NuclearFactorofActivatedT3IsaNegativeRegulatorof Ras-JNK1/2-AP … · these results indicated that NFAT3 inhibited neoplastic transfor-mation through a negative feedback regulation

Nuclear Factor of Activated T3 Is a Negative Regulator of

Ras-JNK1/2-AP-1–Induced Cell Transformation

Ke Yao,1Yong-Yeon Cho,

1H. Robert Bergen III,

2Benjamin J. Madden,

2Bu Young Choi,

1

Wei-Ya Ma,1Ann M. Bode,

1and Zigang Dong

1

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

Abstract

The c-jun-NH2-kinases (JNK) play a critical role in tumorpromoter–induced cell transformation and apoptosis. Here,we showed that the nuclear factor of activated T3 (NFAT3) isphosphorylated by JNK1 or JNK2 at Ser213 and Ser217, which arelocated in the conserved SP motif. The transactivation domainof NFAT3 is found between amino acids (aa) 113 and 260 andincludes the phosphorylation targets of JNK1 and JNK2.NFAT3 transactivation activity was suppressed in JNK1�/� orJNK2�/� mouse embryonic fibroblast (MEF) cells comparedwith wild-type MEF cells. Moreover, a 3xNFAT-luc reportergene assay indicated that NFAT3 transcriptional activity wasincreased in a dose-dependent manner by JNK1 or JNK2.Double mutations at Ser213 and Ser217 suppressed NFAT3transactivation activity; and SP600125, a JNK inhibitor, sup-pressed NFAT3-induced 3xNFAT-luciferase activity. Knock-down of JNK1 or JNK2 suppressed foci formation in NIH3T3cells. Importantly, ectopic expression of NFAT3 inhibited AP-1activity and suppressed foci formation. Furthermore, knock-down of NFAT3 enhanced Ras-JNK1 or JNK2-induced fociformation in NIH3T3 cells. Taken together, these resultsprovided direct evidence for the anti-oncogenic potential ofthe NFAT3 transcription factor. [Cancer Res 2007;67(18):8725–35]

Introduction

The JNKs signal transduction pathway has been shown to playan important role in coordinating various cellular responsessuch as apoptosis (1), proliferation (2), and neoplastic transforma-tion (3). Mice that are deficient in both jnk1 and jnk2 exhibitembryonic death at E10.5 due to enhanced apoptosis in thehindbrain (4) and forebrain regions (4, 5), which clearly suggeststhat JNK1 and JNK2 are involved in cell survival during develop-ment. Furthermore, specific antisense oligonucleotides againstJNKs inhibited tumor cell growth (6) and jnk2-deficient micedisplayed significant suppression of skin papilloma developmentinduced by 12-O-tetradecanoylphorbol-13-acetate (TPA; ref. 7).These types of observations may have been due to an enhancedapoptosis in jnk-deficient cells and mice (8).

When cells are stimulated by environmental stress, cytokines, ortoxins (9), JNK phosphorylation is increased through MKK4/7 (10),

and the activation signal is transmitted to downstream substrate(s)such as c-Jun (11). JNK1 and JNK2 are well known for the activationand phosphorylation of c-Jun at Ser63 and Ser73. However, otherdownstream target proteins include Elk-1 (12), c-Myc (13), p53 (14),and NFATc2 (15), as well as several members of the apoptosis-related family of proteins, including Bcl-2, Bcl-XL, Bim, and Bad(16–18). These functions of JNKs have been primarily attributed tothe fact that JNKs activate different substrates based on the specificstimulus or cell type.

Although the nuclear factor of activated T cell (NFAT) family oftranscription factors has been primarily identified in immune cells,recent studies indicated that NFAT is functionally active in severalother non-immune cell types, including vascular endothelial cells,embryonic exon cells, and 3T3-L1 fibroblasts (19–22). Four differentisotypes of NFATs, including NFAT1 (1a, 1b, and 1c), NFAT2 (2a and2b), NFAT3, and NFAT4 (4x, 4a, 4b, and 4c) were shown to havedifferential tissue distribution (23). These findings suggested thatdistinct NFAT isotypes play different roles in diverse tissues undervarious physiologic conditions (24). Calcineurin, a Ca2+/calmodu-lin-dependent protein phosphatase that is a downstream target ofintracellular Ca2+ signaling, is a well-known effector of the NFATfamily of transcription factors (NFAT1–4; ref. 23). Classically,calcineurin dephosphorylates NFAT1–4, allowing NFAT to translo-cate to the nucleus, bind to consensus DNA sites, and control genetranscription (24). Upon cessation of the Ca2+ signal, NFAT proteinsare re-phosphorylated by kinases such as GSK-3 (25), resulting inthe translocation of NFAT to the cytoplasm (24). However, recentstudies indicated that the Ras signaling pathway positivelyregulates NFAT3 activity (26) by forming an activation complexto regulate PPARg2 promoter activity, which leads to adipocytedifferentiation (27). In addition, RSK2-mediated phosphorylation ofNFAT3 regulates NFAT3 activity and induces muscle cell differen-tiation (28). Furthermore, the NFAT3 protein forms a complex withCBP to activate transcription machinery (29), and NFATc1 bindswith AP-1 to enhance its transcriptional activity (30). Activation ofNFATc1 was also reported to induce cell transformation (22). Onthe other hand, NFATc2 was shown to repress cyclin-dependentkinase 4 (CDK4), resulting in cell cycle arrest at G0-G1 (20, 31). Inaddition, when the lymphomagenic virus SL3-3 was infected inNFAT4-deficient mice, T-cell lymphoma developed faster and withhigher frequency compared with wild-type mice (20). These reportsstrongly indicate that the oncogenic or anti-oncogenic activitiesof the NFAT proteins are dependent on the isotype and specificphysiologic condition. However, the role of NFAT3 in the tumori-genesis is not yet understood.

In this study, we showed that NFAT3 is a strong binding partnerof JNK1 and JNK2. The phosphorylation of NFAT3 at 213 and 217by JNK1/2 induced NFAT3 transactivation activity. Importantly,overexpression of NFAT3 suppressed RasG12V-JNK1– or -JNK2–induced foci formation by inhibiting AP-1 activity. Taken together,

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

K. Yao and Y-Y. Cho contributed equally to this work.Requests for reprints: Zigang Dong, Hormel Institute, University of Minnesota,

801 16th Avenue NE, Austin, MN 55912. Phone: 507-437-9600; Fax: 507-437-9606;E-mail: [email protected].

I2007 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-06-4788

www.aacrjournals.org 8725 Cancer Res 2007; 67: (18). September 15, 2007

Research Article

Research. on August 4, 2021. © 2007 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

these results indicated that NFAT3 inhibited neoplastic transfor-mation through a negative feedback regulation of JNK1/2-AP-1signaling.

