-mail: [email protected]. G12V I2007American Association for Cancer Research. overexpression of NFAT3 suppressed Ras -JNK1– or -JNK2– doi:10.1158/0008-5472.CAN-06-4788 induced foci formation by inhibiting AP-1 activity. Taken together, www.aacrjournals.org 8725 Cancer Res 2007; 67: (18). September 15, 2007

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Cancer Research these results indicated that NFAT3 inhibited neoplastic transfor- System protocols. In brief, 293 cells were maintained in 10% FBS-DMEM, 4 mation through a negative feedback regulation of JNK1/2-AP-1 seeded into 48-well plates (2.0 Â 10 ), and incubated with 10% FBS-DMEM signaling. for 18 h before transfection. The various DNAs, pACT-TFs, pBIND-JNK1 or pBIND-JNK2, and pG5-luciferease were combined in the same molar ratio (1:1:1), and the total amount of DNA was not more than 100 ng per well. Materials and Methods The transfection was done using jetPEI following the manufacturer’s Reagents and antibodies. DMEM and fetal bovine serum (FBS) were recommended protocols. For the luciferase assay, the cells were disrupted purchased from Life Technologies, Inc. Restriction enzymes and some by the addition of lysis buffer and incubated for 30 min at room modifying enzymes were obtained from New England BioLabs, Inc. The temperature with gentle shaking. The luciferase activity was measured DNA ligation kit (version 2.0) was purchased from TAKATA Bio, Inc. The automatically by the addition of 100 AL substrate buffer, and data were À À LipofectAMINE Plus transfection reagent for NIH3T3, JNKWT, JNK1 / , collected using the Luminoskan Ascent (MTX Lab, Inc.). The relative À À and JNK2 / mouse embryonic fibroblast (MEF) cells were purchased luciferase activity was calculated using the pG5-luciferase basal control and from Invitrogen. The JetPEI transfection reagent for 293 cells was from normalized against Renilla luciferase activity, which included the pBIND Q-Biogene. The pcDNA3.1 plasmid was purchased from Life Technologies, vector as an internal control. Inc. The luciferase assay substrate was from Promega. Antibodies against In vitro kination assay. Wild-type GST-NFAT3 and deletion or point the Flag or v5 epitope were purchased from Sigma-Aldrich or Invitrogen, mutant proteins were used for the in vitro kination assay using an active respectively. Antibodies against JNK1 and NFAT3 were purchased from JNK1 or JNK2 (Upstate Biotechnology, Inc.). Reactions were carried out at Santa Cruz Biotechnology, Inc., and the antibody against JNK2 was from 30jC for 30 min in a mixture containing 50 Amol/L unlabeled ATP and Cell Signaling Technology, Inc. 10 ACi of [g-32P] ATP and then stopped by adding 6Â SDS sample buffer. À À Cell culture. The 293 (human embryonic kidney), JNKWT,JNK1/ , Samples were boiled and then separated by 12% SDS-PAGE and visualized À À and JNK2 / MEF cells were cultured in DMEM supplemented with 10% by autoradiography or Coomassie blue staining. heat-inactivated FBS in a 37jC, 5% CO2 incubator. NIH/3T3 cells were NFAT3 protein domain analysis. Data for analyzing the NFAT3 protein cultured in DMEM supplemented with 10% bovine calf serum in a 37jC, domains were downloaded from the ExPASy Proteomics Server (NiceProt

