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Molecular and Cellular Biology, February 2005, p. 907-920, Vol. 25, No. 3
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.3.907-920.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Teddy T. C. Yang,¹ Qiufang Xiong,¹ Isabella A. Graef,² Gerald R. Crabtree,² and Chi-Wing Chow^1*

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York,¹ Department of Developmental Biology and Pathology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California²

Received 23 August 2004/ Returned for modification 15 September 2003/ Accepted 2 November 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 GSK3ß) 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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents. The NFAT luciferase reporter plasmids and the expression vectors for MAP kinases, RSK, and NFATc4 have been described (20, 21, 37, 39, 48). NFATc4 mutants were generated by PCR. Recombinant ERK and NFATc4 proteins were purified by glutathione affinity chromatography. Antibodies to phospho-Ser⁶⁷⁶ NFATc4 were generated by using synthetic phospho-peptide [Lys-Arg-Ser(P)-Pro-Thr-Gln-Ser-Phe-Arg-Phe-Leu-Pro-Val-Ile-Cys] encoding human NFATc4 and conjugated to ovalbumin. The phospho-specific antibody was affinity purified from rabbit serum as described previously (12).

Cell culture. 3T3/L1, COS, HEK293, and mouse embryonic fibroblasts were cultured in Dulbecco modified Eagle medium. Jurkat and BHK cells were cultured in RPMI 1640 and minimal essential medium, respectively. All media were supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml) (Invitrogen). Cells were transfected by using Lipofectamine (Invitrogen). 3T3/L1 cells were differentiated into lipid-laden adipocytes as described previously (48).

Coupled DNA-binding-in-gel kinase assays. Streptavidin-agarose-precleared cell extracts were incubated with double-stranded, biotinylated PPAR2 proximal NFAT DNA binding element (10 pmol, biotin-ATTACAGGGAAAATATTGCCACACTGTCTC) at 4°C overnight in the presence of 1 µg of poly(dI-dC). Competition was performed by using 10-fold excess of nonbiotinylated NFAT binding element. NFAT-DNA complex was precipitated with 20 µl of streptavidin-agarose at 4°C for an additional 2 h. After three washes with Triton-lysis buffer, the bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to in-gel kinase assays as described previously (30) by using recombinant NFATc4 (0.5 mg/ml) and myelin basic protein (0.5 mg/ml) as substrates. In brief, recombinant proteins were copolymerized with acrylamide gel matrix, and the prepared gel were used to resolve protein kinases associated with the NFAT-DNA precipitates. Resolved gels were subjected to SDS removal (in 20% iso-propanol and 50 mM Tris for 1 h at room temperature), and the gel-bound protein kinases were denaturated (in 6 M guanidine-HCl in 50 mM Tris and 5 mM 2-mercaptoethanol for 1 h at room temperature) and renaturated (in 0.04% Tween 40 in 50 mM Tris and 5 mM 2-mercaptoethanol at 4°C overnight) before incubation in kinase reaction buffer (50 µM [-³²P]ATP in 40 mM HEPES [pH 8.0], 2 mM dithiothreitol, 0.1 mM EGTA, and 5 mM MgCl₂) for 1 h at room temperature. After multiple washes (in 5% trichloroacetic acid and 1% sodium pyrophosphate at room temperature), phosphorylation was examined by autoradiography and phosphorimaging. Biotinylated, mutated PPAR2 proximal NFAT DNA-binding element (biotin-ATTACAGGCATTATATTGCCACACTGTCTC) was used as a control. Binding with biotinylated, wild-type (5'-TTCTTAAATGGAAAACTTAAATCTCTTGCT-3') and mutated (5'-TTCTTAAATGCATTACTTAAATCTCTTGCT-3') PPAR2 distal NFAT DNA was also examined.

Binding assays. Cell extracts prepared by using Triton-lysis buffer (20 mM Tris [pH 7.4], 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 25 mM ß-glycerophosphate, 1 mM sodium vanadate, 2 mM sodium pyrophosphate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg of leupeptin/ml) were incubated (5 h at 4°C) with antibodies prebound to 20 µl of protein G-Sepharose. For binding assays with recombinant proteins (5 µg), 20 µl of glutathione-Sepharose were used. After three washes with Triton-lysis buffer, the bound proteins were separated by SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane (Millipore). Antibodies to NFATc4 (Santa Cruz catalog no. sc-13036), phospho-ERK (sc-7383), ERK (sc-94), phospho-RSK (sc-17033), and RSK (BD Transduction Laboratories catalog no. R23820) were used for immunoblot analysis. Enhanced chemiluminescence was performed to visualize NFAT, RSK, and ERK.

Kinase assays. Hemagglutinin epitope (HA)-tagged ERK, JNK1, and p38 were coexpressed with or without constitutive-active MEK1 (MEK1 N3+S²¹⁸E+S²²²D), MLK3, and constitutive-active MKK6 (MKK6 S²⁰⁷E+T²¹¹E), respectively, in COS cells. HA-tagged RSK were expressed in COS cells and stimulated or not with 100 nM phorbol myristate acetate (PMA). Cell extracts were prepared with Triton-lysis buffer 48 h after transfection, and immunecomplex kinase assays were performed as described previously (48).

Luciferase assays. The NFAT luciferase reporter plasmid (0.3 µg) was cotransfected with the control plasmid pRSV ß-galactosidase (0.1 µg) and NFAT (0.3 µg) or MEK1 (0.1 µg) expression plasmids as indicated. Luciferase and ß-galactosidase activity were measured 48 h after transfection. Cells were stimulated with ionomycin (2 µM), serum (20%), and/or PMA (100 nM) as indicated. The data were presented as the relative luciferase activity, calculated as the ratio of the luciferase activity to the activity of ß-galactosidase (mean ± the standard error [n = 4]).