Materials and Methods

Reagents and antibodies. DMEM and fetal bovine serum (FBS) werepurchased from Life Technologies, Inc. Restriction enzymes and some

modifying enzymes were obtained from New England BioLabs, Inc. The

DNA ligation kit (version 2.0) was purchased from TAKATA Bio, Inc. The

LipofectAMINE Plus transfection reagent for NIH3T3, JNKWT, JNK1�/�,and JNK2�/� mouse embryonic fibroblast (MEF) cells were purchased

from Invitrogen. The JetPEI transfection reagent for 293 cells was from

Q-Biogene. The pcDNA3.1 plasmid was purchased from Life Technologies,

Inc. The luciferase assay substrate was from Promega. Antibodies againstthe Flag or v5 epitope were purchased from Sigma-Aldrich or Invitrogen,

respectively. Antibodies against JNK1 and NFAT3 were purchased from

Santa Cruz Biotechnology, Inc., and the antibody against JNK2 was fromCell Signaling Technology, Inc.

Cell culture. The 293 (human embryonic kidney), JNKWT, JNK1�/�,and JNK2�/� MEF cells were cultured in DMEM supplemented with 10%

heat-inactivated FBS in a 37jC, 5% CO2 incubator. NIH/3T3 cells werecultured in DMEM supplemented with 10% bovine calf serum in a 37jC,5% CO2 incubator. The cells were maintained by splitting at 80% to 90%

confluence, and media were changed every 3 days.

Construction of expression and siRNA vectors. For the mammaliantwo-hybrid system assay, the pACT-transcription factors (TF), pBIND-JNK1

and -JNK2, and pcDNA3.1-v5-JNK1 and -JNK2 were constructed as

previously described (28). The pcDNA3.1-FLAG-NFAT3 and various deletionmutants of NFAT3 from the COOH-terminal end (residues 1–853, 1–580,

1–450, 1–365, 1–260, 1–219, and 1–112) were described (28). The deletion

GST-NFAT3 fusion vectors (1–112, 113–260, 261–365, 366–450, 451–580,

581–853, 853–902) were generated by PCR using wild-type NFAT3 fulllength (FL) subcloned into the BamHI/XbaI site of the pGEX-5X-C vector.

The mutation of Ser213 and Ser217 to alanine was carried out using the

QuickChange II Site–Directed Mutagenesis Kit (Strategene) according to

recommended protocols. The deletion mutants of NFAT3-1–112, -113–260and -261–902, NFAT3-113–260 and NFAT3-113-260S213,217A, and NFAT3-

S213A,217A were also subcloned into the pcDNA4 (Invitrogen) or pGEX-

5X-C vector. The pU6pro vector (provided by David L. Turner, University

of Michigan, Ann Arbor, MI) was used to construct small interfering RNAJNK1 (si-JNK1), siRNA JNK2 (si-JNK2), and siRNA NFAT3 (si-NFAT3)

following the recommended protocols.3 All of the constructs were con-

firmed by restriction enzyme mapping and DNA sequencing.Western blotting. The proteins were extracted with NP40 cell lysis

buffer with freezing and thawing. The same amount of protein was resolved

by SDS-PAGE, transferred onto polyvinylidene difluoride membranes,

hybridized with appropriate antibodies, and then visualized using theenhanced chemiluminescence (ECL) detection kit (Amersham Biosciences).

NFAT3 activity assay. The 293 cells (2.0 � 104) or JNKWT, JNK1�/�, andJNK2�/� MEFs (4.0 � 104) were seeded into 48- or 12-well plates and

cultured in 10% FBS-DMEM for 18 h before transfection, respectively. The3xNFAT-luc reporter plasmid, which was constructed by fusion of three

NFAT binding consensus sequences on the 5¶ upstream end of the minimal

interleukin 2 promoter-luciferase reporter plasmid, was transfected withvarious recombinant plasmids as indicated in the respective figures. The

cells were cultured for 36 h and were or were not stimulated with UVB or

specific chemicals and then cultured for an additional 12 h. At each time

point, cells were disrupted and analyzed for firefly luciferase activity. The3xNFAT-luc luciferase activity was normalized against Renilla luciferase

activity (pRL-SV40).

Mammalian two-hybrid assay. For the mammalian two-hybrid (M2H)

assay, we followed the Promega Checkmate Mammalian Two-Hybrid

System protocols. In brief, 293 cells were maintained in 10% FBS-DMEM,

seeded into 48-well plates (2.0 � 104), and incubated with 10% FBS-DMEM

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.

The transfection was done using jetPEI following the manufacturer’s

recommended protocols. For the luciferase assay, the cells were disrupted

by the addition of lysis buffer and incubated for 30 min at room

temperature with gentle shaking. The luciferase activity was measured

automatically by the addition of 100 AL substrate buffer, and data were

collected using the Luminoskan Ascent (MTX Lab, Inc.). The relative

luciferase activity was calculated using the pG5-luciferase basal control and

normalized against Renilla luciferase activity, which included the pBIND

vector as an internal control.In vitro kination assay. Wild-type GST-NFAT3 and deletion or point

mutant proteins were used for the in vitro kination assay using an active

JNK1 or JNK2 (Upstate Biotechnology, Inc.). Reactions were carried out at

30jC for 30 min in a mixture containing 50 Amol/L unlabeled ATP and

10 ACi of [g-32P] ATP and then stopped by adding 6� SDS sample buffer.

Samples were boiled and then separated by 12% SDS-PAGE and visualized

by autoradiography or Coomassie blue staining.

NFAT3 protein domain analysis. Data for analyzing the NFAT3 proteindomains were downloaded from the ExPASy Proteomics Server (NiceProt

view of Swiss-Prot: Q14934). Putative phosphorylation sites were predicted

by the Netphospho 2.0 server.4

Construction and expression of GST-NFAT3 fusion proteins. TheNFAT3 wild-type and deletion mutants were constructed as described (28).

To express and purify the glutathione S-transferase (GST)-fusion NFAT3

fusion proteins, the BL21 bacterial strain was used. A single colony for each

vector was cultured, and the GST-fusion protein was induced by isopropyl-

L-thio-h-D-galactopyranoside ( final concentration, 0.5 mmol/L) treatment

at 25jC for 5 h. The protein was then partially purified with 1� IB wash

buffer (Novagen) for disruption, binding to glutathione Sepharose 4B, and

elution with 10 mmol/L reduced glutathione, followed by dialysis. Purified

proteins were stored �70jC until used.

