Recruitment of the Extracellular Signal-Regulated Kinase/Ribosomal S6 Kinase Signaling Pathway to the NFATc4 Transcription Activation Complex

Transcription machinery mediates the molecular basis of eukaryotic gene expression. Multiple transcription factors are assembled on critical cis-acting DNA enhancers to form an enhancer complex (enhanceosome) in order to control the spatial, temporal, and magnitude of gene expression (reviewed in references 2 and 7). Cooperative interactions within the enhanceosome are further promoted by transcription coregulators (activators or repressors) and architectural proteins (e.g., high-mobility-group DNA-binding proteins) to facilitate the formation of transcription activation complex. In addition, various extracellular stimuli elicit distinct enhanceosomes in specific cells, suggesting that signaling molecules are also involved in the making of the transcription activation complex. For example, recruitment of the mitogen-activated protein (MAP) kinase to the initiation mediator complex (31, 40) and transcriptional regulation by histone deacetylase through protein phosphatase 1 and CK2 protein kinase (6, 43) demonstrate integration of phosphotransferases in transcription activation. Thus, understanding the organization, regulation, and function of cis-acting DNA enhancers is important to elucidate the molecular basis of complex eukaryotic gene transcription. The nuclear factor of activated T cells (NFAT) group of proteins were first characterized as transcription factors that bind to the antigen receptor response element (ARRE) of the interleukin-2 (IL-2) gene promoter (reviewed in references 14 and 22). Characterization of the ARRE indicates that a cytoplasmic (NFATc) and a nuclear (NFATn) component cooperate to regulate gene transcription. The NFATc component was subsequently identified as a family of transcription factors (NFATc1 to NFATc4) involve in multiple biological processes. The Fos and Jun group of transcription factors (AP1 proteins) were identified as the NFATn component in the IL-2 gene. Molecular and structural analyses further demonstrate intimate associations to promote cooperative interaction of NFAT and AP-1 (8, 29). Other partners have also been identified as critical NFATn components, including CCAAT/enhancer binding proteins (C/EBP) and transcription factor GATA (32, 47). Thus, formation of a composite enhancer complex is required for NFAT-mediated gene transcription. Current models suggest that two signaling pathways are converged onto NFAT (14, 22). NFAT is located in the cytosol of resting cells. A calcium-mediated signaling pathway is involved to activate the calcineurin phosphatase, which binds to the conserved NH2-terminal NFAT homology domain and dephosphorylates NFAT. Presumably, dephosphorylation induces conformational changes, which then exposes nuclear localization sequences and promotes NFAT nuclear accumulation. Inducible (e.g., stress-activated MAP kinases) and constitutive-active (e.g., CK1 and GSK3β) protein kinases have been indicated to phosphorylate NFAT, in concert or in sequential manners, to oppose nuclear accumulation (4, 13, 34, 48, 49). In addition, differential regulation of subcellular distribution of specific NFAT members by distinct protein kinases has been proposed. Thus, phosphorylation at the conserved NH2-terminal NFAT homology domain impedes NFAT nuclear accumulation and contributes negatively on NFAT activation. In addition to the calcium-mediated NFAT dephosphorylation, a second signaling pathway—the Ras-mediated ERK MAP kinase—is involved in phosphorylation and activation of the NFAT partners. Activation mechanisms include promoting nuclear accumulation, DNA binding, and transcription activation of the NFAT partners. Therefore, in contrast to the negative role of phosphorylation that opposes NFAT nuclear accumulation, phosphorylation promotes NFAT activation, in part, by targeting the NFAT partners. Hence, a balance of phosphotransferases activity is required for optimal NFAT function. The purpose of the present study was to examine the molecular basis of NFAT transcription activation. We ask whether protein kinases are associated with the activation complex, and positively contribute to NFAT transcription. We report that ribosomal p90 S6 protein kinase (RSK) is recruited to the activated NFAT-DNA complex. RSK interacts with the COOH-terminal REL homology DNA-binding domain and phosphorylates Ser676 of NFATc4. Phosphorylation of Ser676 promotes NFATc4 DNA binding by escalating NFAT-DNA association. Ser676 of NFATc4 is also targeted by the ERK MAP kinase. Thus, integration of the ERK/RSK signaling pathway provides an additional means to modulate NFATc4 transcription activity.