Gel mobility shift assays. Nuclear extracts were prepared from cultured cells as described previously (48). Double-stranded oligonucleotides for gel mobility shift assays were labeled with [-³²P]dCTP. The sequences for the PPAR2 proximal and distal NFAT elements are described above. The sequence for the IL-2 ARRE NFAT was 5'-AGAAAGGAGGAAAAACTGTTTCATACAGAAGG-3'. The binding reactions were carried out at room temperature in gel shift buffer [1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES (pH 7.9), 50 mM NaCl, 15 mM ß-mercaptoethanol, 10% glycerol, 0.1 mg of bovine serum albumin/ml, and 1 mg of poly(dI-dC)/ml] for 30 min. The protein-DNA complexes were separated in 5% nondenaturing polyacrylamide gels in Tris-glycine-EDTA buffer (25 mM Tris, 200 mM glycine, 1 mM EDTA) and were visualized by autoradiography. For supershift analysis, antibody was preincubated with nuclear extract for 30 min at room temperature before addition of the labeled probe. For association analysis, NFAT-DNA complex was assembled for the indicated time and loaded into the running nondenaturing polyacrylamide gel. For dissociation analysis, an excess amount of unlabeled oligonucleotides (50 pmol) was added to the preassembled NFAT-DNA complex for the indicated times. The amount of NFAT-DNA complex was quantitated by phosphorimager analysis and plotted against the duration of association or dissociation. For saturation analysis, increasing amount of labeled probe (50, 100, 150, and 200 fmol) was incubated with a constant amount of nuclear extract. The amount of NFAT-DNA complexes and unbound probe were quantitated by phosphorimager analysis. The bound/free ratio was plotted against the amount of bound probe to assess relative binding affinity.

Chromatin immunoprecipitations. Nuclear factors that were associated with chromatin in differentiated and undifferentiated 3T3/L1 cells were cross-linked to DNA by using formaldehyde (1%). Cells were harvested and cross-linked chromatin was sheared by sonication. Sonicated cell lysate was immunoprecipitated by using NFAT or RSK antibodies. DNA present in the immunoprecipitated chromatin was isolated, after reversed cross-link and proteinase K digestion, and PCR was performed (5'-GAATTGGCTGGCACTGTCCT-3'; 5'-ATAGACTTGTTGAATAAATC-3') to examine the presence of PPAR2 gene promoter. The presence of GAPDH (for glyceraldehyde-3-phosphate dehydrogenase) promoter (5'-GGCTCTCTGCTCCTCCCTGTTCC-3' and 5'-TCAATGAAGGGGTCGTTGATGGC-3') was also examined.

Top Abstract Introduction Materials and Methods Results Discussion References

 
RSK associates with activated NFAT-DNA complex. To test whether protein kinases are involved in NFAT transcription activation, we performed DNA affinity isolation to avidly pull down activated NFAT and its associated proteins. We reasoned that associated proteins such as protein kinase exhibit enzymatic activity and, hence, we performed in-gel kinase assays to detect the presence of phosphotransferases in the NFAT-DNA precipitates. Coupled DNA-binding-in-gel kinase assays demonstrated that two protein kinases (70 and 90 kDa) are present in the NFAT-DNA precipitates (Fig. 1A). Association of these protein kinases in the NFAT-DNA precipitates is dependent on stimulation with calcium ionophore ionomycin and phorbol ester (PMA). Competition with excess nonbiotinylated NFAT DNA binding element reduced the p70 and p90 protein kinases (70 and 90 kDa, respectively) in the NFAT-DNA precipitates, demonstrating the specificity of the interaction. Thus, these data demonstrate that protein kinases are recruited to the NFAT transcription complex upon activation.

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FIG. 1. RSK associates with activated NFAT-DNA complex. (A) Identification of protein kinases present in the activated NFAT-DNA complex. Biotinylated PPAR2 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 (-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 PPAR2 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 PPAR2 distal NFAT DNA element was also examined.

We suspected that the p90 kinase might be RSK because of the similar mass and the reported function of RSK as a downstream effector kinase in the ERK signaling pathway (17). To test this hypothesis, we performed immunodepletion prior to DNA binding with an antibody to RSK. Coupled DNA-binding-in-gel kinase assays demonstrated that depletion of RSK reduced the p90 but not the p70 protein kinase in the NFAT-DNA precipitates (Fig. 1B). These data indicate that RSK might be involved in the NFAT transcription activation complex.

To confirm the presence of RSK in the NFAT activation complex, we performed immunoblot analysis subsequent to the DNA-binding assays. Immunoblot analysis demonstrated the presence of endogenous RSK in the NFAT-DNA precipitates (Fig. 1C). Importantly, phosphorylated RSK is preferentially recruited upon NFAT activation, and the interaction with NFAT-DNA is reduced by MEK inhibitor U0126. Moreover, recruitment of activated RSK is independent of NFAT partners, as demonstrated by the presence of RSK in both PPAR2 proximal and distal NFAT DNA-binding elements, which form different NFAT complexes (48). Recruitment of RSK upon NFAT activation is further corroborated by coupled DNA-binding-immunoblot analysis with cell extracts expressing RSK2 (Fig. 1D). Together, these data demonstrate that activated RSK is recruited to the NFAT-DNA complex.

Requirement of NFAT in the recruitment of RSK. Previous studies indicated that bound NFAT determines the formation of a ternary activation complex with NFAT partners and DNA (24, 25, 47). To further examine the recruitment of RSK to the NFAT activation complex, we performed mutagenesis to abolish the NFAT binding element. Mutational removal of the NFAT binding element reduced the presence of RSK in the NFAT-DNA precipitates (Fig. 2A). These data indicate that NFAT is required for the recruitment of RSK.

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FIG. 2. Requirement of NFAT in the recruitment of RSK. (A) Biotinylated PPAR2 proximal or distal NFAT DNA-binding elements were incubated with ionomycin and phorbol ester-treated cell extracts prepared from mouse embryonic fibroblasts. Endogenous RSK in the cell extracts and bound to the immobilized wild-type (WT) NFAT DNA elements was detected by immunoblot assays. Binding of RSK to the mutated (MUT) PPAR2 proximal or distal NFAT DNA element was also examined. (B) Biotinylated PPAR2 proximal NFAT DNA-binding elements were incubated with untreated (–) or ionomycin (Ion; 2 µM)- and phorbol ester (PMA; 100 nM)-treated (+) cell extracts prepared from wild-type (WT) and Nfatc2–/– Nfatc4–/– (DKO) mouse embryonic fibroblasts. Endogenous RSK in the cell extracts and bound to the immobilized NFAT DNA element was detected by immunoblot assays. Expression of NFAT proteins was also examined.