5% CO2 incubator. The cells were maintained by splitting at 80% to 90% view of Swiss-Prot: Q14934). Putative phosphorylation sites were predicted confluence, and media were changed every 3 days. by the Netphospho 2.0 server.4 Construction of expression and siRNA vectors. For the mammalian Construction and expression of GST-NFAT3 fusion proteins. The two-hybrid system assay, the pACT-transcription factors (TF), pBIND-JNK1 NFAT3 wild-type and deletion mutants were constructed as described (28). and -JNK2, and pcDNA3.1-v5-JNK1 and -JNK2 were constructed as To express and purify the glutathione S-transferase (GST)-fusion NFAT3 previously described (28). The pcDNA3.1-FLAG-NFAT3 and various deletion fusion proteins, the BL21 bacterial strain was used. A single colony for each mutants of NFAT3 from the COOH-terminal end (residues 1–853, 1–580, vector was cultured, and the GST-fusion protein was induced by isopropyl- 1–450, 1–365, 1–260, 1–219, and 1–112) were described (28). The deletion L-thio-h-D-galactopyranoside (final concentration, 0.5 mmol/L) treatment GST-NFAT3 fusion vectors (1–112, 113–260, 261–365, 366–450, 451–580, at 25jC for 5 h. The protein was then partially purified with 1Â IB wash 581–853, 853–902) were generated by PCR using wild-type NFAT3 full buffer (Novagen) for disruption, binding to glutathione Sepharose 4B, and length (FL) subcloned into the BamHI/XbaI site of the pGEX-5X-C vector. elution with 10 mmol/L reduced glutathione, followed by dialysis. Purified The mutation of Ser213 and Ser217 to alanine was carried out using the proteins were stored À70jC until used. QuickChange II Site–Directed Mutagenesis Kit (Strategene) according to LTQ Orbitrap hybrid mass spectrometer analysis. Protein phosphor- recommended protocols. The deletion mutants of NFAT3-1–112, -113–260 ylation site assignment was carried out using ingel trypsin digest and and -261–902, NFAT3-113–260 and NFAT3-113-260S213,217A, and NFAT3- nanoLC-ESI-tandem hybrid mass spectrometry with a ThermoFinnigan S213A,217A were also subcloned into the pcDNA4 (Invitrogen) or pGEX- LTQ Orbitrap Hybrid Mass Spectrometer. Briefly, the Coomassie blue– 5X-C vector. The pU6pro vector (provided by David L. Turner, University stained protein bands were excised from the gel and cut into pieces no of Michigan, Ann Arbor, MI) was used to construct small interfering RNA larger than 2 mm2. Before digestion, the gel pieces were destained with JNK1 (si-JNK1), siRNA JNK2 (si-JNK2), and siRNA NFAT3 (si-NFAT3) 50% acetonitrile/50 mmol/L Tris-HCl (pH, 8.1) until clear. Gel pieces were following the recommended protocols.3 All of the constructs were con- then reduced with 20 mmol/L DTT/50 mmol/L Tris-HCl (pH, 8.1) at 55jC firmed by restriction enzyme mapping and DNA sequencing. for 30 min and alkylated with 40 mmol/L iodoacetamide at room Western blotting. The proteins were extracted with NP40 cell lysis temperature for 30 min in the dark. Proteins were digested in situ with buffer with freezing and thawing. The same amount of protein was resolved 30 AL (0.004 Ag/AL) trypsin (Promega) in 20 mmol/L Tris-HCl (pH, 8.1) by SDS-PAGE, transferred onto polyvinylidene difluoride membranes, at 37jC overnight, followed by peptide extraction with 60 ALof2% hybridized with appropriate antibodies, and then visualized using the trifluoroacetic acid followed by 60 AL of acetonitrile. The pooled extracts enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences). were concentrated to <5 AL on a SpeedVac spinning concentrator (Savant À À NFAT3 activity assay. The 293 cells (2.0 Â 104) or JNKWT, JNK1 / , and Instruments) and then brought up in 0.1% formic acid for protein À À JNK2 / MEFs (4.0 Â 104) were seeded into 48- or 12-well plates and identification by nanoflow liquid chromatography electrospray tandem cultured in 10% FBS-DMEM for 18 h before transfection, respectively. The mass spectrometry (nanoLC-ESI-MS/MS) using a ThermoFinnigan LTQ 3xNFAT-luc reporter plasmid, which was constructed by fusion of three Orbitrap Hybrid Mass Spectrometer (ThermoElectron Bremen) coupled to NFAT binding consensus sequences on the 5¶ upstream end of the minimal an Eksigent nanoLC-2D HPLC system (Eksigent). The peptide mixture is interleukin 2 promoter-luciferase reporter plasmid, was transfected with loaded onto a 250-nL OPTI-PAK trap (Optimize Technologies) packed various recombinant plasmids as indicated in the respective figures. The with Michrom Magic C8 solid phase (Michrom Bioresources) and eluted cells were cultured for 36 h and were or were not stimulated with UVB or with a 0.1% formic acid/acetonitrile gradient through a Michrom packed specific chemicals and then cultured for an additional 12 h. At each time tip capillary Magic C18 column (75 Am Â 150 mm). The LTQ Orbitrap point, cells were disrupted and analyzed for firefly luciferase activity. The mass spectrometer experiment was set to perform an Fourier transform 3xNFAT-luc luciferase activity was normalized against Renilla luciferase (FT) full scan from 380 to 1,600 m/z with resolving power set at 60,000 activity (pRL-SV40). (400 m/z), followed by linear ion trap MS/MS scans on the top three ions. Mammalian two-hybrid assay. For the mammalian two-hybrid (M2H) The ambient air polycyclodimethylsiloxane 391 m/z ion was used as an assay, we followed the Promega Checkmate Mammalian Two-Hybrid internal lock mass for the FT full scans giving 2 ppm or better mass

3 http://sitemaker.umich.edu/dlturner.vectors 4 http://www.cbs.dtu.dk/services/NetPhos

