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 calcium-mediated 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 Ser⁶⁷⁶ and potentiates NFATc4 DNA binding by escalating NFAT-DNA association. Ser⁶⁷⁶ 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.

ACKNOWLEDGMENTS

We thank R. J. Davis, T. Hoey, J. A. Smith, T. R. Soderling, and T. W. Sturgill for providing reagents. We thank C. S. Rubin and Z.-Y. Zhang for critical reading of the manuscript. We also thank Paul Shore for his insightful discussion and the Diabetes Research and Training Center at AECOM for their support.

This research was supported, in part, by grants from the NIH/NIDDK and the American Heart Association.