LTQ Orbitrap hybrid mass spectrometer analysis. Protein phosphor-

ylation site assignment was carried out using ingel trypsin digest andnanoLC-ESI-tandem hybrid mass spectrometry with a ThermoFinnigan

LTQ Orbitrap Hybrid Mass Spectrometer. Briefly, the Coomassie blue–

stained protein bands were excised from the gel and cut into pieces nolarger than 2 mm2. Before digestion, the gel pieces were destained with

50% acetonitrile/50 mmol/L Tris-HCl (pH, 8.1) until clear. Gel pieces were

then reduced with 20 mmol/L DTT/50 mmol/L Tris-HCl (pH, 8.1) at 55jCfor 30 min and alkylated with 40 mmol/L iodoacetamide at roomtemperature for 30 min in the dark. Proteins were digested in situ with

30 AL (0.004 Ag/AL) trypsin (Promega) in 20 mmol/L Tris-HCl (pH, 8.1)

at 37jC overnight, followed by peptide extraction with 60 AL of 2%

trifluoroacetic acid followed by 60 AL of acetonitrile. The pooled extractswere concentrated to <5 AL on a SpeedVac spinning concentrator (Savant

Instruments) and then brought up in 0.1% formic acid for protein

identification by nanoflow liquid chromatography electrospray tandemmass spectrometry (nanoLC-ESI-MS/MS) using a ThermoFinnigan LTQ

Orbitrap Hybrid Mass Spectrometer (ThermoElectron Bremen) coupled to

an Eksigent nanoLC-2D HPLC system (Eksigent). The peptide mixture is

loaded onto a 250-nL OPTI-PAK trap (Optimize Technologies) packedwith Michrom Magic C8 solid phase (Michrom Bioresources) and eluted

with a 0.1% formic acid/acetonitrile gradient through a Michrom packed

tip capillary Magic C18 column (75 Am � 150 mm). The LTQ Orbitrap

mass spectrometer experiment was set to perform an Fourier transform(FT) full scan from 380 to 1,600 m/z with resolving power set at 60,000

(400 m/z), followed by linear ion trap MS/MS scans on the top three ions.

The ambient air polycyclodimethylsiloxane 391 m/z ion was used as an

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

Cancer Research

Cancer Res 2007; 67: (18). September 15, 2007 8726 www.aacrjournals.org



accuracy. To detect phosphorylated peptides, a MS scan is triggered if oneof the top three ions in the MS/MS scan corresponds to the neutral loss of

one or two phosphoric acid moieties with and without water for [M + 2H]2+

(49, 58, 98, and 107 m/z) and [M+3H]3+ (32.7 and 65.4 m/z) precursor ions.

Dynamic exclusion was set to 2, and selected ions were placed on anexclusion list for 90 s. The MS/MS raw data were converted to DTA files

using ThermoElectron Bioworks 3.2 and correlated to theoretical fragmen-

tation patterns of tryptic peptide sequences from the Swissprot databases

using both SEQUEST (ThermoElectron) and Mascot (Matrix Sciences)search algorithms running on 10 node clusters. All searches were conducted

with fixed cysteine modifications of +57 for carboxamidomethyl-cysteines

and variable modifications allowing +16 with methionines for methionine

sulfoxide, +42 for protein NH2-terminal acetylation, +80 at serine, threo-nine, and tyrosine for phosphorylation and �18 for serine and threonine

H3PO4 loss. The search was restricted to trypsin-generated peptides

allowing for two missed cleavages and was left open to all species. Anadditional search was done using only the expected sequence with no

enzyme. Peptide mass search tolerances are set to 20 ppm, and fragment

mass tolerance was set to F0.8 Da. Matches to phosphorylated peptides

were further interpreted manually to assign the site of phosphorylationusing both +80 and �18 diagnostic ions. Preferences were given to matches

of the more abundant ions.

Focus forming assay. Transformation of NIH3T3 cells was conducted

according to standard protocols (32). Cells were transiently transfected withvarious combinations of H-RasG12V (50 ng), JNK1 or JNK2 (450 ng), siRNA

vector DNA (450 ng), and pcDNA3-mock (compensation for equal amount

of DNA) as indicated in figures and then cultured in 5% FBS-DMEMfor 2 weeks. Foci were fixed with methanol, stained with 0.5% crystal violet,

and then counted with a microscope and the Image-Pro PLUS software

program.

Results

NFAT3 is a binding partner of JNK1 and JNK2 in vivo . Toidentify novel binding or substrate protein(s) for JNKs, weintroduced JNK1 or JNK2 cDNA, including the open reading frame(ORF), into the pBIND mammalian two-hybrid system vector(pBIND-JNK1 or pBIND-JNK2) as bait. The ORF of each TF wasamplified by PCR and then introduced into the pACT mammaliantwo-hybrid system vector (pACT-TFs). Each individual pACT-TFplus the pG5-luciferase reporter plasmid was cotransfected into 293cells with pBIND-JNK1 or -JNK2. Each interaction activity wascompared against the pG5-luc/pBIND-JNK1 or -JNK2 as the basallevel (Fig. 1A, lane 1 or 7 , respectively). Elk-1, c-Jun, and activat-ing transcription factor 2 (ATF2) were used as positive controls(Fig. 1A, lanes 4–6 and 10–12). NFAT3 and NFAT4 showed stronginteracting activity with pBIND-JNK1 or -JNK2 compared with thepG5-luc/pBIND-JNK1 or -JNK2 control (Fig. 1A, lanes 2 and 3versus 1 or 8 and 9 versus 7 , respectively). NFAT4 has been iden-tified previously as a binding partner for JNKs (33), and therefore,we selected NFAT3 for further study. To verify the interactionof NFAT3 with JNK1 and JNK2, we introduced the pcDNA3-v5-JNK1or -JNK2 construct and the pcDNA3-Flag-NFAT3 construct into293 cells. Results of immunoprecipitation (IP) experiments showedthat NFAT3 could bind with JNK1 or JNK2 in vivo (Fig. 1B).

To identify the NFAT3 domain that is involved in the interactionof NFAT3 with JNK1 or JNK2, we constructed pcDNA3-Flag-NFAT3deletion mutants (Supplementary Fig. S1A) that were thenindividually transfected into 293 cells with pcDNA3-v5-JNK1 or-JNK2, and expression was confirmed (Supplementary Fig. S1B).IP results using a v5 monoclonal antibody showed that the FLNFAT3 and deletion mutants from the COOH-terminal to aa 365coprecipitated with JNK1 or JNK2 (Fig. 1C and D, lanes 2–6).However, when aa 365–260 were deleted, the NFAT3 protein was

not detected, and any mutants with deletions to aa 219 or 112 didnot coprecipitate with the JNK1 or JNK2 protein (Fig. 1C and D,lanes 7–9). Interestingly, when aa 450 to 580 were deleted, thebinding affinity of NFAT3 with JNK1 or JNK2 was increased (Fig. 1Cand D, lane 5–6). In addition, the immunoprecipitated levels ofJNK1 or JNK2 were almost the same (Fig. 1C and D, bottom). Theseresults suggested that aa 260 to 365 of NFAT3 are important forbinding with JNK1 and JNK2.NFAT3 is a substrate of JNK1 and JNK2 in vitro . To identify