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Anti-oncogenic Potential of NFAT3 accuracy. To detect phosphorylated peptides, a MS scan is triggered if one not detected, and any mutants with deletions to aa 219 or 112 did of the top three ions in the MS/MS scan corresponds to the neutral loss of not coprecipitate with the JNK1 or JNK2 protein (Fig. 1C and D, one or two phosphoric acid moieties with and without water for [M + 2H]2+ 3+ lanes 7–9). Interestingly, when aa 450 to 580 were deleted, the (49, 58, 98, and 107 m/z) and [M+3H] (32.7and 65.4 m/z) precursor ions. binding affinity of NFAT3 with JNK1 or JNK2 was increased (Fig. 1C Dynamic exclusion was set to 2, and selected ions were placed on an and D, lane 5–6). In addition, the immunoprecipitated levels of exclusion list for 90 s. The MS/MS raw data were converted to DTA files using ThermoElectron Bioworks 3.2 and correlated to theoretical fragmen- JNK1 or JNK2 were almost the same (Fig. 1C and D, bottom). These tation patterns of tryptic peptide sequences from the Swissprot databases results suggested that aa 260 to 365 of NFAT3 are important for using both SEQUEST (ThermoElectron) and Mascot (Matrix Sciences) binding with JNK1 and JNK2. search algorithms running on 10 node clusters. All searches were conducted NFAT3 is a substrate of JNK1 and JNK2 in vitro. To identify with fixed cysteine modifications of +57for carboxamidomethyl-cysteines the target region of NFAT3 that harbors potential phosphorylation and variable modifications allowing +16 with methionines for methionine sites for JNK1 or JNK2, we constructed a series of GST-NFAT3 sulfoxide, +42 for protein NH2-terminal acetylation, +80 at serine, threo- deletion fragments (Fig. 2A, vector maps). These proteins were À nine, and tyrosine for phosphorylation and 18 for serine and threonine expressed partially purified as described in Materials and Methods H3PO4 loss. The search was restricted to trypsin-generated peptides and then subjected to an in vitro phosphorylation assay. The results allowing for two missed cleavages and was left open to all species. An indicated that JNK1 and JNK2 phosphorylated NFAT3 in the region additional search was done using only the expected sequence with no enzyme. Peptide mass search tolerances are set to 20 ppm, and fragment spanning aa 113 to 260 (Fig. 2A, top and middle), which includes a mass tolerance was set to F0.8 Da. Matches to phosphorylated peptides calcineurin binding site and two SP repeat regions. The p38 kinase were further interpreted manually to assign the site of phosphorylation is a well-known upstream kinase of NFAT3 that phosphorylates 168 170 using both +80 and À18 diagnostic ions. Preferences were given to matches NFAT3 at Ser and Ser (34). To determine whether the phos- of the more abundant ions. phorylation sites for JNKs and p38 were identical, we constructed Focus forming assay. Transformation of NIH3T3 cells was conducted an NFAT3113-260 protein with mutations to replace Ser168 and according to standard protocols (32). Cells were transiently transfected with Ser170 with alanine (NFAT3-113-260S168,170A) and conducted the G12V various combinations of H-Ras (50 ng), JNK1 or JNK2 (450 ng), siRNA in vitro phosphorylation assay (Fig. 2B). Results indicated that the vector DNA (450 ng), and pcDNA3-mock (compensation for equal amount phosphorylation of the mutant NFAT3-113-260S168,170A by JNK1 of DNA) as indicated in figures and then cultured in 5% FBS-DMEM or JNK2 was the same as for wild-type NFAT3-113-260 (Fig. 2B, for 2 weeks. Foci were fixed with methanol, stained with 0.5% crystal violet, 168 170 and then counted with a microscope and the Image-Pro PLUS software left and middle, lane 2 versus 3), suggesting that Ser and Ser of program. NFAT3 are not the phosphorylation targets of JNK1 or JNK2. Identification of the NFAT3 sites phosphorylated by JNK1 and JNK2. To determine the specific site(s) of NFAT3-113-260 that Results are phosphorylated by JNK1 and JNK2, we used the human protein NFAT3 is a binding partner of JNK1 and JNK2 in vivo. To reference database5 to compare the amino acid similarity among identify novel binding or substrate protein(s) for JNKs, we various JNK substrates. Results showed that JNK1 and JNK2 introduced JNK1 or JNK2 cDNA, including the open reading frame recognized and phosphorylated various substrates at the SP/TP (ORF), into the pBIND mammalian two-hybrid system vector consensus sequences (Supplementary Fig. S2A). Moreover, we (pBIND-JNK1 or pBIND-JNK2) as bait. The ORF of each TF was found that NFAT3-113-260 contains 13 SP/TP motifs (Supplemen- amplified by PCR and then introduced into the pACT mammalian tary Fig. S2B), including Ser168 and Ser170, which are phosphory- two-hybrid system vector (pACT-TFs). Each individual pACT-TF lated by p38 (34), but not by JNK1 or JNK2 (Fig. 2B). Therefore, plus the pG5-luciferase reporter plasmid was cotransfected into 293 we next used the LTQ Orbitrap hybrid mass spectrometer to cells with pBIND-JNK1 or -JNK2. Each interaction activity was identify the NFAT3 sites that are phosphorylated by JNK1 and compared against the pG5-luc/pBIND-JNK1 or -JNK2 as the basal JNK2 as described in Materials and Methods. The results indicated level (Fig. 1A, lane 1 or 7, respectively). Elk-1, c-Jun, and activat- that JNK1 and JNK2 phosphorylated two sites on NFAT3 at Ser213 ing transcription factor 2 (ATF2) were used as positive controls and Ser217 (Supplementary Fig. S2B and C). To confirm these (Fig. 1A, lanes 4–6 and 10–12). NFAT3 and NFAT4 showed strong phosphorylation sites, we constructed mutants of GST-NFAT3- interacting activity with pBIND-JNK1 or -JNK2 compared with the 113-260, replacing Ser213 and/or Ser217 with alanine (GST-NFAT3- pG5-luc/pBIND-JNK1 or -JNK2 control (Fig. 1A, lanes 2 and 3 113-260S217A or GST-NFAT3-113-260S213,217A; Fig. 2C). These versus 1 or 8 and 9 versus 7, respectively). NFAT4 has been iden- proteins were expressed and partially purified and then directly tified previously as a binding partner for JNKs (33), and therefore, subjected to the in vitro phosphorylation assay using active JNK1 we selected NFAT3 for further study. To verify the interaction or JNK2. Results confirmed that mutation of NFAT3-113-260 at of NFAT3 with JNK1 and JNK2, we introduced the pcDNA3-v5-JNK1 Ser217 caused a marked decrease in phosphorylation by JNK1 or -JNK2 construct and the pcDNA3-Flag-NFAT3 construct into (Fig. 2C, left, lane 3) or JNK2 (Fig. 2C, middle, lane 3). Further- 293 cells. Results of immunoprecipitation (IP) experiments showed more, mutations at both Ser213 and Ser217 totally blocked phos- that NFAT3 could bind with JNK1 or JNK2 in vivo (Fig. 1B). phorylation of NFAT3-113-260 by either JNK1 (Fig. 2C, left, lane 4) To identify the NFAT3 domain that is involved in the interaction or JNK2 (Fig. 2C, middle, lane 4). These results showed that of NFAT3 with JNK1 or JNK2, we constructed pcDNA3-Flag-NFAT3 Ser213 and Ser217 are the amino acids targeted for phosphorylation deletion mutants (Supplementary Fig. S1A) that were then by JNK1 and JNK2. individually transfected into 293 cells with pcDNA3-v5-JNK1 or JNK1 and JNK2 regulate NFAT3 activity. To examine the role -JNK2, and expression was confirmed (Supplementary Fig. S1B). of JNK1 and JNK2 in the regulation of NFAT3, we first developed IP results using a v5 monoclonal antibody showed that the FL pGal4-NFAT3-1-902 and pGal4-NFAT3-261-902 constructs (Fig. 3A, NFAT3 and deletion mutants from the COOH-terminal to aa 365 coprecipitated with JNK1 or JNK2 (Fig. 1C and D, lanes 2–6). However, when aa 365–260 were deleted, the NFAT3 protein was 5 http://www.hprd.org www.aacrjournals.org 8727 Cancer Res 2007; 67: (18). September 15, 2007

