FIG. 1.

RSK associates with activated NFAT-DNA complex. (A) Identification of protein kinases present in the activated NFAT-DNA complex. Biotinylated PPARγ2 proximal NFAT DNA-binding element was incubated with untreated (−) or ionomycin (Ion; 2 μM)- and phorbol ester (PMA; 100 nM)-treated (+, Ion+PMA) 3T3/L1 cell extracts. Protein kinases in the extracts and bound to the immobilized NFAT DNA element were detected by in-gel kinase assays and visualized by autoradiography. The specificity of the recruited protein kinases was examined by competition with excess nonbiotinylated NFAT binding element. (B) The 90-kDa NFAT-associated kinase belongs to the p90 RSK family. Extracts prepared from untreated (−) and treated (+, Ion+PMA) 3T3/L1 cells were preincubated with antibody against RSK (α-RSK). RSK precleared (+) or not (−) extracts were subjected to coupled DNA-binding-in-gel kinase assays. The amount of phosphorylation was quantitated by phosphorimager analysis. (C and D) Endogenous RSK is recruited to the NFAT-DNA complex upon activation. Biotinylated PPARγ2 proximal NFAT DNA-binding element was incubated with untreated (−) or treated (+, Ion+PMA) cell extracts prepared from mouse embryonic fibroblasts (C). Effect of MEK1 kinase inhibitor U0126 was also examined. Recruitment of RSK is also assessed by using extracts prepared from treated (+, Ion+PMA) or untreated (−) HEK293 cells transfected with epitope-tagged RSK2 (D). Activated (p-RSK) and total RSK (RSK) in the cell extracts and bound to the immobilized NFAT DNA element were detected by immunoblot assays. Binding of RSK to the PPARγ2 distal NFAT DNA element was also examined.