the target region of NFAT3 that harbors potential phosphorylationsites for JNK1 or JNK2, we constructed a series of GST-NFAT3deletion fragments (Fig. 2A, vector maps). These proteins wereexpressed partially purified as described in Materials and Methodsand then subjected to an in vitro phosphorylation assay. The resultsindicated that JNK1 and JNK2 phosphorylated NFAT3 in the regionspanning aa 113 to 260 (Fig. 2A, top and middle), which includes acalcineurin binding site and two SP repeat regions. The p38 kinaseis a well-known upstream kinase of NFAT3 that phosphorylatesNFAT3 at Ser168 and Ser170 (34). To determine whether the phos-phorylation sites for JNKs and p38 were identical, we constructedan NFAT3113-260 protein with mutations to replace Ser168 andSer170 with alanine (NFAT3-113-260S168,170A) and conducted thein vitro phosphorylation assay (Fig. 2B). Results indicated that thephosphorylation of the mutant NFAT3-113-260S168,170A by JNK1or JNK2 was the same as for wild-type NFAT3-113-260 (Fig. 2B,left and middle, lane 2 versus 3), suggesting that Ser168 and Ser170 ofNFAT3 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 thatare phosphorylated by JNK1 and JNK2, we used the human proteinreference database5 to compare the amino acid similarity amongvarious JNK substrates. Results showed that JNK1 and JNK2recognized and phosphorylated various substrates at the SP/TPconsensus sequences (Supplementary Fig. S2A). Moreover, wefound that NFAT3-113-260 contains 13 SP/TP motifs (Supplemen-tary Fig. S2B), including Ser168 and Ser170, which are phosphory-lated by p38 (34), but not by JNK1 or JNK2 (Fig. 2B). Therefore,we next used the LTQ Orbitrap hybrid mass spectrometer toidentify the NFAT3 sites that are phosphorylated by JNK1 andJNK2 as described in Materials and Methods. The results indicatedthat JNK1 and JNK2 phosphorylated two sites on NFAT3 at Ser213

and Ser217 (Supplementary Fig. S2B and C). To confirm thesephosphorylation sites, we constructed mutants of GST-NFAT3-113-260, replacing Ser213 and/or Ser217 with alanine (GST-NFAT3-113-260S217A or GST-NFAT3-113-260S213,217A; Fig. 2C). Theseproteins were expressed and partially purified and then directlysubjected to the in vitro phosphorylation assay using active JNK1or JNK2. Results confirmed that mutation of NFAT3-113-260 atSer217 caused a marked decrease in phosphorylation by JNK1(Fig. 2C, left, lane 3) or JNK2 (Fig. 2C, middle, lane 3). Further-more, mutations at both Ser213 and Ser217 totally blocked phos-phorylation of NFAT3-113-260 by either JNK1 (Fig. 2C, left, lane 4)or JNK2 (Fig. 2C, middle, lane 4). These results showed thatSer213 and Ser217 are the amino acids targeted for phosphorylationby JNK1 and JNK2.JNK1 and JNK2 regulate NFAT3 activity. To examine the role

of JNK1 and JNK2 in the regulation of NFAT3, we first developedpGal4-NFAT3-1-902 and pGal4-NFAT3-261-902 constructs (Fig. 3A,

5 http://www.hprd.org

Anti-oncogenic Potential of NFAT3




vector map) and analyzed the transactivation activity of NFAT3using cotransfection of each of these constructs with the 5xGal4-luciferase reporter plasmid into JNK wild-type (JNKWT) MEF cells.The transactivation activity of NFAT3 was significantly increasedin cells transfected with Gal4-NFAT3-1-902 compared with cellstransfected with the pGal4-mock control (Fig. 3A, lane 2). On theother hand, the NH2-terminal–deleted Gal4-NFAT3-261-902 trun-cated form markedly suppressed the transactivation activity ofNFAT3 (Fig. 3A, lane 3). These results indicated that thetransactivation activity might be regulated in the NH2-terminalarea spanning aa 1 to 260 of the NFAT3 protein. Next, wetransfected the pGal4-NFAT3-1-902 plasmid into JNKWT, JNK1knock-out (JNK1�/�), or JNK2 knock-out (JNK2�/�) MEF cells. Theresulting 5xGal4-luciferase activity indicated that the increasedtransactivation activity of Gal4-NFAT3-1-902 observed in JNKWT

MEFs was significantly suppressed in JNK1�/� or JNK2�/� MEFs(Fig. 3B), suggesting that JNK1 and JNK2 might be positiveregulators of the transactivation activity of NFAT3. To further

delineate the role of JNK1 and JNK2 in the regulation of NFAT3transactivation, we created Gal4-NFAT3-1-112 and Gal4-NFAT3-113-260 constructs and cotransfected each with p5xGal4-luc andanalyzed NFAT3 transactivation activity in JNKWT, JNK1�/�, andJNK2�/� MEFs (Fig. 3C). Results indicated that the transactivationactivity of NFAT3-113-260 was strongly increased compared withthe pGal4 mock control, but NFAT3-1-112 did not induce trans-activation (Fig. 3C). Furthermore, the increased transactivationactivity of Gal4-NFAT3-113-260 was significantly inhibited inJNK1�/� or JNK2�/� MEFs similar to that observed for NFAT3-1-902 in these same cells (Fig. 3B and C). To further verify a role forJNKs in regulating the transcriptional activity of NFAT3, wetransfected increasing amounts of pcDNA3-Flag-NFAT3/pcDNA3.1-v5-JNK1 or pcDNA3-Flag-NFAT3/pcDNA3.1-v5-JNK2 with the3xNFAT-luciferase reporter plasmid and analyzed luciferase acti-vity (Fig. 3D). Results indicated that NFAT3-mediated 3xNFAT-luciferase activity was increased in a JNK1 or JNK2 dose-dependentmanner (Fig. 3D). Taken together, these results indicated that JNK1

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 withpACT-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 Renillaluciferase 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 togetherwith 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 anegative 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 visualizedby 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 withpcDNA3-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. NFAT3was 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 wereindividually cotransfected with pcDNA3-v5-JNK2 into 293 cells.

Cancer Research




and JNK2 phosphorylate NFAT3 at Ser213 and Ser217 and positivelyregulate NFAT3 transcriptional activity.Ser213 and Ser217 of NFAT3 are involved in transactivation

but not with the interaction of NFAT3 with JNK1 or JNK2. Toexamine the role of Ser213 and Ser217 in NFAT3 activity, we firstanalyzed the effect of SP600125, a JNK inhibitor. The p3xNFAT-luciferase plasmid was transfected into JNKWT MEFs either withor without pcDNA3-Flag-NFAT3, and then cells were treated with10 Amol/L SP600125. The results indicated that SP600125 sup-pressed NFAT3-induced 3xNFAT-luciferase activity (Fig. 4A). Next,we analyzed the binding affinity of JNK1 and wild-type NFAT3 ormutant NFAT3 (S213,217A) by the mammalian two-hybrid assay.

