1: J Biol Chem. 2005 May 27;280(21):20867-78. Epub 2005 Mar 2. c-Jun N-terminal kinase (JNK) positively regulates NFATc2 transactivation through phosphorylation within the N-terminal regulatory domain. Ortega-Perez I, Cano E, Were F, Villar M, Vazquez J, Redondo JM. Centro Nacional de Investigaciones Cardiovasculares (CNIC), Ronda de Poniente 5, Tres Cantos, Madrid 28760, Spain. The nuclear factor of activated T cells (NFAT) family of transcription factors regulates the transcription of cytokine genes and other genes involved in the regulation and function of the immune system. NFAT activity is regulated by the phosphatase calcineurin, which binds and dephosphorylates the NFAT N-terminal regulatory domain, a critical step required for nuclear translocation and transcriptional activity. Here we show that the mitogen-activated protein kinase (MAPK) JNK activates NFATc2-dependent transcription. Mass spectrometry revealed that JNK phosphorylates at least six residues within the NFATc2 regulatory domain in vitro. Transfection of cells with a chimeric construct encoding the GAL-4 DNA binding domain linked to wild-type NFATc2 showed that JNK stimulates the NFATc2 transactivation domain in activated Jurkat T lymphocytes, an effect that is inhibited by dominant-negative versions of JNK. Likewise, the mutation of the phosphorylation sites identified revealed that Thr(116) and Ser(170) are critical for the transactivation of NFATc2 by JNK. In addition, clustered mutation of the SP-conserved motifs of NFATc2 showed that SP1 and SP2, but not SP3, are also important for the inducible transactivation of NFATc2. Furthermore, mass spectrometry analysis of NFATc2-transfected cells indicated that the activation of the JNK pathway results in the in vivo phosphorylation of Thr(116). Our results indicate that, unlike other NFAT members, the transcriptional activity of NFATc2 is up-regulated by JNK. JNK-mediated phosphorylation of NFATs thus appears to play a differential physiological role among NFAT family members. PMID: 15743762 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: J Clin Invest. 2004 Aug;114(4):475-84. Comment in: J Clin Invest. 2004 Aug;114(4):463-5. Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. Ikeda F, Nishimura R, Matsubara T, Tanaka S, Inoue J, Reddy SV, Hata K, Yamashita K, Hiraga T, Watanabe T, Kukita T, Yoshioka K, Rao A, Yoneda T. Department of Biochemistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan. Receptor activator of NF-kappaB ligand (RANKL) plays an essential role in osteoclast formation and bone resorption. Although genetic and biochemical studies indicate that RANKL regulates osteoclast differentiation by activating receptor activator of NF-kappaB and associated signaling molecules, the molecular mechanisms of RANKL-regulated osteoclast differentiation have not yet been fully established. We investigated the role of the transcription factor c-Jun, which is activated by RANKL, in osteoclastogenesis using transgenic mice expressing dominant-negative c-Jun specifically in the osteoclast lineage. We found that the transgenic mice manifested severe osteopetrosis due to impaired osteoclastogenesis. Blockade of c-Jun signaling also markedly inhibited soluble RANKL-induced osteoclast differentiation in vitro. Overexpression of nuclear factor of activated T cells 1 (NFAT1) (NFATc2/NFATp) or NFAT2 (NFATc1/NFATc) promoted differentiation of osteoclast precursor cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated osteoclast-like cells even in the absence of RANKL. Overexpression of NFAT1 also markedly transactivated the TRAP gene promoter. These osteoclastogenic activities of NFAT were abrogated by overexpression of dominant-negative c-Jun. Importantly, osteoclast differentiation and induction of NFAT2 expression by NFAT1 overexpression or soluble RANKL treatment were profoundly diminished in spleen cells of the transgenic mice. Collectively, these results indicate that c-Jun signaling in cooperation with NFAT is crucial for RANKL-regulated osteoclast differentiation. PMID: 15314684 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: J Exp Med. 2004 Jun 7;199(11):1513-22. Nuclear factor of activated T cells balances angiogenesis activation and inhibition. Zaichuk TA, Shroff EH, Emmanuel R, Filleur S, Nelius T, Volpert OV. Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. It has been demonstrated that vascular endothelial cell growth factor (VEGF) induction of angiogenesis requires activation of the nuclear factor of activated T cells (NFAT). We show that NFATc2 is also activated by basic fibroblast growth factor and blocked by the inhibitor of angiogenesis pigment epithelial-derived factor (PEDF). This suggests a pivotal role for this transcription factor as a convergence point between stimulatory and inhibitory signals in the regulation of angiogenesis. We identified c-Jun NH2-terminal kinases (JNKs) as essential upstream regulators of NFAT activity in angiogenesis. We distinguished JNK-2 as responsible for NFATc2 cytoplasmic retention by PEDF and JNK-1 and JNK-2 as mediators of PEDF-driven NFAT nuclear export. We identified a novel NFAT target, caspase-8 inhibitor cellular Fas-associated death domain-like interleukin 1beta-converting enzyme inhibitory protein (c-FLIP), whose expression was coregulated by VEGF and PEDF. Chromatin immunoprecipitation showed VEGF-dependent increase of NFATc2 binding to the c-FLIP promoter in vivo, which was attenuated by PEDF. We propose that one possible mechanism of concerted angiogenesis regulation by activators and inhibitors may be modulation of the endothelial cell apoptosis via c-FLIP controlled by NFAT and its upstream regulator JNK. PMID: 15184502 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: J Mol Biol. 2003 Dec 12;334(5):1009-22. Structure of NFAT bound to DNA as a monomer. Stroud JC, Chen L. Department of Chemistry and Biochemistry, University of Colorado at Boulder, Campus Box 215, Boulder, CO 80309-0437, USA. The nuclear factor of activated T cells (NFAT) is a calcium-dependent transcription factor that cooperates with a myriad of partner transcription factors to regulate distinct transcription programs. Transcription activation by NFAT without the cooperation of co-stimulatory signals in lymphocytes can also impose a genetic program of anergy. Although the ternary NFAT1/Fos-Jun/DNA complex has been structurally characterized, how NFAT1 recognizes DNA in the absence of cooperative partners and how such a binary NFAT/DNA complex may lead to the assembly of distinct high-order NFAT transcription complexes are still poorly understood. We have determined the crystal structure of the entire Rel homology region (RHR) of human NFAT1 (NFATc2) bound to DNA as a monomer. We also present footprinting evidence that corroborates the protein-DNA contacts observed in the crystal structure. Our structural and biochemical studies reveal the mechanism by which the monomeric Rel protein NFAT recognizes its cognate DNA site. A remarkable feature of the binary NFAT/DNA complex is the conformational flexibility exhibited by NFAT1 in the four independent copies of the NFAT/DNA complex in the crystal structure, which may reflect a mechanism by which NFAT1 interacts with a variety of protein partners as it mediates disparate biological responses. PMID: 14643663 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------