We further examined whether lowered levels of total NFAT proteins affected RSK association. We performed coupled DNA-binding-immunoblot assays with extracts prepared from wild-type and Nfatc2^–/– Nfatc4^–/– cells (Fig. 2B). Immunoblot analysis indicated that the loss of NFATc2 and NFATc4 expression in the Nfatc2^–/– Nfatc4^–/– cells, whereas the expression levels of NFATc1 and NFATc3 proteins were similar. The expression levels of RSK was also similar in wild-type and the Nfatc2^–/– Nfatc4^–/– cells. However, the amount of RSK in the NFAT-DNA precipitates was reduced in Nfatc2^–/– Nfatc4^–/– cells. Together, these data demonstrate that RSK targets activated NFAT in the DNA precipitates.

RSK binds to NFATc4. Next, we examined the interaction of RSK to NFAT. Coimmunoprecipitation assays demonstrated the presence of endogenous RSK in the NFATc4 precipitates (Fig. 3A). The interaction between RSK and NFATc4 is further confirmed by coexpression and subsequent immunoprecipitation and immunoblot analysis with either RSK or NFATc4 antibody (Fig. 3B). These data demonstrate that RSK binds to NFATc4.

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FIG. 3. RSK binds to NFATc4. (A) Endogenous NFATc4 were immunoprecipitated (IP) from untreated (–) or treated (+, Ion+PMA) mouse embryonic fibroblast cell extracts. Endogenous RSK in the immunoprecipitates was detected by immunoblot (IB) analysis. Activation and expression of RSK and NFATc4 in the cell extracts is also shown. (B) Extracts prepared from COS cells transfected with epitope-tagged NFATc4 and/or RSK2 were immunoprecipitated (IP) with NFATc4 or RSK2 antibodies. Associated RSK2 or NFATc4 was detected by immunoblot (IB) analysis. (C) Identification of RSK binding site on NFATc4. Various COOH-terminal truncated NFATc4 proteins (residues 1 to 902, 1 to 853, 1 to 581, 1 to 450, 1 to 365, 1 to 308, and 1 to 260) were coexpressed with epitope-tagged RSK2 in COS cells. NFATc4 proteins in the extracts and immunoprecipitated (IP) with RSK2 were detected by immunoblot (IB) analysis with M2 monoclonal antibody to the FLAG epitope. A schematic illustration of the NFATc4 protein and the RSK binding site is also shown.

To delineate the RSK binding site on NFAT, we examined the interaction between various NFATc4 deletion mutants and RSK. Immunoblot analysis demonstrated similar expression of various NFATc4 proteins (Fig. 3C). Deletion of the most COOH-terminal end of NFATc4 (to residue 853) had a minimal effect on RSK binding. However, deletion to remove the COOH-terminal end of the NFATc4 REL homology domain (to residue 581) reduced RSK binding. Further deletion to remove the NH₂-terminal end of the NFATc4 REL homology domain (to residue 450) abolished RSK binding. These data demonstrate that RSK binds to the NH₂-terminal end of the NFATc4 REL homology domain.

ERK binds to NFATc4. Previous studies established that RSK is a prominent downstream effector of the ERK MAP kinase signaling pathway (17). In addition, both RSK and ERK have been demonstrated to phosphorylate the same substrate upon activation (5, 19, 44, 45). ERK binds to and phosphorylates multiple targets by initially docking to the conserved sequence motifs on its targets (reviewed in references 38 and 42). Two docking motifs—FxF and LxL sequences—have been indicated to facilitate ERK interaction. The FxF motif seems to be specific for the ERK substrates, whereas the LxL sequence provides a common recognition site for other MAP kinases, including the c-Jun NH₂-terminal protein kinases (JNK) (23). Sequence analysis indicates that there is a potential FxF motif (Phe^681,683) at the COOH-terminal end of the NFATc4 REL domain (Fig. 4A). Similar sequence is also found in other NFAT members (FxY motifs in NFATc1, NFATc2, and NFATc3). Based on the recent structural analysis (8, 18, 26), the FxF motif on the NFAT REL domain is exposed and possibly allows interaction with ERK.

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FIG. 4. ERK binds to NFATc4. (A) Endogenous NFATc4 interacts with activated ERK. The ERK docking FxF motif on NFAT REL domains is illustrated schematically. The position of the ß-strands (ßf and ßg) of the REL domain, as indicated by the recent structural analysis (8), is also shown. Ser676 of NFATc4 is also highlighted. Endogenous NFATc4 were immunoprecipitated (IP) from untreated or ionomycin (Ion; 2 µM) and phorbol ester (PMA; 100 nM) treated (Ion+PMA) mouse embryonic fibroblast extracts. Endogenous ERK in the immunoprecipitates was detected by immunoblot (IB) analysis. Activation (p-ERK) and expression of ERK in the cell lysate was also shown. (B) Identification of ERK binding site on NFATc4. Various COOH-terminal truncated NFATc4 proteins (residues 1 to 902, 1 to 853, 1 to 581, 1 to 450, 1 to 365, 1 to 308, and 1 to 260) were coexpressed with epitope-tagged ERK in COS cells. NFATc4 proteins in the extracts and immunoprecipitated (IP) with ERK were detected by immunoblot (IB) analysis. A schematic illustration of the NFATc4 protein and the ERK binding site are also shown. (C) Extracts prepared from cells transfected with epitope-tagged NFATc4 344-749 were incubated with recombinant GST-ERK. NFATc4 in the extracts and bound to the immobilized GST-ERK were detected by immunoblot analysis. Effect of deletion (NFATc4 344-581) and mutation of the ERK FxF docking site (NFATc4 344-749 FF681,683GG) on NFATc4 interaction was also examined. (D) Recombinant GST and GST-NFATc4 531-749 proteins were incubated with extracts prepared from cells transfected with epitope-tagged ERK. ERK bound to the immobilized GST proteins was detected by immunoblot analysis. The effect of mutation of the ERK FxF docking site (NFATc4 531-749 FF681,683GG) on NFATc4 interaction was also examined.

To investigate whether ERK binds to NFAT, we examined binding of endogenous ERK to NFATc4 by coimmunoprecipitation assays (Fig. 4A). Immunoblot analysis indicated the presence of ERK in the NFATc4 precipitates. Importantly, NFATc4 preferentially interacts with activated ERK. These data demonstrate that NFATc4 interacts with ERK upon activation.