Figure 1. NFAT3 is a binding partner of JNK1 and JNK2 in vivo. A, assessment of the in vivo protein-protein interaction of pBIND-JNK1 or pBIND-JNK2 with pACT-transcription factors (TF) as determined by the mammalian two-hybrid assay. Activity is indicated by relative luminescence units normalized to a negative control (value for cells transfected with only pG5-luc/pBIND-JNK1 = 1.0 or pG5-luc/pBIND-JNK2 = 1.0). The firefly luciferase activity was normalized against the Renilla luciferase activity. Columns, means of values obtained from triplicate experiments; bars, SD. Significant differences were evaluated using the Student’s t test. *, P < 0.01; **, P < 0.0001. B, JNK1 or JNK2 was co-immunoprecipitated with NFAT3. The pcDNA3-v5-JNK1 or -JNK2 plasmid was transfected alone or together with pcDNA3-Flag-NFAT3 into 293 cells and then cultured for 36 h at 37jC in a 5% CO2 incubator. Cells transfected with the pcDNA3-mock vector served as a negative control. The proteins were extracted as described in Materials and Methods, and 100 Ag were used for IP with anti-Flag. JNK1 and JNK2 were visualized by Western blot with anti–v5-horseradish peroxidase using the ECL kit (Amersham Biosciences). C, identification of the NFAT3 domain that binds with JNK1. To identify the domain of NFAT3 where JNK1 binds, the FL and 7 pcDNA3-Flag-NFAT3 deletion constructs (D1–D7) were individually cotransfected with pcDNA3-v5-JNK1 into 293 cells. After culturing for 48 h, cells were disrupted with NP40 cell lysis buffer and immunoprecipitated with a v5 monoclonal antibody. NFAT3 was detected by Western blot using anti–Flag-HRP. D, identification of the NFAT3 domain that binds with JNK2. Details are as for C, except that constructs were individually cotransfected with pcDNA3-v5-JNK2 into 293 cells. vector map) and analyzed the transactivation activity of NFAT3 delineate the role of JNK1 and JNK2 in the regulation of NFAT3 using cotransfection of each of these constructs with the 5xGal4- transactivation, we created Gal4-NFAT3-1-112 and Gal4-NFAT3- luciferase reporter plasmid into JNK wild-type (JNKWT) MEF cells. 113-260 constructs and cotransfected each with p5xGal4-luc and À À The transactivation activity of NFAT3 was significantly increased analyzed NFAT3 transactivation activity in JNKWT, JNK1 / , and À À in cells transfected with Gal4-NFAT3-1-902 compared with cells JNK2 / MEFs (Fig. 3C). Results indicated that the transactivation transfected with the pGal4-mock control (Fig. 3A, lane 2). On the activity of NFAT3-113-260 was strongly increased compared with other hand, the NH2-terminal–deleted Gal4-NFAT3-261-902 trun- the pGal4 mock control, but NFAT3-1-112 did not induce trans- cated form markedly suppressed the transactivation activity of activation (Fig. 3C). Furthermore, the increased transactivation NFAT3 (Fig. 3A, lane 3). These results indicated that the activity of Gal4-NFAT3-113-260 was significantly inhibited in À/À À/À transactivation activity might be regulated in the NH2-terminal JNK1 or JNK2 MEFs similar to that observed for NFAT3-1- area spanning aa 1 to 260 of the NFAT3 protein. Next, we 902 in these same cells (Fig. 3B and C). To further verify a role for transfected the pGal4-NFAT3-1-902 plasmid into JNKWT, JNK1 JNKs in regulating the transcriptional activity of NFAT3, we À À À À knock-out (JNK1 / ), or JNK2 knock-out (JNK2 / ) MEF cells. The transfected increasing amounts of pcDNA3-Flag-NFAT3/pcDNA3.1- resulting 5xGal4-luciferase activity indicated that the increased v5-JNK1 or pcDNA3-Flag-NFAT3/pcDNA3.1-v5-JNK2 with the transactivation activity of Gal4-NFAT3-1-902 observed in JNKWT 3xNFAT-luciferase reporter plasmid and analyzed luciferase acti- À À À À MEFs was significantly suppressed in JNK1 / or JNK2 / MEFs vity (Fig. 3D). Results indicated that NFAT3-mediated 3xNFAT- (Fig. 3B), suggesting that JNK1 and JNK2 might be positive luciferase activity was increased in a JNK1 or JNK2 dose-dependent regulators of the transactivation activity of NFAT3. To further manner (Fig. 3D). Taken together, these results indicated that JNK1

