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

Abstract

Integration of protein kinases into transcription activation complexes influences the magnitude of gene expression. The nuclear factor of activated T cells (NFAT) group of proteins are critical transcription factors that direct gene expression in immune and nonimmune cells. A balance of phosphotransferase activity is necessary for optimal NFAT activation. Activation of NFAT requires dephosphorylation by the calciummediated calcineurin phosphatase to promote NFAT nuclear accumulation, and the Ras-activated extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase, which targets NFAT partners, to potentiate transcription. Whether protein kinases operate on NFAT and contribute positively to transcription activation is not clear. Here, we coupled DNA affinity isolation with in-gel kinase assays to avidly pull down the activated NFAT and identify its associated protein kinases. We demonstrate that p90 ribosomal S6 kinase (RSK) is recruited to the NFAT-DNA transcription complex upon activation. The formation of RSK-NFATc4-DNA transcription complex is also apparent upon adipogenesis. Bound RSK phosphorylates Ser 676 and potentiates NFATc4 DNA binding by escalating NFAT-DNA association. Ser 676 is also targeted by the ERK MAP kinase, which interacts with NFAT at a distinct region than RSK. Thus, integration of the ERK/RSK signaling pathway provides a mechanism to modulate NFATc4 transcription activity.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 coop...

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