The results indicated that NFAT3FL with mutations at Ser213 andSer217 had no effect on the interaction with JNK1 compared withunmodified wild-type NFAT3 (Fig. 4B). In addition, we observedsimilar results for the binding affinity of JNK2 with the NFAT3mutant (data not shown). To determine the effect of phosphory-lation of Ser213 and Ser217 on the transactivation activity of NFAT3,we used JNKWT and JNK1�/� MEFs cotransfected with pGal4-NFAT3-113-260 or pGal4-NFAT3-113-260S213,217A and p5xGal4-luc. The results showed that mutation of NFAT3 at Ser213 and Ser217

inhibited NFAT3 transactivation activity by about 50% in eitherJNKWT or JNK1�/� MEFs (Fig. 4C). These results also revealed thatSer213 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 andschematic diagrams of GST-NFAT3 fusion constructs (1–8 ). To identify the phosphorylation target domain of NFAT3 for active JNK1 or JNK2, each GST-NFAT3 fusionprotein (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-proteinsincluding 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 andthen visualized by autoradiography. *, respective GST-fusion protein (1–3 ) in the JNK1 autoradiograph (left), JNK2 autoradiograph (middle ), and the Coommassieblue–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 activeJNK1 (left ) or JNK2 (middle ), and the results were visualized by autoradiography or Coomassie blue (right ). For A–C , GST mock served as the respective negativecontrol. *, respective GST-fusion protein in the Coommassie blue stained gel. Other bands, which are not marked, are nonspecific bands.





JNK2 and play an important role in the positive regulation ofNFAT3 activity.Knockdown of JNK1 and JNK2 inhibits cell transformation.

JNK1 and JNK2 are critical upstream kinases of c-Jun, which is a

major component of the AP-1 complex. When c-Jun is phosphor-ylated by JNK1 or JNK2 at Ser63 and Ser73, c-Jun DNA bindingaffinity is increased, and the expression of many target genes isincreased many times, resulting in increased cell proliferation and

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 andpGal4-NFAT3-261-902 expression vectors were constructed as indicated. Each construct was cotransfected with the p5xGal4-luciferase reporter plasmid into JNKwild-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 constructwas transfected into JNKWT, JNK1�/�, or JNK2�/� MEF cells with the 5xGal4-luciferase reporter plasmid, respectively, and then firefly luciferase activity wasanalyzed. 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 constructwas transfected with the 5xGal4-luciferase reporter plasmid, respectively, and then firefly luciferase activity was analyzed. Activity is indicated by relative luminescenceunits 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 NFAT3transcriptional activity. The pcDNA3-Flag-NFAT3 and p3xNFAT-luciferase plasmids were cotransfected with increasing amounts of pcDNA3-v5-JNK1 or -JNK2 andthen firefly luciferase activity was analyzed. Activity is indicated by relative luminescence units normalized to a negative control (value for cells transfected with onlyp3xNFAT3-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.

Cancer Research




cell transformation (35–39). Therefore, we proposed that phos-phorylation and activation of NFAT3 by JNK1 and JNK2 might playan important role in cell proliferation as well as cell transforma-tion. To elucidate the physiologic significance of JNK1/2-NFAT3signaling, we constructed siRNA vectors against JNK1 (si-JNK1) andJNK2 (si-JNK2; Fig. 5A). These si-constructs were transfected intoNIH3T3 cells, and knockdown of endogenous JNK1 (f75%) andJNK2 (f95%) was confirmed by Western blotting (Fig. 5B). Toexamine the effect of JNK1 and/or JNK2 knockdown on celltransformation, we introduced various combinations of RasG12V,JNK1, JNK2, NFAT3, si-JNK1, and si-JNK2 expression vectors intoNIH3T3 cells and conducted a focus-forming assay (Fig. 5C). Asexpected, constitutively active Ras (RasG12V) induced cell transfor-mation in NIH3T3 cells (Fig. 5C and graph lane 2). Moreover,overexpression of JNK1 or JNK2 combined with RasG12V resulted inan increased foci formation in NIH3T3 cells (Fig. 5C and graphlanes 3 and 4). Notably, si-JNK1 or si-JNK2 almost completelyblocked 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 notincrease, but actually suppressed foci formation in NIH3T3 cellscompared 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 ofsi-JNK1 and si-JNK2 might be mediated by AP-1 and NFAT3because AP-1 luciferase activity (Fig. 5D) and transactivation acti-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, weconstructed siRNA against NFAT3 (Supplementary Fig. S3A). Thisplasmid was transfected into NIH3T3 cells, and knockdown ofendogenous NFAT3 protein level by about 90% was confirmed byWestern blot (Supplementary Fig. S3B). We then introducedvarious combinations of RasG12V, JNK1, JNK2, NFAT3, and si-NFAT3 into NIH3T3 cells and conducted a focus-forming assay.

As before, RasG12V alone induced foci formation, and JNK1 orJNK2 enhanced RasG12V-induced foci formation (Fig. 6A andgraph lanes 2–4). Interestingly, overexpression of NFAT3 sup-pressed RasG12V/JNK1- or RasG12V/JNK2-induced foci formation inNIH3T3 cells (Fig. 6A and graph lanes 5 and 6). Very importantly,si-NFAT3 enhanced foci formation compared with RasG12V alone,RasG12V/JNK1, or RasG12V/JNK2 in NIH3T3 cells (Fig. 6A and graphlanes 7 and 8). Moreover, the colony size in si-NFAT3–transfectedcells seemed to be larger than RasG12V alone, RasG12V/JNK1,or RasG12V/JNK2 (Fig. 6A). We also found that UVB (4 kJ/m2)

Figure 4. Ser213 and Ser217 of NFAT3 are required for transactivation but not forbinding with JNK1 or JNK2. A, SP600125 inhibits NFAT3-mediated luciferaseactivity. The pcDNA3-Flag-NFAT3-1-902 construct was transfected with the3xNFAT-luciferase reporter plasmid into JNKWT MEFs. Cells were cultured for36 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 luciferaseactivity was analyzed. Activity is indicated by relative luminescence unitsnormalized to a negative control (value for cells transfected with onlyp3xNFAT-luc/pcDNA3-mock = 1.0). B, mutation of NFAT3 at Ser213 andSer217 does not affect NFAT3 binding activity with JNK1 or JNK2. ThepACT-NFAT3-113-260, pACT-NFAT3-113-260-S213,217A, pACT-NFAT3-1-902, or pACT-NFAT3-1-902-S213,217A constructs were individuallycotransfected with pBIND-JNK1 and the pG5-luciferase reporter plasmid intoJNKWT MEFs and then firefly luciferase activity for binding was analyzed. Activityis indicated by relative luminescence units normalized to a negative control(value for cells transfected with only pG5-luc/pBIND-JNK1 = 1.0). The fireflyluciferase activity was normalized against the Renilla luciferase activity.Columns, means of values from triplicate experiments; bars, SD. Significantdifferences were evaluated using the Student’s t test; *, P < 0.005. C, mutationof NFAT3 at Ser213 and Ser217 inhibits NFAT3 transactivation. The pGal4-NFAT3-113-260 and pGal4-NFAT3-113-260S213,217A constructs were eachcotransfected with the p5xGal4-luciferase reporter plasmid into JNKWT orJNK1�/� MEF cells, and then firefly luciferase activity was analyzed. Activity isindicated 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 theRenilla luciferase (pRL-SV40) activity. Columns, means of values from triplicateexperiments; bars, SD. Significant differences were evaluated using theStudent’s t test; *, P < 0.05.





treatment increased AP-1–luciferase activity as well as 3xNFAT-luciferase activity compared with untreated control NIH3T3 cells(Fig. 6B). Moreover, mutation of NFAT3 at Ser213 and Ser217

suppressed 3xNFAT-luciferase activity under a variety of cultureconditions as well as after UVB stimulation (Fig. 6C). Importantly,AP-1 luciferase activity was significantly inhibited by overexpres-sion of NFAT3 in NIH3T3 cells (Fig. 6D).

