Recruitment of the extracellular signal-regulated kinase/ribosomal S6 kinase signaling pathway to the NFATc4 transcription activation complex.

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 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.

Phosphorylation of NFATc4 by p38 mitogen-activated protein kinases.

Nuclear factor of activated T cells (NFAT) is implicated in multiple biological processes, including cytokine gene expression, cardiac hypertrophy, and adipocyte differentiation. A conserved NFAT homology domain is identified in all NFAT members. Dephosphorylation of the NFAT homology region is critical for NFAT nuclear translocation and transcriptional activation. Here we demonstrate that NFATc4 is phosphorylated by p38 mitogen-activated protein (MAP) kinase but not by JNK. The p38 MAP kinase phosphorylates multiple residues, including Ser(168) and Ser(170), in the NFAT homology domain of NFATc4. Replacement of Ser(168,170) with Ala promotes nuclear localization of NFATc4 and increases NFAT-mediated transcription activity. Stable expression of Ala(168,170) NFATc4, but not of wild-type NFATc4, in NIH 3T3 cells promotes adipocyte formation under differentiation conditions. Molecular analysis indicates that peroxisome proliferator-activated receptor gamma 2 (PPAR gamma 2) is a target of NFAT. Two distinct NFAT binding elements are located in the PPAR gamma 2 gene promoter. Stable expression of Ala(168,170) NFATc4, but not of wild-type NFATc4, increases the expression of PPAR gamma, which contributes in part to increased adipocyte formation. Thus, NFAT regulates PPAR gamma gene expression and has a direct role in adipocyte differentiation.