To delineate the ERK binding site on NFAT, we examined the interaction between various NFATc4 deletion mutants and ERK (Fig. 4B). Deletion of the most COOH-terminal end of NFATc4 (to residue 853) reduced ERK binding. Further deletion to remove the COOH-terminal end of the NFATc4 REL homology domain (to residue 581) abolished ERK binding. These data indicate that ERK binds to the COOH terminus of the NFATc4 REL homology domain, which encompasses the conserved FxF motif.

Next, we tested whether the FxF motif in NFATc4 is important for interaction by using recombinant ERK (Fig. 4C). Binding assays indicated that the NFATc4 REL domain (NFATc4 344-749) was found in the ERK precipitates. Deletion to residue 581 of NFATc4 (NFATc4 344-581) to eliminate the FxF motif or mutation removal of Phe^681,683 of the FxF motif with Gly (NFATc4 344-749 FF^681,683GG) abolished ERK binding. Similarly, with the wild-type and mutated [FF^681,683GG] recombinant NFATc4 proteins in binding assays, replacement of Phe^681,683 with Gly reduced interaction with ERK (Fig. 4D). These data demonstrate that Phe^681,683 of NFATc4 is important for ERK interaction. Together with the mapping of RSK interaction, these data demonstrate that RSK and ERK bind to different regions of the NFATc4 REL homology domain.

RSK and ERK phosphorylates Ser⁶⁷⁶ of the NFATc4 REL homology domain. Binding of RSK and ERK may promote NFATc4 phosphorylation. Hence, we mapped the phosphorylation sites on NFATc4 targeted by RSK and/or ERK. Previous studies indicated that RSK phosphorylation sites (Ser or Thr) are frequently located adjacent to Arg or Lys residues (15, 16). On the other hand, Ser/Thr-Pro motifs are ERK phosphorylation sites, which are often located within 20 amino acids from the ERK binding motif (38). Sequence analysis indicates that there is a Ser-Pro motif (Ser⁶⁷⁶) locates near the NFATc4 FxF motif (Fig. 4A). Thus, Ser⁶⁷⁶ is a potential ERK target. Interestingly, four consecutive Arg/Lys residues (Arg⁶⁷²Arg⁶⁷³Lys⁶⁷⁴Arg⁶⁷⁵) precede Ser⁶⁷⁶, suggesting that Ser⁶⁷⁶ may be an RSK phosphorylation site as well. In addition, a similar Ser residue is found in other NFAT members (Fig. 4A).

Next, we performed in vitro kinase assays to test whether RSK phosphorylates Ser⁶⁷⁶ of NFATc4 (Fig. 5A). Immune complex kinase assays indicated that RSK phosphorylates NFATc4 upon activation. Replacement of Ser⁶⁷⁶ with Ala eliminated RSK phosphorylation. These data demonstrate that RSK phosphorylates Ser⁶⁷⁶.

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FIG. 5. RSK and ERK phosphorylates Ser676 of the NFATc4 REL homology domain. (A) Phosphorylation of Ser676 of NFATc4 by RSK2. Recombinant NFATc4 REL homology domain (GST-NFATc4 531-749) is illustrated schematically. Replacement of Ser676 with Ala is also indicated. Epitope-tagged RSK2 was transfected into COS cells, and extracts were prepared from PMA-treated cells (+; 100 nM) or untreated cells (–) for immune complex kinase assays. Phosphorylation of NFATc4 by RSK2 was quantitated by phosphorimager analysis. The effect of mutation of Ser676 (NFATc4 531-749 S676A) on NFATc4 phosphorylation was also examined. (B) Phosphorylation of Ser676 of NFATc4 by ERK. Epitope-tagged ERK, which was activated (+) or not activated (–) by coexpression with constitutive-active MEK1, was immunoprecipitated and incubated with recombinant GST-NFATc4 531-749 proteins. Phosphorylation of GST-NFATc4 531-749 by ERK was examined by immune complex kinase assays and quantitated by phosphorimager analysis. The effect of mutation at Ser676 (NFATc4 531-749 S676A) on NFATc4 phosphorylation was also examined. (C) Epitope-tagged JNK, ERK, and p38 MAPK, which were activatedor not by coexpression with MLK3, constitutive-active MEK1, and constitutive-active MKK6, respectively, were immunoprecipitated and incubated with recombinant GST-NFATc4 531-749 proteins. Phosphorylation of NFATc4 DNA-binding domain was examined by autoradiography.

We also tested whether ERK phosphorylates Ser⁶⁷⁶ (Fig. 5B). Immune complex kinase assays indicated that ERK phosphorylates NFATc4 and, to a lesser extent, Ala⁶⁷⁶ NFATc4. These data demonstrate that ERK phosphorylates multiple sites, including residue Ser⁶⁷⁶, of the NFATc4 REL domain.

Similar to the ERK MAP kinase, JNK and p38 MAP kinases are also Pro-directed Ser/Thr kinase. To ascertain whether phosphorylation of NFATc4 REL homology domain is specific to the ERK/RSK signaling pathway, we examined phosphorylation of Ser⁶⁷⁶ by JNK and p38 MAP kinases (Fig. 5C). Immune complex kinase assays indicated that activated ERK phosphorylated the NFATc4 REL domain. However, neither JNK nor p38 MAP kinases phosphorylated the NFATc4 REL domain. These data demonstrate that the ERK/RSK signaling pathway mediates phosphorylation at the NFATc4 REL homology domain.

Phospho-Ser⁶⁷⁶ antibodies reveal NFATc4 phosphorylation. We further examined phosphorylation of Ser⁶⁷⁶ of NFATc4 by generating phospho-Ser⁶⁷⁶ antibodies. Immunoblotting analysis indicated that in vitro ERK phosphorylated NFATc4 REL domain was detected by the phospho-Ser⁶⁷⁶ antibodies (Fig. 6A). Preincubation of the antibodies with the phospho-Ser⁶⁷⁶ peptide abolished the recognition (data not shown). In addition, the phospho-Ser⁶⁷⁶ antibodies detected the wild-type NFATc4 proteins but not the Ser⁶⁷⁶ mutated NFATc4 proteins (Fig. 6B). Importantly, detection of the wild-type NFATc4 by the phospho-Ser⁶⁷⁶ antibodies is dependent upon stimulation with PMA. Pretreatment with MEK inhibitor U0126 reduced NFATc4 Ser⁶⁷⁶ phosphorylation. These data confirm phosphorylation of Ser⁶⁷⁶ of NFATc4.