Discussion

Because NFAT transcription factors are found in a wide range ofcell types and tissues and regulate genes involved in cell cycleprogression, cell development, and differentiation and angiogenesis

(19–22, 40, 41), NFAT3 is also now believed to play an important

role in tumorigenesis or to possess antitumorigenic functions.

Recent research reports have indicated that constitutively active

NFATc1 induces a transformation phenotype in 3T3L1 fibroblasts

(22). However, NFATc2 negatively regulates CDK4 gene expression,

resulting in cells re-entering into the resting stage of the cell cycle

(20). NFAT4 has also been reported to act as a tumor suppressor forthe development of murine T-cell lymphomas induced by theretrovirus SL3-3 (31).

The mitogen-activated protein kinase p38 phosphorylates NFAT3at Ser168 and Ser170 and induces cytoplasmic localization. Whencalcineurin is activated, Ser168 and Ser170 are dephosphorylated, and

Figure 5. Knockdown of JNK1 or JNK2 inhibits cell transformation. A, nucleotidesequences for siRNA JNK1 (si-JNK1) and siRNA JNK2 (si-JNK2) primers.B, knockdown efficiency of si-JNK1 and si-JNK2. NIH3T3 cells were transfectedwith si-JNK1 or si-JNK2 and cultured 36 h. The proteins were extracted withNP40 cell lysis buffer, and total JNK1 or JNK2 protein level was visualized byWestern blot using specific antibodies. h-Actin was used as an internal control toverify equal protein loading. C, knockdown of JNK1 or JNK2 suppressesRasG12V-induced foci formation. Various combinations of expression vectorswere transfected into NIH3T3 cells as indicated, and a foci formation assay wasdone following standard protocols as described in Materials and Methods.Graph, average number of foci; columns, means of values from triplicateexperiments; bars, SD. Significant differences were evaluated using theStudent’s t test. *, P < 0.05; **, P < 0.01; and ***, P < 0.005. D , AP-1 luciferaseactivity is suppressed in JNK1�/� or JNK2�/� MEF cells. The AP-1–luciferasereporter plasmid was transfected into JNKWT, JNK1�/�, or JNK2�/� MEF cellsand 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 fireflyluciferase 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.

Cancer Research




NFAT3 is localized to the nucleus. However, we found that JNK1/2phosphorylated Ser213 and Ser217, and the mutation of Ser213 andSer217 to alanine abolished NFAT3 transactivation and transcrip-tional activity (Figs. 4C and 6C). Furthermore, the NFAT3transactivation activity was suppressed in JNK1�/� and JNK2�/�

MEF cells (Fig. 3B). In addition, NFAT3-mediated 3xNFAT-luciferase activity was increased by cotransfection of JNK1 orJNK2 (Fig. 3D), indicating that phosphorylation of NFAT3 atSer213 and Ser217 by JNK1/2 might induce nuclear localization.These results strongly indicated that distinct phosphorylationsite(s) of NFAT3 might be involved in a different regulatorymechanism for NFAT3.

When NFAT3 was overexpressed with a combination of RasG12V,JNK1, or JNK2 in NIH3T3 cells, foci formation was suppressedcompared with the expression of only RasG12V, RasG12V/JNK1, orRasG12V/JNK2 (Figs. 5C and 6A). In contrast, when siRNA-NFAT3was cotransfected with various combinations of RasG12V, JNK1, andJNK2 into NIH3T3 cells, foci formation was increased comparedwith the expression of only RasG12V, RasG12V/JNK1, or RasG12V/JNK2(Fig. 6A). Furthermore, we found that AP-1 activity was suppressedby ectopic expression of NFAT3 (Fig. 6D), indicating that NFAT3suppressed cell transformation through the inhibition of AP-1activity. Therefore, we hypothesized that NFAT3 is a negativeregulator 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 fociformation. Various combinations of expression vectors were transfected into NIH3T3 cells as indicated, and the foci formation assay was done following standardprotocols as described in Materials and Methods. Graph, average number of foci; columns, means of values from triplicate experiments; bars, SD. Significantdifferences 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-luciferasereporter plasmid was transfected into JNKWT MEF cells. Cells were cultured 36 h, and then firefly luciferase activity was analyzed. Activity is indicated as relativeluminescence 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 atSer213 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 cultured12 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 byrelative luminescence units normalized to a control (value for each cell transfected with only p3xNFAT-luc/pcDNA4-NFAT3 = 100% in each indicated condition). D , AP-1luciferase activity is inhibited by ectopic NFAT3 expression. The pAP-1–luciferase plasmid was transfected with or without pcDNA3-Flag-NFAT3 into JNKWT MEFcells. 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 < 0.05 for C .





Three possibilities could explain the negative regulatory effectmediated by NFAT3. One is a possible competition between c-Junand NFAT3 as a JNK1/2 substrate. When a cell is stimulated by, e.g.,UV, JNK is activated and localized to the nucleus where it thenphosphorylates the c-Jun protein (42). At the same time, JNKphosphorylates and activates NFAT3 because UV stimulation alsoinduces 3xNFAT-luciferase activity (Fig. 6B). Therefore, NFAT3 andc-Jun might compete with JNK1/2 as substrates, resulting in thesuppression of AP-1 activity. However, this is probably unlikelybecause the affinity of the interaction between JNK1/2 and c-Junis very much higher than the potential interaction of JNK1/2and NFAT3 as determined by the mammalian two-hybrid assay(Fig. 1A). The second possibility is that NFAT3 interferes with thenuclear localization of JNK1/2. We found that although JNK nuclearlocalization is induced by stimuli such as UV, only a relatively smallamount of JNK is localized into the nucleus compared with thelarge amount of JNK still remaining in the cytoplasm (42). In cellculture, we found that NFAT3 is predominantly localized in thecytoplasm (28), and when cells are stimulated, JNK1/2 is phos-phorylated and activated in the cytoplasm. After activation,JNK1/2 binds with NFAT3 and phosphorylates NFAT3 also in thecytoplasm. During this process, NFAT3 interfered with JNK1/2localization, resulting in a large amount of JNK1/2 beinglocalized in the cytoplasm. This could at least partially explainwhy the JNK1/2-NFAT3 interaction affinity is lower than theJNK1/2–c-Jun interaction, but could still inhibit AP-1 transcrip-tion activity. However, this idea does not explain the observationthat UV induces a strong and rapid c-Jun phosphorylation atSer63/73. The third possibility involves JDP2 (c-Jun dimerizationprotein 2). JDP2 is a member of the bZIP family of transcriptionfactors and binds with c-Jun and represses AP-1 transcriptionalactivity (43). JDP2 also binds with core transcription machinerysuch as ATF2 and CBP (44). The NFAT3 protein forms a complexwith CBP (29), suggesting that a complex composed of NFAT3and JDP2 is also possible, and that this complex might suppressAP-1 activity as well as induce cell differentiation. This idea issupported by findings showing that (a) JDP2 inhibited Ras-mediated cell transformation in NIH3T3 cells and suppressedtumor development in the xenograft severe combined immuno-

