Phosphorylation of NFATc4 by p38 mitogen-activated protein kinases.

Nuclear factor of activated T cells (NFAT) is a group of transcription factors that was first identified to play an important role in cytokine gene expression (22). Subsequent studies demonstrated that NFATs are present in numerous tissues (33, 34, 40). The wide tissue distribution of the NFAT isoforms suggests that NFAT may participate in multiple physiological processes. Recently, NFAT activity has been implicated in adipocyte differentiation, cardiac hypertrophy, and learning and memory (30, 32, 41). Thus, elucidation of mechanisms that regulate NFAT is critical for understanding these biological processes. Four distinct genes encoding closely related NFAT proteins (NFATc1/NFATc/NFAT2, NFATc2/NFATp/NFAT1, NFATc3/NFAT4/NFATx, and NFATc4/NFAT3) have been identified (reviewed in references 18 and 47). Alternative mRNA splicing of these four genes further generates at least 10 different NFAT polypeptides. The function of these alternatively spliced NFAT isoforms remains elusive. However, all NFAT members contain a highly conserved NH2-terminal regulatory NFAT homology domain and a COOH-terminal Rel homology region for DNA binding. Thus, understanding the function of these conserved domains will provide new insights on NFAT regulation. The NH2-terminal NFAT homology domain encodes several distinct sequences, including the PXIXIT motif, the Ser-rich region (SRR), and the Ser-Pro (SP)-rich boxes for NFAT regulation (18, 47). These sequences are found in all NFAT members. The PXIXIT motif is recognized by the calcineurin phosphatase (3, 15), which dephosphorylates NFAT upon activation. Sequestration of the calcineurin phosphatase by overexpression of the PXIXIT motif blocks NFAT activation. The SRR and the SP boxes are major targets for NFAT phosphorylation (4-6, 12-14, 44, 46, 58). Dephosphorylation of Ser residues in the SRR and the SP boxes promotes nuclear localization of NFAT. Thus, dephosphorylation of the NFAT homology domain, which is mediated by the calcineurin phosphatase, plays an important role in NFAT activation. Once NFAT is dephosphorylated and translocated into the nucleus, activated NFAT interacts with other transcription factors to induce gene expression. The interaction of NFAT with Fos-Jun (AP-1 complex), GATA, and MEF2 suggests that NFAT often functions at composite DNA elements (7, 41, 43, 54, 56). Formation of a ternary complex induces expression of NFAT targets, such as interleukin-2 (IL-2), IL-4, IL-5, and tumor necrosis factor alpha. However, physiological function of NFAT in nonimmune tissues remains to be established. Multiple protein kinases, including the mitogen-activated protein (MAP) kinase group (ERK, JNK, and p38 kinase), glycogen synthase kinase 3β (GSK3β), protein kinase A (PKA), and casein kinase 1α (CK1α), have been shown to phosphorylate NFAT (4, 6, 12-14, 29, 46, 58). NFAT is phosphorylated on multiple Ser residues located in the conserved SRR and the SP boxes. Phosphorylation of these Ser residues opposes nuclear localization of NFAT either by promoting nuclear export or by impeding nuclear import. For example, phosphorylation at Ser269 of NFATc1 (6) and Ser289 of NFATc4 (12) could be mediated by PKA. Sequence comparisons indicate that Ser269 of NFATc1 corresponds to Ser289 of NFATc4. Phosphorylation at Ser269 of NFATc1 promotes its subsequent phosphorylation mediated by GSK3β, which is critical to enhance NFATc1 nuclear export. Phosphorylation at Ser289 of NFATc4, in conjunction with phosphorylation at Ser272, recruits 14-3-3, a signaling modulator, to mask the function of an adjacent nuclear localization sequence; thus, phosphorylated NFATc4 is located in the cytosol. Additional phosphorylation at other Ser residues present in the NFAT homology domain may promote intramolecular interactions to mask the nuclear localization sequence (6, 14, 58) or to obstruct calcineurin binding (12) and hence maintain NFAT in a phosphorylated and inactive state. The MAP kinase group of signaling proteins also phosphorylates members of the NFAT family. NFATc3 was identified as a substrate for JNK in a yeast two-hybrid assay (14). Phosphorylation of Ser163 and Ser165 of NFATc3 by JNK opposes calcineurin-mediated nuclear localization. Ser172 of NFATc1, which is located in a position analogous to that of Ser165 of NFATc3, is also phosphorylated by JNK (13, 46). Replacement of Ser172 with Ala, to prevent JNK phosphorylation, promotes nuclear localization of NFATc1. Importantly, gene targeting studies that disrupt the JNK1 locus also promote NFATc1 nuclear localization and enhance the expression of IL-4, a target of NFAT in T cells (21). Thus, JNK negatively regulates NFATc1 and NFATc3 but not other NFAT isoforms. The purpose of this study was to examine the phosphorylation and function of NFATc4. Since NFATc4 is primarily expressed in nonimmune tissues and has been implicated in multiple biological processes, understanding the regulation of NFATc4 phosphorylation is an important goal. We report that NFATc4 is differentially phosphorylated by MAP kinases. Ser168 and Ser170 of NFATc4, which are analogously located at Ser172 of NFATc1 and Ser163,165 of NFATc3, are targets of the p38 MAP kinase but not of the JNK MAP kinase. Replacement of Ser168 and Ser170 with Ala promotes NFATc4 nuclear localization and enhances NFAT-mediated transcription activity. Stable expression of Ala168,170 NFATc4 (but not wild-type NFATc4) in NIH 3T3 cells promotes adipocyte formation under differentiation conditions. Molecular analysis indicates that NFATc4 binds to two distinct DNA elements on the peroxisome proliferator-activated receptor γ2 (PPARγ2) promoter. Increased PPARγ expression caused by NFATc4 accounts, in part, for the increased adipocyte differentiation.