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FIG. 6. Phospho-Ser676 antibodies reveal NFATc4 phosphorylation. (A) Phosphorylation of NFATc4 Ser676 by ERK in vitro. Recombinant NFATc4 (GST-NFATc4 531-749) was phosphorylated by ERK in vitro by using immune complex kinase assays. Phosphorylation of NFATc4 Ser676 was detected by immunoblot analysis (IB) with anti-phosphoNFATc4 (p-Ser676) and GST antibodies. ns, nonspecific cross-reaction. (B) Phosphorylation of Ser676 by ERK is susceptible to U0126. HA-tagged NFATc4 REL domain (NFATc4 344-749) was transfected into COS cells, and extracts were prepared from PMA-treated (+; 100 nM) or untreated (–) cells for immunoblot analysis with anti-phosphoNFATc4 (p-Ser676) and HA antibodies. The expression of activated (p-ERK) and total ERK is indicated. The effect of replacement of Ser676 with Ala and pretreatment with MEK inhibitor U0126 were also examined. (C) Phosphorylation of Ser676 by RSK2. NFATc4 was coexpressed with constitutive-active RSK2 or constitutive-active MEK1 in COS cells. Cell extracts were examined by immunoblot analysis with anti-phosphoNFATc4 (p-Ser676) and HA antibodies. Expression of RSK2 and MEK1 was indicated. (D) Phosphorylation of Ser676 of endogenous NFATc4. Endogenous NFATc4 were immunoprecipitated from untreated (–) or PMA-treated (+) Jurkat cell extracts. DNA affinity isolation was also performed by using the PPAR2 proximal NFAT element to precipitate endogenous NFAT. Phosphorylation of Ser676 of NFATc4 (p-Ser676) in the immunoprecipitates and DNA precipitates was detected by immunoblot analysis. The effect of pretreatment with MEK inhibitor U0126 was also examined. Activation (p-RSK) and expression of RSK in the cell lysate was also shown.

We also tested whether RSK phosphorylates Ser⁶⁷⁶. Constitutive-active RSK2 (36) was coexpressed with NFATc4, thus circumventing the requirement for activated ERK (Fig. 6C). Immunoblot analysis with the phospho-Ser⁶⁷⁶ antibodies indicated that, similar to coexpression with MEK1, constitutive-active RSK2 phosphorylated NFATc4. These data demonstrate that both RSK and ERK phosphorylate NFATc4.

To ascertain phosphorylation of Ser⁶⁷⁶ in endogenous NFATc4, we performed immunoprecipitation with NFATc4 antibodies and subsequent immunoblotting analysis to identify phospho-Ser⁶⁷⁶ NFATc4. PMA stimulation increased NFATc4 phosphorylation at Ser⁶⁷⁶ (Fig. 6D). Administration of MEK1 inhibitor U0126, however, reduced Ser⁶⁷⁶ phosphorylation. Furthermore, we performed coupled DNA-binding-immunoblot assays with phospho-Ser⁶⁷⁶ NFATc4 antibodies. Similar activation-dependent of NFATc4 phosphorylation of Ser⁶⁷⁶ was revealed (Fig. 6D). Together, these data establish that Ser⁶⁷⁶ of NFATc4 is targeted by the MEK/ERK/RSK signaling pathway.

Activation of RSK and ERK potentiates NFAT-mediated gene transcription. Previous studies indicated that mutational removal of Ser^168,170 at the NH₂-terminal of NFATc4 with Ala causes constitutive nuclear localization, which leads to increase in NFAT-mediated gene transcription (48). Thus, phosphorylation at the NH₂-terminal of NFATc4 inhibits NFAT activation. We suspected that phosphorylation at Ser⁶⁷⁶ of the COOH-terminal NFAT REL homology domain, however, might positively regulate NFATc4 because of the activation-dependent recruitment and nuclear localization of RSK and ERK. To test this hypothesis, we examined whether MEK inhibitor U0126 blocked NFAT-mediated transcription (Fig. 7A). Luciferase reporter gene assays indicated that pretreatment of U0126 reduced NFAT-mediated gene transcription. These data support the view that the ERK/RSK signaling pathway positively regulates NFAT.

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FIG. 7. Activation of RSK and ERK potentiates NFAT-mediated gene transcription. (A) MEK inhibitor U0126 reduces NFAT-mediated transcription. Cells transfected with NFAT-luciferase reporter were pretreated or not with MEK inhibitor U0126 for 2 h. The cells were then stimulated without (untreated) and with serum (20%) plus ionomycin (Ion; 2 µM) for 16 h before harvest. Transfection efficiency was monitored by measurement of ß-galactosidase activity. (B) Expression constructs for full-length, wild-type NFATc4, constitutive nuclear NFATc4 (NFATc4 SS168,170AA), and constitutive nuclear, Ser676 phosphorylation-defective NFATc4 (NFATc4 SS168,170AA+S676A) were coexpressed, in the presence or absence of constitutive-active MEK1, with the NFAT-luciferase reporter plasmid. Cells were harvested 36 h after transfection. The luciferase and ß-galactosidase activities were measured. (C) Expression constructs for NFATc4 REL DNA-binding domain (NFATc4 344-749), ERK binding-defective NFATc4 344-749 FF681,683GG, and Ser676 phosphorylation-defective NFATc4 344-749 S676A were coexpressed, in the presence or absence of constitutive-active MEK1, with the NFAT-luciferase reporter plasmid. Cells were harvested 36 h after transfection. Luciferase and ß-galactosidase activities were measured. (D) Expression constructs for NFATc4 REL DNA-binding domain (NFATc4 344-749) and cooperation-defective NFATc4 (NFATc4 344-749 RN474,475AA, T541G) were coexpressed with C/EBPß and, in the presence or absence of constitutive-active MEK1. Compound mutation to abolish Ser676 phosphorylation and NFAT cooperative interaction (NFATc4 344-749 RN474,475AA, T541G+S676A) was also examined. Cells were harvested 36 h after transfection. NFAT-mediated luciferase activity and control of ß-galactosidase activity were measured. (E) Expression constructs for full-length, constitutive nuclear NFATc4 (NFATc4 SS168,170AA), and constitutive nuclear, Ser676 phosphorylation-defective NFATc4 (NFATc4 SS168,170AA+S676A) were coexpressed, in the presence or absence of wild-type RSK2, constitutive-active RSK2 (active RSK2) or dead RSK2, with the NFAT-luciferase reporter plasmid. Cells were harvested 36 h after transfection. The luciferase and ß-galactosidase activities were measured.