deficiency mouse model (45); (b) ectopic expression of JDP2 inmyoblasts and rhabdomyosarcoma tumor cells strongly promot-ed muscle cell differentiation (46); (c) phosphorylation of NFAT3(Ser281, Ser285, Ser289, and Ser344) by RSK2 induced NFAT3transcriptional activity, resulting in multinucleated myotubedifferentiation of C2C12 myoblasts (28); and (d) siRNA againstNFAT3 enhanced RasG12V/JNK-mediated foci formation, andectopic expression of NFAT3 suppressed RasG12V/JNK-mediatedfoci formation (Fig. 6A). Furthermore, although NFAT1 and AP-1are cooperative for gene expression of many cytokine genes (30),no report has shown that NFAT3 and AP-1 bind to each otherand cooperate directly. Therefore, the detailed molecularmechanism of AP-1 activity inhibition by NFAT3 requires furtherinvestigation.

Based on our observation that NFAT3 is a substrate of JNK1/2,resulting in increased NFAT3 activity, we hypothesized that JNK1/2-NFAT3 function might be involved in cell transformation as wellas oncogenesis because JNK1/2 signaling plays a pivotal role forcell proliferation through the regulation of AP-1 activity. Surpris-ingly, we found that JNK1/2-mediated NFAT3 activation signalingsuppressed RasG12V-mediated NIH3T3 cell transformation as wellas AP-1 luciferase activity (Figs. 5C and 6D). In contrast,knockdown of NFAT3 induced RasG12V-mediated NIH3T3 celltransformation (Fig. 6A). Therefore, we concluded that NFAT3might play a key modulator function to control proliferation anddifferentiation through the regulation of AP-1 activity. Our resultsare the first to show an anti-oncogenic function for NFAT3 that ismediated through the JNK signaling pathway.

Acknowledgments

Received 1/2/2007; revised 4/2/2007; accepted 5/17/2007.Grant support: The Hormel Foundation and NIH grants CA77646, CA81064,

CA27502, and CA111356.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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. R.J. Davis (Department of Biochemistry and Molecular Biology,University of Massachusetts Medical School, Worcester, MA) for the pcDNA3-Flag-JNK1, Dr. C.W. Chow (Department of Molecular Pharmacology, Albert Einstein Collegeof Medicine, Bronx, NY) for the pcDNA3-Flag-NFAT3 deletion mutants, and AndriaHansen for secretarial assistance.

Cancer Research


References

1. Ip YT, Davis RJ. Signal transduction by the c-Jun N-terminal kinase (JNK)-from inflammation to develop-ment. Curr Opin Cell Biol 1998;10:205–19.2. Bost F, McKay R, Dean N, Mercola D. The JUN kinase/stress-activated protein kinase pathway is required forepidermal growth factor stimulation of growth ofhuman A549 lung carcinoma cells. J Biol Chem 1997;272:33422–9.3. Rodrigues GA, Park M, Schlessinger J. Activation of theJNK pathway is essential for transformation by the Metoncogene. EMBO J 1997;16:2634–45.4. Sabapathy K, Jochum W, Hochedlinger K, Chang L,Karin M, Wagner EF. Defective neural tube morpho-genesis and altered apoptosis in the absence of bothJNK1 and JNK2. Mech Dev 1999;89:115–24.5. Kuan CY, Yang DD, Samanta Roy DR, Davis RJ,Rakic P, Flavell RA. The Jnk1 and Jnk2 protein kinasesare required for regional specific apoptosis during earlybrain development. Neuron 1999;22:667–76.6. Bost F, McKay R, Bost M, Potapova O, Dean NM,Mercola D. The Jun kinase 2 isoform is preferentiallyrequired for epidermal growth factor-induced transfor-

mation of human A549 lung carcinoma cells. Mol CellBiol 1999;19:1938–49.7. Chen N, Nomura M, She QB, et al. Suppression of skintumorigenesis in c-Jun NH(2)-terminal kinase-2–defi-cient mice. Cancer Res 2001;61:3908–12.8. Liu J, Lin A. Role of JNK activation in apoptosis: adouble-edged sword. Cell Res 2005;15:36–42.9. Whitmarsh AJ, Davis RJ. Transcription factor AP-1regulation by mitogen-activated protein kinase signaltransduction pathways. J Mol Med 1996;74:589–607.10. Fleming Y, Armstrong CG, Morrice N, Paterson A,Goedert M, Cohen P. Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase(SAPK1/JNK) isoforms by mitogen-activated proteinkinase kinase 4 (MKK4) and MKK7. Biochem J 2000;352 Pt 1:145–54.11. Hibi M, Lin A, Smeal T, Minden A, Karin M.Identification of an oncoprotein- and UV-responsiveprotein kinase that binds and potentiates the c-Junactivation domain. Genes Dev 1993;7:2135–48.12. Fuchs SY, Xie B, Adler V, Fried VA, Davis RJ, Ronai Z.c-Jun NH2-terminal kinases target the ubiquitination oftheir associated transcription factors. J Biol Chem 1997;272:32163–8.