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Anti-oncogenic Potential of NFAT3 and JNK2 phosphorylate NFAT3 at Ser213 and Ser217 and positively The results indicated that NFAT3FL with mutations at Ser213 and regulate NFAT3 transcriptional activity. Ser217 had no effect on the interaction with JNK1 compared with Ser213 and Ser217 of NFAT3 are involved in transactivation unmodified wild-type NFAT3 (Fig. 4B). In addition, we observed but not with the interaction of NFAT3 with JNK1 or JNK2. To similar results for the binding affinity of JNK2 with the NFAT3 examine the role of Ser213 and Ser217 in NFAT3 activity, we first mutant (data not shown). To determine the effect of phosphory- analyzed the effect of SP600125, a JNK inhibitor. The p3xNFAT- lation of Ser213 and Ser217 on the transactivation activity of NFAT3, À À luciferase plasmid was transfected into JNKWT MEFs either with we used JNKWT and JNK1 / MEFs cotransfected with pGal4- or without pcDNA3-Flag-NFAT3, and then cells were treated with NFAT3-113-260 or pGal4-NFAT3-113-260S213,217A and p5xGal4- 10 Amol/L SP600125. The results indicated that SP600125 sup- luc. The results showed that mutation of NFAT3 at Ser213 and Ser217 pressed NFAT3-induced 3xNFAT-luciferase activity (Fig. 4A). Next, inhibited NFAT3 transactivation activity by about 50% in either À À we analyzed the binding affinity of JNK1 and wild-type NFAT3 or JNKWT or JNK1 / MEFs (Fig. 4C). These results also revealed that mutant NFAT3 (S213,217A) by the mammalian two-hybrid assay. Ser213 and Ser217 of NFAT3 are the target amino acids for JNK1 and

Figure 2. Identification of the NFAT3 site that is phosphorylated by JNK1 or JNK2. A, NFAT3 is a substrate of JNK1 or JNK2 in vitro. Top, structure and schematic diagrams of GST-NFAT3 fusion constructs (1–8). To identify the phosphorylation target domain of NFAT3 for active JNK1 or JNK2, each GST-NFAT3 fusion protein (1–8) was partially purified, directly subjected to an in vitro phosphorylation assay with active JNK1 or JNK2, and results were visualized by autoradiography. *, the respective GST-fusion protein in the Commassie blue-stained gel (bottom). B, Ser168 and Ser170 of NAFT3 are not phosphorylated by JNK1/2. GST-proteins including mutant GST-NFAT3-113-260S168,170A (1–3, top) were partially purified, directly subjected to an in vitro phosphorylation assay with active JNK1 or JNK2 and then visualized by autoradiography. *, respective GST-fusion protein (1–3) in the JNK1 autoradiograph (left), JNK2 autoradiograph (middle), and the Coommassie blue–stained gel (right). C, Ser213 and Ser217 of NFAT3 are the phosphorylation targets of JNK1 and JNK2. GST-mock, GST-NFAT3-113-260, GST-NFAT3-113- 260S217A, and GST-NFAT3-113-260S213,217A GST proteins (1–4, top) were partially purified and directly subjected to an in vitro phosphorylation assay with active JNK1 (left) or JNK2 (middle), and the results were visualized by autoradiography or Coomassie blue (right). For A–C, GST mock served as the respective negative control. *, respective GST-fusion protein in the Coommassie blue stained gel. Other bands, which are not marked, are nonspecific bands. www.aacrjournals.org 8729 Cancer Res 2007; 67: (18). September 15, 2007

Figure 3. JNK1 and JNK2 regulate NFAT3 activity. A, the NH2-terminal region of NFAT3-1-260 is important for transactivation. The pGal4-NFAT3-1-902 and pGal4-NFAT3-261-902 expression vectors were constructed as indicated. Each construct was cotransfected with the p5xGal4-luciferase reporter plasmid into JNK wild-type (JNKWT) MEF cells, and then firefly luciferase activity was analyzed. Activity is indicated by the relative luminescence units normalized to a negative control (value for cells transfected with only p5xGal4-luc/pGal4-mock = 1.0). B, JNK1 and JNK2 regulate transactivation of NFAT3. The pGal4-NFAT3-1-902 construct was transfected into JNKWT, JNK1À/À, or JNK2À/À MEF cells with the 5xGal4-luciferase reporter plasmid, respectively, and then firefly luciferase activity was analyzed. Activity is indicated by relative luminescence units normalized to a negative control (value for cells transfected with only p5xGal4-luc/pGal4-mock = 1.0). C, determination of the transactivation domain of NFAT3. The pGal4-NFAT3-1-112 and pGal4-NFAT3-113-260 plasmids were constructed as indicated. Each construct was transfected with the 5xGal4-luciferase reporter plasmid, respectively, and then firefly luciferase activity was analyzed. Activity is indicated by relative luminescence units normalized to a negative control (value for cells transfected with only p5xGal4-luc/pGal4-mock = 1.0). D, JNK1 and JNK2 are positive regulators for NFAT3 transcriptional activity. The pcDNA3-Flag-NFAT3 and p3xNFAT-luciferase plasmids were cotransfected with increasing amounts of pcDNA3-v5-JNK1 or -JNK2 and then firefly luciferase activity was analyzed. Activity is indicated by relative luminescence units normalized to a negative control (value for cells transfected with only p3xNFAT3-luc/pcDNA3-mock = 1.0). For all experiments (A–D), the firefly luciferase activity was normalized against Renilla luciferase activity (pRL-SV40). Columns, means of values from triplicate experiments; bars, SD. Significant differences were evaluated using the Student’s t test. *, P < 0.05; **, P < 0.005.