We further examined whether MEK1 affects NFAT-mediated gene transcription by Ser⁶⁷⁶ phosphorylation (Fig. 7B). Luciferase reporter gene assays indicated that expression of wild-type NFATc4 increased transcription activity. Expression of constitutive nuclear NFATc4 SS^168,170AA led to an additional increase in transcription activity. In the absence of MEK1, NFATc4 SS^168,170AA and NFATc4 SS^168,170AA+S⁶⁷⁶A mediated transcription to a similar extent. Coexpression of MEK1 with either wild-type or SS^168,170AA NFATc4 further potentiated NFATc4-mediated transcription. Mutational removal of Ser⁶⁷⁶, however, reduced MEK1-mediated transcription potentiation. Replacement of Ser⁶⁷⁶ with Glu could not mimic phospho-Ser⁶⁷⁶ and increase NFATc4-mediated transcription (data not shown). These data indicate that activation mediated by the ERK signaling pathway requires Ser⁶⁷⁶ phosphorylation, which further augments the elevated transcription upon dephosphorylation at Ser^168,170 of NFATc4.

We further examined the effect of removal of ERK binding on MEK1-induced NFAT gene transcription (Fig. 7C). Similar to the Ser⁶⁷⁶ phosphorylation-defective NFATc4, mutational removal of the ERK binding site (NFATc4 344-749 FF^681,683GG) reduced MEK1-mediated transcription potentiation. These data demonstrate that binding and phosphorylation is required for the effect of ERK on NFAT activation.

Previous studies established that replacement of Arg⁴⁷⁴ and Asn⁴⁷⁵ with Ala and Thr⁵⁴¹ with Gly of NFATc4 abolished cooperative interaction between NFAT and NFAT partners (8, 29, 47). To ascertain that MEK1-mediated NFAT activation channels to both NFAT and NFAT partners, we examined the effect of MEK1 on NFAT mutant (NFATc4 344-749 RN^474,475AA, T⁵⁴¹G) that is defective in cooperative interaction with AP-1 or C/EBP (Fig. 7D). Similar to the Ser⁶⁷⁶ phosphorylation-defective NFATc4 and ERK binding-defective (NFATc4 344-749 FF^681,683GG) NFATc4, mutational removal to abolish NFAT cooperative interaction (NFATc4 344-749 RN^474,475AA, T⁵⁴¹G) reduced MEK1-mediated transcriptionpotentiation. Compound mutation to abolish Ser⁶⁷⁶ phosphorylation and NFAT cooperative interaction (NFATc4 344-749 RN^474,475AA, T⁵⁴¹G + S⁶⁷⁶A) further reduced MEK1-mediated transcription. These data demonstrate that the MEK/ERK/RSK signaling pathway regulates both NFAT and NFAT partners to achieve optimal NFAT activity.

We also examined the effect of RSK on transcription mediated by the full-length, constitutive nuclear NFATc4 (NFATc4 SS^168,170AA) (Fig. 7E). Expression of constitutive-active RSK2, but not wild-type or dead RSK2, further increased NFATc4 SS^168,170AA-mediated gene transcription. However, mutational removal of Ser⁶⁷⁶ reduced transcription potentiation mediated by constitutive-active RSK2. These data demonstrate that RSK also positively regulates NFATc4 activation.

Phosphorylation of Ser⁶⁷⁶ potentiates NFATc4 DNA binding. NFATc4, ERK, and RSK are located in the nucleus upon activation (11, 14, 22, 28, 35). Since the interaction with NFATc4 is activation dependent, ERK and RSK may regulate NFAT nuclear function to potentiate gene transcription. Two nuclear events of NFAT—DNA binding and transcription activation—could be regulated by ERK and RSK. First, we tested whether activation of the ERK and RSK signaling pathway regulates NFAT transactivation. We performed Gal4-luciferase reporter gene assays with the NH₂- and the COOH-terminal NFATc4 transactivation domains (46). Coexpression of MEK1 had minimal effect on the transcription mediated by the NH₂- or the COOH-terminal NFATc4 transactivation domains (data not shown).

Next, we tested whether the DNA binding of NFAT is affected by ERK activation by using gel mobility shift assays. Stimulation with serum plus ionomycin increased NFAT DNA binding (Fig. 8A). Administration of U0126, however, reduced NFAT DNA binding. Thus, these data demonstrate that the ERK/RSK signaling pathway regulates NFAT DNA binding.

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FIG. 8. Phosphorylation of Ser676 potentiates NFATc4 DNA binding. (A) U0126 attenuates DNA binding of NFAT. 3T3/L1 cells were serum starved and pretreated with MEK inhibitor U0126 for 2 h before stimulation with serum (20%) and ionomycin (2 µM) for 30 min. Nuclear extracts were prepared and DNA binding of endogenous NFAT was examined by gel mobility shift assays with the IL-2 canonical NFAT binding element. (B and C) NFATc4 REL DNA-binding domain (NFATc4 344-749) and Ser676 phosphorylation-defective NFATc4 344-749 S676A were expressed in the presence or absence of constitutive-active MEK1. Nuclear extracts were prepared, and gel mobility shift assays were performed to examine NFATc4 DNA binding using the proximal and distal PPAR2 NFAT elements (B). Expression of NFATc4 proteins was also shown. Nuclear extracts were also incubated with increasing amount of 32P-labeled IL-2 NFAT element (50, 100, 150, and 200 fmol) to examine saturation binding of NFATc4 (C). The amounts of NFATc4-DNA complexes and remaining unbound free probe were quantitated by phosphorimager analysis. The ratio of bound to free probe was plotted against the amount of bound probe to assess the relative binding affinity. (D and E) Kinetic parameters of formation and dissociation of NFATc4-DNA complexes. Nuclear extracts prepared from wild-type and S676A NFATc4, in the presence or absence of MEK1, were incubated with 32P-labeled IL-2 NFAT element for the indicated times to determine the formation of NFATc4-DNA complexes (D). For dissociation analysis, excess amounts of unlabeled oligonucleotides (50 pmol) were added to the preassembled NFAT-DNA complex for the indicated times (E). The amount of NFATc4-DNA complex was quantitated by phosphorimager analysis.