13. Noguchi K, Kitanaka C, Yamana H, Kokubu A,Mochizuki T, Kuchino Y. Regulation of c-Myc throughphosphorylation at Ser-62 and Ser-71 by c-Jun N-terminal kinase. J Biol Chem 1999;274:32580–7.14. Fuchs SY, Adler V, Pincus MR, Ronai Z. MEKK1/JNKsignaling stabilizes and activates p53. Proc Natl Acad SciU S A 1998;95:10541–6.15. Ortega-Perez I, Cano E, Were F, Villar M, Vazquez J,Redondo JM. c-Jun N-terminal kinase (JNK) positivelyregulates NFATc2 transactivation through phosphoryla-tion within the N-terminal regulatory domain. J BiolChem 2005;280:20867–78.16. Yu C, Minemoto Y, Zhang J, et al. JNK suppressesapoptosis via phosphorylation of the proapoptotic Bcl-2family protein BAD. Mol Cell 2004;13:329–40.17. Putcha GV, Le S, Frank S, et al. JNK-mediated BIMphosphorylation potentiates BAX-dependent apoptosis.Neuron 2003;38:899–914.18. She QB, Ma WY, Zhong S, Dong Z. Activation of JNK1,RSK2, and MSK1 is involved in serine 112 phosphory-lation of Bad by ultraviolet B radiation. J Biol Chem2002;277:24039–48.19. Hernandez GL, Volpert OV, Iniguez MA, et al.Selective inhibition of vascular endothelial growth





factor-mediated angiogenesis by cyclosporin A: roles ofthe nuclear factor of activated T cells and cyclo-oxygenase 2. J Exp Med 2001;193:607–20.20. Baksh S, Widlund HR, Frazer-Abel AA, et al. NFATc2-mediated repression of cyclin-dependent kinase 4expression. Mol Cell 2002;10:1071–81.21. Graef IA, Wang F, Charron F, et al. Neurotrophinsand netrins require calcineurin/NFAT signaling to sti-mulate outgrowth of embryonic axons. Cell 2003;113:657–70.22. Neal JW, Clipstone NA. A constitutively activeNFATc1 mutant induces a transformed phenotype in3T3-1 fibroblasts. J Biol Chem 2003;278:17246–54.23. Rao A, Luo C, Hogan PG. Transcription factors ofthe NFAT family: regulation and function. Annu RevImmunol 1997;15:707–47.24. Horsley V, Pavlath GK. NFAT: ubiquitous regulator ofcell differentiation and adaptation. J Cell Biol 2002;156:771–4.25. Graef IA, Chen F, Crabtree GR. NFAT signaling invertebrate development. Curr Opin Genet Dev 2001;11:505–12.26. Ichida M, Finkel T. Ras regulates NFAT3 activity incardiac myocytes. J Biol Chem 2001;276:3524–30.27. Yang TT, Xiong Q, Graef IA, Crabtree GR, Chow CW.Recruitment of the extracellular signal-regulated kinase/ribosomal S6 kinase signaling pathway to the NFATc4transcription activation complex. Mol Cell Biol 2005;25:907–20.28. Cho YY, Yao K, Bode AM, et al. RSK2 mediates musclecell differentiation through regulation of NFAT3. J BiolChem 2007;282:8380–92.29. Yang T, Davis RJ, Chow CW. Requirement of two

NFATc4 transactivation domains for CBP potentiation.J Biol Chem 2001;276:39569–76.30. Macian F, Garcia-Rodriguez C, Rao A. Gene expres-sion elicited by NFAT in the presence or absence ofcooperative recruitment of Fos and Jun. EMBO J 2000;19:4783–95.31. Glud SZ, Sorensen AB, Andrulis M, et al. A tumor-suppressor function for NFATc3 in T-cell lympho-magenesis by murine leukemia virus. Blood 2005;106:3546–52.32. Colburn NH, Wendel EJ, Abruzzo G. Dissociation ofmitogenesis and late-stage promotion of tumor cellphenotype by phorbol esters: mitogen-resistant variantsare sensitive to promotion. Proc Natl Acad Sci U S A1981;78:6912–6.33. Chow CW, Rincon M, Cavanagh J, Dickens M, DavisRJ. Nuclear accumulation of NFAT4 opposed by theJNK signal transduction pathway. Science 1997;278:1638–41.34. Yang TT, Xiong Q, Enslen H, Davis RJ, Chow CW.Phosphorylation of NFATc4 by p38 mitogen-activatedprotein kinases. Mol Cell Biol 2002;22:3892–904.35. Binetruy B, Smeal T, Karin M. Ha-Ras augments c-Junactivity and stimulates phosphorylation of its activationdomain. Nature 1991;351:122–7.36. Papavassiliou AG, Treier M, Bohmann D. Intramo-lecular signal transduction in c-Jun. EMBO J 1995;14:2014–9.37. Pulverer BJ, Kyriakis JM, Avruch J, Nikolakaki E,Woodgett JR. Phosphorylation of c-jun mediated byMAP kinases. Nature 1991;353:670–4.38. Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M.Oncogenic and transcriptional cooperation with Ha-Ras

requires phosphorylation of c-Jun on serines 63 and 73.Nature 1991;354:494–6.39. Behrens A, Sibilia M, Wagner EF. Amino-terminalphosphorylation of c-Jun regulates stress-induced apo-ptosis and cellular proliferation. Nat Genet 1999;21:326–9.40. Caetano MS, Vieira-de-Abreu A, Teixeira LK, WerneckMB, Barcinski MA, Viola JP. NFATC2 transcription factorregulates cell cycle progression during lymphocyteactivation: evidence of its involvement in the control ofcyclin gene expression. FASEB J 2002;16:1940–2.41. Zaichuk TA, Shroff EH, Emmanuel R, Filleur S, NeliusT, Volpert OV. Nuclear factor of activated T cellsbalances angiogenesis activation and inhibition. J ExpMed 2004;199:1513–22.42. Choi BY, Choi HS, Ko K, et al. The tumor suppressorp16(INK4a) prevents cell transformation through inhi-bition of c-Jun phosphorylation and AP-1 activity. NatStruct Mol Biol 2005;12:699–707.43. Aronheim A, Zandi E, Hennemann H, Elledge SJ,Karin M. Isolation of an AP-1 repressor by a novelmethod for detecting protein-protein interactions. MolCell Biol 1997;17:3094–102.44. Broder YC, Katz S, Aronheim A. The ras recruitmentsystem, a novel approach to the study of protein-proteininteractions. Curr Biol 1998;8:1121–4.45. Heinrich R, Livne E, Ben-Izhak O, Aronheim A. Thec-Jun dimerization protein 2 inhibits cell transformationand acts as a tumor suppressor gene. J Biol Chem 2004;279:5708–15.46. Ostrovsky O, Bengal E, Aronheim A. Induction ofterminal differentiation by the c-Jun dimerizationprotein JDP2 in C2 myoblasts and rhabdomyosarcomacells. J Biol Chem 2002;277:40043–54.



2007;67:8725-8735. Cancer Res Ke Yao, Yong-Yeon Cho, H. Robert Bergen III, et al.

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

Updated version

http://cancerres.aacrjournals.org/content/67/18/8725

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2007/09/12/67.18.8725.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/67/18/8725.full#ref-list-1

This article cites 46 articles, 27 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/67/18/8725.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://cancerres.aacrjournals.org/content/67/18/8725To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/content/suppl/2007/09/12/67.18.8725.DC1

http://cancerres.aacrjournals.org/content/67/18/8725.full#ref-list-1

http://cancerres.aacrjournals.org/content/67/18/8725.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Documents

NuclearFactorofActivatedT3IsaNegativeRegulatorof Ras-JNK1/2-AP … · these results indicated that NFAT3 inhibited neoplastic transfor-mation through a negative feedback regulation