JNK2 and play an important role in the positive regulation of major component of the AP-1 complex. When c-Jun is phosphor- NFAT3 activity. ylated by JNK1 or JNK2 at Ser63 and Ser73, c-Jun DNA binding Knockdown of JNK1 and JNK2 inhibits cell transformation. affinity is increased, and the expression of many target genes is JNK1 and JNK2 are critical upstream kinases of c-Jun, which is a increased many times, resulting in increased cell proliferation and

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Anti-oncogenic Potential of NFAT3 cell transformation (35–39). Therefore, we proposed that phos- As before, RasG12V alone induced foci formation, and JNK1 or phorylation and activation of NFAT3 by JNK1 and JNK2 might play JNK2 enhanced RasG12V-induced foci formation (Fig. 6A and an important role in cell proliferation as well as cell transforma- graph lanes 2–4). Interestingly, overexpression of NFAT3 sup- tion. To elucidate the physiologic significance of JNK1/2-NFAT3 pressed RasG12V/JNK1- or RasG12V/JNK2-induced foci formation in signaling, we constructed siRNA vectors against JNK1 (si-JNK1) and NIH3T3 cells (Fig. 6A and graph lanes 5 and 6). Very importantly, JNK2 (si-JNK2; Fig. 5A). These si-constructs were transfected into si-NFAT3 enhanced foci formation compared with RasG12V alone, NIH3T3 cells, and knockdown of endogenous JNK1 (f75%) and RasG12V/JNK1, or RasG12V/JNK2 in NIH3T3 cells (Fig. 6A and graph JNK2 (f95%) was confirmed by Western blotting (Fig. 5B). To lanes 7 and 8). Moreover, the colony size in si-NFAT3–transfected examine the effect of JNK1 and/or JNK2 knockdown on cell cells seemed to be larger than RasG12V alone, RasG12V/JNK1, transformation, we introduced various combinations of RasG12V, or RasG12V/JNK2 (Fig. 6A). We also found that UVB (4 kJ/m2) JNK1, JNK2, NFAT3, si-JNK1, and si-JNK2 expression vectors into NIH3T3 cells and conducted a focus-forming assay (Fig. 5C). As expected, constitutively active Ras (RasG12V) induced cell transformation in NIH3T3 cells (Fig. 5C and graph lane 2). Moreover, overexpression of JNK1 or JNK2 combined with RasG12V resulted in an increased foci formation in NIH3T3 cells (Fig. 5C and graph lanes 3 and 4). Notably, si-JNK1 or si-JNK2 almost completely blocked foci formation induced by RasG12V alone or RasG12V combined with JNK1 or JNK2 (Fig. 5C and graph lanes 5–8). Interestingly, we found that RasG12V combined with NFAT3 did not increase, but actually suppressed foci formation in NIH3T3 cells compared with RasG12V alone (Fig. 5C and graph lane 9 versus 2). Foci formation was even further suppressed by si-JNK1 or si-JNK2 (Fig. 5C and graph lanes 10 and 11). The suppressive effect of si-JNK1 and si-JNK2 might be mediated by AP-1 and NFAT3 because AP-1 luciferase activity (Fig. 5D) and transactivation acti- À À À À vity of NFAT3 (Fig. 3B) were suppressed in JNK1 / and JNK2 / MEF cells compared with JNKWT MEF cells. NFAT3 negatively regulates cell transformation mediated by JNK1 and JNK2. To more fully examine the JNK1- and JNK2- mediated physiologic function of NFAT3 in cell transformation, we constructed siRNA against NFAT3 (Supplementary Fig. S3A). This plasmid was transfected into NIH3T3 cells, and knockdown of endogenous NFAT3 protein level by about 90% was confirmed by Western blot (Supplementary Fig. S3B). We then introduced various combinations of RasG12V, JNK1, JNK2, NFAT3, and si- NFAT3 into NIH3T3 cells and conducted a focus-forming assay.

Figure 4. Ser213 and Ser217 of NFAT3 are required for transactivation but not for binding with JNK1 or JNK2. A, SP600125 inhibits NFAT3-mediated luciferase activity. The pcDNA3-Flag-NFAT3-1-902 construct was transfected with the 3xNFAT-luciferase reporter plasmid into JNKWT MEFs. Cells were cultured for 36 h and then treated or 12 h with the JNK inhibitor, SP600125 (10 Amol/L). The cells were disrupted with luciferase lysis buffer, and firefly luciferase activity was analyzed. Activity is indicated by relative luminescence units normalized to a negative control (value for cells transfected with only p3xNFAT-luc/pcDNA3-mock = 1.0). B, mutation of NFAT3 at Ser213 and Ser217 does not affect NFAT3 binding activity with JNK1 or JNK2. The pACT-NFAT3-113-260, pACT-NFAT3-113-260-S213,217A, pACT-NFAT3-1- 902, or pACT-NFAT3-1-902-S213,217A constructs were individually cotransfected with pBIND-JNK1 and the pG5-luciferase reporter plasmid into JNKWT MEFs and then firefly luciferase activity for binding was analyzed. Activity is indicated by relative luminescence units normalized to a negative control (value for cells transfected with only pG5-luc/pBIND-JNK1 = 1.0). The firefly luciferase activity was normalized against the Renilla luciferase activity. Columns, means of values from triplicate experiments; bars, SD. Significant differences were evaluated using the Student’s t test; *, P < 0.005. C, mutation of NFAT3 at Ser213 and Ser217 inhibits NFAT3 transactivation. The pGal4- NFAT3-113-260 and pGal4-NFAT3-113-260S213,217A constructs were each cotransfected with the p5xGal4-luciferase reporter plasmid into JNKWT or JNK1À/À MEF cells, and then firefly luciferase activity was analyzed. Activity is indicated by relative luminescence units normalized to a negative control (value for cells transfected with only p5xGal4-luc/pGal4-NFAT3-113-260 = 100%). For A and C, the firefly luciferase activity was normalized against the Renilla luciferase (pRL-SV40) activity. Columns, means of values from triplicate experiments; bars, SD. Significant differences were evaluated using the Student’s t test; *, P < 0.05. www.aacrjournals.org 8731 Cancer Res 2007; 67: (18). September 15, 2007