We further examined whether phosphorylation at Ser⁶⁷⁶ affects DNA binding (Fig. 8B). We prepared nuclear extracts from cells transfected with wild-type and mutated (S⁶⁷⁶A) NFATc4 REL domain for gel mobility shift assays. Immunoblot analysis indicated similar expression of NFATc4 proteins. Expression of the NFATc4 REL proteins promotes specific protein-DNA complexes as indicated by supershift analysis with antibody detects NFAT. Coexpression of MEK1 increased formation of NFATc4-DNA complexes. Saturation analysis indicated that there is approximately threefold increase in NFATc4 DNA binding upon ERK/RSK activation by MEK1 expression (Fig. 8C). On the other hand, mutational removal of Ser⁶⁷⁶ with Ala, to prevent ERK/RSK phosphorylation, reduced NFATc4 DNA binding approximately sixfold. Importantly, phosphorylation at Ser⁶⁷⁶ of NFATc4 increased DNA association in three different NFAT DNA binding elements (Fig. 8B and C). Together, these data demonstrate that phosphorylation of Ser⁶⁷⁶ is critical for ERK/RSK-potentiated NFATc4 DNA binding.

Next, we examined the kinetic parameters of formation of NFATc4-DNA complexes upon Ser⁶⁷⁶ phosphorylation (Fig. 8D). Phosphorylation at Ser⁶⁷⁶ increased the on-rate of NFATc4 DNA binding. In approximately 4 min, 50% of phosphorylated NFATc4 was DNA bound. On the contrary, approximately 7 min and >20 min were required for 50% DNA binding for wild-type and S⁶⁷⁶A NFATc4, respectively.

We also examined the dissociation of the NFATc4-DNA complexes (Fig. 8E). Both wild-type and S⁶⁷⁶A NFATc4 were dissociated from the DNA at a similar rate. ERK/RSK activation had minimal effect on NFATc4-DNA dissociation. Together, these data demonstrate that Ser⁶⁷⁶ phosphorylation increases DNA binding by promoting association of NFATc4 with DNA.

Recruitment of RSK in NFAT transcription complex upon adipogenesis. Previous studies demonstrated that NFAT cooperates with C/EBP and regulates PPAR2 gene transcription in adipocyte differentiation (47, 48). In addition, RSK phosphorylates NFAT (Fig. 5) and C/EBP (5, 19). Hence, we sought to determine whether RSK is associated with the NFAT transcription complex upon adipocyte differentiation. Chromatin immunoprecipitations indicated that NFATc4 is associated with the PPAR2 gene promoter in differentiated, but not the undifferentiated, 3T3/L1 adipocytes (Fig. 9) (47). RSK is also recruited to the PPAR2 promoter complex upon differentiation (Fig. 9). Importantly, phospho-RSK is present in the PPAR2 promoter complex. Thus, these data demonstrate that active RSK is recruited to the NFAT transcription complex to mediate PPAR2 gene expression upon adipocyte differentiation.

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FIG. 9. Recruitment of RSK in NFAT transcription complex upon adipogenesis. The binding of NFAT and RSK to the PPAR2 promoter upon adipocyte differentiation was evaluated. 3T3/L1 cells were subjected to adipocyte differentiation in the presence of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. Chromatin immunoprecipitations (ChIP) were performed to precipitate NFATc4-, RSK-, and phospho-RSK-bound promoters. The DNA isolated was amplified by PCR with oligonucleotides encoding the PPAR2 and GAPDH promoter. Unimmunized rabbit immunoglobulin (IgG) was used as a control.

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Phosphorylation and dephosphorylation in NFAT activation. NFAT is located in the cytosol of resting cells. Dephosphorylation at the NH₂-terminal NFAT homology domain promotes NFAT nuclear accumulation. Indeed, replacement of the conserved NH₂-terminal phosphorylation sites with Ala, to mimic dephosphorylation, increases nuclear localization and NFAT-mediated transcription (3, 13, 33, 48). Plausibly, the mechanism of activation is by mass accumulation of nuclear NFAT. Thus, phosphorylation at the NH₂-terminal end impedes NFAT activation. We demonstrate here that phosphorylation at the COOH-terminal REL homology domain by the ERK/RSK signaling pathway increases NFATc4 DNA binding and potentiates transcription activation. Hence, dephosphorylation at the NH₂-terminal NFAT homology domain only partially activates NFAT. Further transcription potentiation by increasing DNA binding leads to optimal NFAT activation. Therefore, a balance of dephosphorylation and phosphorylation is required for NFAT function.

Requirement of a second signal to mediate NFAT phosphorylation by ERK/RSK suggests a stepwise activation mechanism to achieve optimal transcription. In addition to regulating DNA binding of nuclear NFAT, phosphorylation of Ser⁶⁷⁶ by ERK/RSK may indirectly act as a nuclear retention signal and prolong nuclear accumulation by NFAT association with DNA. Hence, nuclear NFAT is secluded, by DNA or DNA matrix, and escaped from rephosphorylation that leads to shuttling to the cytosol.

ERK/RSK phosphorylation may also modulate the duration of NFAT-DNA association and elicit various levels of activation, by recruiting diverse NFAT partners and/or coactivators, to attain a different threshold of NFAT-mediated transcription. For example, increased duration of NFAT-DNA association may favor recruitment of transcription coactivators and modulate the induction of NFAT targets, especially since NFATc4 interacts with CBP coactivator at two sites (46). Thus, increased duration of NFAT-DNA association may sustain the expression of specific NFAT targets.

Alternatively, ERK/RSK phosphorylation may modulate interaction of NFAT on the DNA (e.g., regulate formation of NFAT dimers versus NFAT partners) (8, 18, 26). Structural analysis indicates that the surrounding residues of Ser⁶⁷⁶ of NFAT are important for intramolecular interaction and/or binding to the minor groove of DNA. Phosphorylation at Ser⁶⁷⁶ may achieve specific conformations by providing additional salt bridges between NFAT and its interacting molecules, including DNA, NFAT-partners, and NFAT itself. In addition, phosphorylation at Ser⁶⁷⁶ may induce an S-switch type of conformational change, which has been demonstrated in the similar immunoglobulin fold Runt DNA-binding domain (1, 41), and promote NFAT DNA binding. Hence, a specific transcription profile mediated by distinct NFAT ternary complex may be accomplished upon ERK/RSK activation.

