NuclearFactorofActivatedT3IsaNegativeRegulatorof Ras-JNK1/2-AP-1–Induced Cell Transformation 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 Dong 1 1 Hormel Institute, University of Minnesota, Austin, Minnesota and 2 Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, Minnesota Abstract The c-jun-NH 2 -kinases (JNK) play a critical role in tumor promoter–induced cell transformation and apoptosis. Here, we showed that the nuclear factor of activated T3 (NFAT3) is phosphorylatedbyJNK1orJNK2atSer 213 andSer 217 ,whichare locatedintheconservedSPmotif.Thetransactivationdomain of NFAT3 is found between amino acids (aa) 113 and 260 and includes the phosphorylation targets of JNK1 and JNK2. NFAT3 transactivation activity was suppressed in JNK1 À/À or JNK2 À/À mouse embryonic fibroblast (MEF) cells compared with wild-type MEF cells. Moreover, a 3xNFAT-luc reporter gene assay indicated that NFAT3 transcriptional activity was increased in a dose-dependent manner by JNK1 or JNK2. Double mutations at Ser 213 and Ser 217 suppressed NFAT3 transactivation activity; and SP600125, a JNK inhibitor, sup- pressed NFAT3-induced 3xNFAT-luciferase activity. Knock- down of JNK1 or JNK2 suppressed foci formation in NIH3T3 cells. Importantly, ectopic expression of NFAT3 inhibited AP-1 activity and suppressed foci formation. Furthermore, knock- down of NFAT3 enhanced Ras-JNK1 or JNK2-induced foci formation in NIH3T3 cells. Taken together, these results provided direct evidence for the anti-oncogenic potential of theNFAT3transcriptionfactor. [Cancer Res 2007;67(18):8725–35] Introduction The JNKs signal transduction pathway has been shown to play an important role in coordinating various cellular responses such as apoptosis (1), proliferation (2), and neoplastic transforma- tion (3). Mice that are deficient in both jnk1 and jnk2 exhibit embryonic death at E10.5 due to enhanced apoptosis in the hindbrain (4) and forebrain regions (4, 5), which clearly suggests that JNK1 and JNK2 are involved in cell survival during develop- ment. Furthermore, specific antisense oligonucleotides against JNKs inhibited tumor cell growth (6) and jnk2 -deficient mice displayed significant suppression of skin papilloma development induced by 12-O -tetradecanoylphorbol-13-acetate (TPA; ref. 7). These types of observations may have been due to an enhanced apoptosis in jnk -deficient cells and mice (8). When cells are stimulated by environmental stress, cytokines, or toxins (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 activation and phosphorylation of c-Jun at Ser 63 and Ser 73 . However, other downstream 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-X L , Bim, and Bad (16–18). These functions of JNKs have been primarily attributed to the fact that JNKs activate different substrates based on the specific stimulus or cell type. Although the nuclear factor of activated T cell (NFAT) family of transcription factors has been primarily identified in immune cells, recent studies indicated that NFAT is functionally active in several other non-immune cell types, including vascular endothelial cells, embryonic exon cells, and 3T3-L1 fibroblasts (19–22). Four different isotypesof NFATs, including NFAT1 (1a, 1b, and 1c), NFAT2 (2a and 2b), NFAT3, and NFAT4 (4x, 4a, 4b, and 4c) were shown to have differential tissue distribution (23). These findings suggested that distinct NFAT isotypes play different roles in diverse tissues under various physiologic conditions (24). Calcineurin, a Ca 2+ /calmodu- lin-dependent protein phosphatase that is a downstream target of intracellular Ca 2+ signaling, is a well-known effector of the NFAT family 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 gene transcription (24). Upon cessation of the Ca 2+ signal, NFAT proteins are re-phosphorylated by kinases such as GSK-3 (25), resulting in the translocation of NFAT to the cytoplasm (24). However, recent studies indicated that the Ras signaling pathway positively regulates NFAT3 activity (26) by forming an activation complex to regulate PPARg2 promoter activity, which leads to adipocyte differentiation (27). In addition, RSK2-mediated phosphorylation of NFAT3 regulates NFAT3 activity and induces muscle cell differen- tiation (28). Furthermore, the NFAT3 protein forms a complex with CBP to activate transcription machinery (29), and NFATc1 binds with AP-1 to enhance its transcriptional activity (30). Activation of NFATc1 was also reported to induce cell transformation (22). On the other hand, NFATc2 was shown to repress cyclin-dependent kinase 4 (CDK4), resulting in cell cycle arrest at G 0 -G 1 (20, 31). In addition, when the lymphomagenic virus SL3-3 was infected in NFAT4-deficient mice, T-cell lymphoma developed faster and with 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- genesis is not yet understood. In this study, we showed that NFAT3 is a strong binding partner of JNK1 and JNK2. The phosphorylation of NFAT3 at 213 and 217 by JNK1/2 induced NFAT3 transactivation activity. Importantly, overexpression of NFAT3 suppressed Ras G12V -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: zgdong@hi.umn.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-4788 www.aacrjournals.org 8725 CancerRes2007;67:(18).September15,2007 Research Article Downloaded from http://aacrjournals.org/cancerres/article-pdf/67/18/8725/2572570/8725.pdf by guest on 11 October 2023