Figure 5. Knockdown of JNK1 or JNK2 inhibits cell transformation. A, nucleotide sequences for siRNA JNK1 (si-JNK1) and siRNA JNK2 (si-JNK2) primers. B, knockdown efficiency of si-JNK1 and si-JNK2. NIH3T3 cells were transfected with si-JNK1 or si-JNK2 and cultured 36 h. The proteins were extracted with NP40 cell lysis buffer, and total JNK1 or JNK2 protein level was visualized by Western blot using specific antibodies. h-Actin was used as an internal control to verify equal protein loading. C, knockdown of JNK1 or JNK2 suppresses RasG12V-induced foci formation. Various combinations of expression vectors were transfected into NIH3T3 cells as indicated, and a foci formation assay was done following standard protocols as described in Materials and Methods. Graph, average number of foci; columns, means of values from triplicate experiments; bars, SD. Significant differences were evaluated using the Student’s t test. *, P < 0.05; **, P < 0.01; and ***, P < 0.005. D, AP-1 luciferase activity is suppressed in JNK1À/À or JNK2À/À MEF cells. The AP-1–luciferase reporter plasmid was transfected into JNKWT,JNK1À/À,orJNK2À/À MEF cells and cultured for 36 h, and then firefly luciferase activity was analyzed. Activity is indicated by relative luminescence units normalized to a control (value for JNKWT MEFs transfected with only AP-1–luc = 100%). The firefly luciferase activity was normalized against the Renilla luciferase (pRL-SV40) activity. Columns, means of values from triplicate experiments; bars, SD. Significant differences were evaluated using the Student’s t test; *, P < 0.05.

treatment increased AP-1–luciferase activity as well as 3xNFAT- (19–22, 40, 41), NFAT3 is also now believed to play an important luciferase activity compared with untreated control NIH3T3 cells role in tumorigenesis or to possess antitumorigenic functions. 213 217 (Fig. 6B). Moreover, mutation of NFAT3 at Ser and Ser Recent research reports have indicated that constitutively active suppressed 3xNFAT-luciferase activity under a variety of culture NFATc1 induces a transformation phenotype in 3T3L1 fibroblasts conditions as well as after UVB stimulation (Fig. 6C). Importantly, (22). However, NFATc2 negatively regulates CDK4 gene expression, AP-1 luciferase activity was significantly inhibited by overexpres- resulting in cells re-entering into the resting stage of the cell cycle sion of NFAT3 in NIH3T3 cells (Fig. 6D). (20). NFAT4 has also been reported to act as a tumor suppressor for the development of murine T-cell lymphomas induced by the Discussion retrovirus SL3-3 (31). Because NFAT transcription factors are found in a wide range of The mitogen-activated protein kinase p38 phosphorylates NFAT3 cell types and tissues and regulate genes involved in cell cycle at Ser168 and Ser170 and induces cytoplasmic localization. When progression, cell development, and differentiation and angiogenesis calcineurin is activated, Ser168 and Ser170 are dephosphorylated, and

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NFAT3 is localized to the nucleus. However, we found that JNK1/2 When NFAT3 was overexpressed with a combination of RasG12V, phosphorylated Ser213 and Ser217, and the mutation of Ser213 and JNK1, or JNK2 in NIH3T3 cells, foci formation was suppressed Ser217 to alanine abolished NFAT3 transactivation and transcrip- compared with the expression of only RasG12V, RasG12V/JNK1, or tional activity (Figs. 4C and 6C). Furthermore, the NFAT3 RasG12V/JNK2 (Figs. 5C and 6A). In contrast, when siRNA-NFAT3 À À À À transactivation activity was suppressed in JNK1 / and JNK2 / was cotransfected with various combinations of RasG12V, JNK1, and MEF cells (Fig. 3B). In addition, NFAT3-mediated 3xNFAT- JNK2 into NIH3T3 cells, foci formation was increased compared luciferase activity was increased by cotransfection of JNK1 or with the expression of only RasG12V, RasG12V/JNK1, or RasG12V/JNK2 JNK2 (Fig. 3D), indicating that phosphorylation of NFAT3 at (Fig. 6A). Furthermore, we found that AP-1 activity was suppressed Ser213 and Ser217 by JNK1/2 might induce nuclear localization. by ectopic expression of NFAT3 (Fig. 6D), indicating that NFAT3 These results strongly indicated that distinct phosphorylation suppressed cell transformation through the inhibition of AP-1 site(s) of NFAT3 might be involved in a different regulatory activity. Therefore, we hypothesized that NFAT3 is a negative mechanism for NFAT3. regulator of Ras-JNK1/2-AP-1–induced cell transformation.

Figure 6. NFAT3 negatively regulates cell transformation mediated by JNK1 or JNK2. A, knockdown of NFAT3 enhances RasG12V/JNK1 or RasG12V/JNK2-induced foci formation. Various combinations of expression vectors were transfected into NIH3T3 cells as indicated, and the foci formation assay was done following standard protocols as described in Materials and Methods. Graph, average number of foci; columns, means of values from triplicate experiments; bars, SD. Significant differences were evaluated using the Student’s t test. *, P < 0.05; **, P < 0.001. B, UVB enhances AP-1 and NFAT activity. The AP-1–luciferase or p3xNFAT-luciferase reporter plasmid was transfected into JNKWT MEF cells. Cells were cultured 36 h, and then firefly luciferase activity was analyzed. Activity is indicated as relative luminescence units normalized to a control (value for cells not exposed to UVB and transfected with only pAP-1-luc or p3xNFAT3-luc = 1.0). C, mutations of NFAT3 at Ser213 and Ser217 suppress NFAT3 transcriptional activity. The pcDNA4-NFAT3-FL or pcDNA4-NFAT3-FL-S213,217A plasmid was transfected with p3xNFAT- luciferase into JNKWT MEF cells, and cells were cultured for 36 h. The cells were starved in 0.1% FBS-DMEM for 24 h and then exposed to 4 kJ/m2 UVB and cultured 12 h more. At each indicated time point, the cells were disrupted with luciferase cell lysis buffer, and firefly luciferase activity was analyzed. Activity is indicated by relative luminescence units normalized to a control (value for each cell transfected with only p3xNFAT-luc/pcDNA4-NFAT3 = 100% in each indicated condition). D, AP-1 luciferase activity is inhibited by ectopic NFAT3 expression. The pAP-1–luciferase plasmid was transfected with or without pcDNA3-Flag-NFAT3 into JNKWT MEF cells. Cells were cultured 36 h, and then firefly luciferase activity was analyzed. Activity is expressed as relative luminescence units normalized to a negative control (value for cells transfected with only pAP-1–luc/pcDNA3-mock = 1.0). For B–D, the firefly luciferase activity was normalized against Renilla luciferase (pRL-SV40) activity. Columns, means of values from triplicate experiments; bars, SD. Significant differences were evaluated using the Student’s t test. *, P < 0.005 for B and D; *, P <0.05forC. www.aacrjournals.org 8733 Cancer Res 2007; 67: (18). September 15, 2007