Role of ERK/RSK signaling pathway in NFAT activation. We show that both ERK and RSK interact with the NFAT REL domain. Although ERK and RSK target different regions of the REL domain, their interactions with NFAT are activation dependent. Current models suggest that elevated levels of intracellular calcium and subsequent dephosphorylation mediated by the calcineurin phosphatase are required to promote conformational changes and exposure of nuclear localization sequences for NFAT nuclear entry. On the contrary, mitogenic agonists promote dimerization and nuclear localization of the ERK kinase (28, 35), whereas RSK is disinhibited upon phosphorylation to release the autoinhibitory domain from the catalytic domain (17). Activated ERK/RSK signaling pathway targets NFAT REL domain and NFAT partners. Hence, the dual requirements of calcium and ERK activation may ensure that both calcineurin and ERK/RSK are in concerted to mediate expression of critical NFAT targets, such as IL-2 and tumor necrosis factor alpha cytokines in inflammation and PPAR in terminated adipocyte differentiation.

Coupled DNA-binding-in-gel kinase assays indicate that RSK, but not ERK, is associated with the DNA-bound NFAT. Mapping of the ERK binding sites to the COOH-terminal end of the REL domain, which mediates NFAT-DNA binding, supports the release of ERK before DNA interaction. Since the ERK-NFAT interaction is activation dependent and ERK is translocated to the nucleus upon activation, ERK is likely to bind to NFAT after calcineurin-mediated dephosphorylation and ERK activation but before NFAT binds to DNA. If so, interaction with nuclear-bound activated ERK, which also process nuclear localization sequence, may further facilitate the nuclear entry of NFAT.

Similar to ERK, RSK is also translocated into nucleus upon activation. However, RSK remains associated with NFAT-DNA and thus may target additional proteins in the NFAT transcription activation complex. For example, analogous to the effect of JNK on phosphorylation of Jun protein complex (27), association of RSK in the NFAT transcription activation complex may trans-phosphorylate NFAT partners, such as C/EBP and Fos, upon activation (10, 19). trans-Phosphorylation of NFAT partners may contribute to the requirement of dual signals for optimal NFAT activity. trans-Phosphorylation of NFAT-associated transcription coactivators, such as CBP and chromatin-bound histones, may further modulate NFAT-mediated gene transcription.

Assembly of the NFAT transcription activation complex. We demonstrate here that two protein kinases (p70 and p90) are recruited to the NFAT transcription complex in an activation-dependent manner. We demonstrate that the p90 kinase is the ERK-activated RSK kinase. The identity of the p70 NFAT-associated kinase remains to be sought. Association of at least two distinct protein kinases upon NFAT activation suggests that, in addition to DNA binding, phosphorylation modulates additional functions of nuclear NFAT. Analogous to the role of protein kinase A on NF-B activation (9), one function could be phosphorylation-dependent recruitment of CBP coactivator to the NFAT activation complex. Such phosphorylation may then provide additional means to modulate NFAT activity.

NFAT is phosphorylated under basal, unstimulated conditions. Termination of NFAT activity required nuclear export, which is mediated by multiple protein kinases, including GSK3ß, CK1, JNK, and p38 MAP kinases. These inhibiting protein kinases phosphorylate NFAT at the NH₂-terminal end and oppose nuclear accumulation. We demonstrate here that ERK and RSK interact with activated NFAT and promote DNA binding. Hence, distinct protein kinases are associated with NFAT in the basal resting state and in the active form. Dynamic modulation of multiple protein kinases in the NFAT transcription complex suggests that NFAT may be the transducer in relaying activation signal from the plasma membrane and cytosol, as well as the regulator to initiate the expression of NFAT targets in the nucleus. If so, NFAT is acting as a recruitment platform to nucleate the assembly of a transcription complex. Thus, identification of other components of the composite enhancer complex will be essential to understand the molecular bases of NFAT-mediated transcription.

Assembly of the NFAT transcription activation complex is dependent on, in part, the interaction between NFAT, NFAT-partners, and DNA. Association and dissociation of NFAT-DNA contributes to the steady-state formation of the transcription activation complex. We demonstrate here that phosphorylation at Ser⁶⁷⁶ promotes NFATc4-DNA association, supporting the potential role of NFAT to nucleate assembly of a functional transcription activation complex. Phosphorylation mediated by the MEK/ERK signaling pathway also acts on NFAT partners and contributes to the final output. Our previous results demonstrated that dissociation of the NFATc4-DNA complex is regulated by the NFAT partners (47), suggesting that NFAT partners modulates termination of the transcription activation complex. Hence, in addition to understand NFAT proteins per se, elucidation of the NFAT partners is equally important to reveal the molecular mechanisms of NFAT-mediated transcription.

The coupled DNA-binding-in-gel kinase assays provide a resourceful avenue for identifying protein kinases present in the NFAT activation complex. This protocol could be extended to identify protein kinases in other transcription factor activation complexes by using distinct consensus DNA binding sequence. Furthermore, this protocol could be modified to allow identification of other NFAT-associated proteins that process enzymatic activity. For example, DNA-modifying enzymes, such as histone acetyltransferases, histone deacetylases, and histone methyltransferases, may also be present in the NFAT transcription complex. Biochemical assays to detect these DNA-modifying enzymes are established. A goal for further research will be to elucidate whether extracellular stimuli will recruit distinct protein kinases and/or DNA modifying enzymes to the NFAT transcription complex.

In conclusion, we have demonstrated that the ERK/RSK signaling pathway is recruited to the NFAT transcription complex upon activation. Phosphorylation by the ERK/RSK kinases promotes NFAT DNA binding. Identification and characterization of the organization, regulation, and function of components of the NFAT activation complex will provide the molecular bases to understand NFAT-mediated gene transcription.

 
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.

 
* Corresponding author. Mailing address: Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-2716. Fax: (718) 430-8922. E-mail: cchow{at}aecom.yu.edu.

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Molecular and Cellular Biology, February 2005, p. 907-920, Vol. 25, No. 3
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.3.907-920.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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