Three possibilities could explain the negative regulatory effect deficiency mouse model (45); (b) ectopic expression of JDP2 in mediated by NFAT3. One is a possible competition between c-Jun myoblasts and rhabdomyosarcoma tumor cells strongly promot- and NFAT3 as a JNK1/2 substrate. When a cell is stimulated by, e.g., ed muscle cell differentiation (46); (c) phosphorylation of NFAT3 UV, JNK is activated and localized to the nucleus where it then (Ser281, Ser285, Ser289, and Ser344) by RSK2 induced NFAT3 phosphorylates the c-Jun protein (42). At the same time, JNK transcriptional activity, resulting in multinucleated myotube phosphorylates and activates NFAT3 because UV stimulation also differentiation of C2C12 myoblasts (28); and (d) siRNA against induces 3xNFAT-luciferase activity (Fig. 6B). Therefore, NFAT3 and NFAT3 enhanced RasG12V/JNK-mediated foci formation, and c-Jun might compete with JNK1/2 as substrates, resulting in the ectopic expression of NFAT3 suppressed RasG12V/JNK-mediated suppression of AP-1 activity. However, this is probably unlikely foci formation (Fig. 6A). Furthermore, although NFAT1 and AP-1 because the affinity of the interaction between JNK1/2 and c-Jun are cooperative for gene expression of many cytokine genes (30), is very much higher than the potential interaction of JNK1/2 no report has shown that NFAT3 and AP-1 bind to each other and NFAT3 as determined by the mammalian two-hybrid assay and cooperate directly. Therefore, the detailed molecular (Fig. 1A). The second possibility is that NFAT3 interferes with the mechanism of AP-1 activity inhibition by NFAT3 requires further nuclear localization of JNK1/2. We found that although JNK nuclear investigation. localization is induced by stimuli such as UV, only a relatively small Based on our observation that NFAT3 is a substrate of JNK1/2, amount of JNK is localized into the nucleus compared with the resulting in increased NFAT3 activity, we hypothesized that JNK1/ large amount of JNK still remaining in the cytoplasm (42). In cell 2-NFAT3 function might be involved in cell transformation as well culture, we found that NFAT3 is predominantly localized in the as oncogenesis because JNK1/2 signaling plays a pivotal role for cytoplasm (28), and when cells are stimulated, JNK1/2 is phos- cell proliferation through the regulation of AP-1 activity. Surpris- phorylated and activated in the cytoplasm. After activation, ingly, we found that JNK1/2-mediated NFAT3 activation signaling JNK1/2 binds with NFAT3 and phosphorylates NFAT3 also in the suppressed RasG12V-mediated NIH3T3 cell transformation as well cytoplasm. During this process, NFAT3 interfered with JNK1/2 as AP-1 luciferase activity (Figs. 5C and 6D). In contrast, localization, resulting in a large amount of JNK1/2 being knockdown of NFAT3 induced RasG12V-mediated NIH3T3 cell localized in the cytoplasm. This could at least partially explain transformation (Fig. 6A). Therefore, we concluded that NFAT3 why the JNK1/2-NFAT3 interaction affinity is lower than the might play a key modulator function to control proliferation and JNK1/2–c-Jun interaction, but could still inhibit AP-1 transcrip- differentiation through the regulation of AP-1 activity. Our results tion activity. However, this idea does not explain the observation are the first to show an anti-oncogenic function for NFAT3 that is that UV induces a strong and rapid c-Jun phosphorylation at mediated through the JNK signaling pathway. Ser63/73. The third possibility involves JDP2 (c-Jun dimerization protein 2). JDP2 is a member of the bZIP family of transcription Acknowledgments factors and binds with c-Jun and represses AP-1 transcriptional activity (43). JDP2 also binds with core transcription machinery Received 1/2/2007; revised 4/2/2007; accepted 5/17/2007. Grant support: The Hormel Foundation and NIH grants CA77646, CA81064, such as ATF2 and CBP (44). The NFAT3 protein forms a complex CA27502, and CA111356. with CBP (29), suggesting that a complex composed of NFAT3 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance and JDP2 is also possible, and that this complex might suppress with 18 U.S.C. Section 1734 solely to indicate this fact. AP-1 activity as well as induce cell differentiation. This idea is We thank Dr. R.J. Davis (Department of Biochemistry and Molecular Biology, supported by findings showing that (a) JDP2 inhibited Ras- University of Massachusetts Medical School, Worcester, MA) for the pcDNA3-Flag- JNK1, Dr. C.W. Chow (Department of Molecular Pharmacology, Albert Einstein College mediated cell transformation in NIH3T3 cells and suppressed of Medicine, Bronx, NY) for the pcDNA3-Flag-NFAT3 deletion mutants, and Andria tumor development in the xenograft severe combined immuno- Hansen for secretarial assistance.

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