PMC

Fig. 7. Two parallel Ca²⁺-dependent signaling pathways leading to Nur77 expression. Abbreviations: TCR, T-cell receptor; CaM, calmodulin; CN, calcineurin. The black diamond represents the antigen–MHC complex on antigen-presenting cells, and the P contained in the pink circle represents phosphate groups.

Fig. 6. Reconstitution of signaling pathways leading to endogenous Nur77 expression. (A) Co-expression of MEF2D, NFAT, p300 and constitutively active calcineurin is sufficient to induce expression of the endogenous Nur77. Nur77 protein was detected by a combination of immunoprecipitation and western blotting. (B) Co-expression of MEF2D, NFAT, p300 and constitutively active calcineurin induces the transcription of endogenous Nur77 mRNA, but not that of IL-2. RT–PCR was performed with cDNAs synthesized from total RNAs purified from each of transfected DO11.10. cells.

Fig. 3. Mapping of NFATp–MEF2D interacting domains. (A and B) NFATp binds to MEF2D through the MADS domain of MEF2D. In vitro transcription/translation products of various [³⁵S]MEF2D deletion mutants were mixed with Jurkat cell lysates containing Flag-tagged NFATp and immunoprecipitated with anti-Flag antibody. Bound [³⁵S]MEF2D mutants were visualized by autoradiography. (C and D) MEF2D associates with the C-terminal fragment of NFATp. Jurkat cells (1.5 × 10⁷) were transfected with 15 µg of pSGMEF2D along with 20 µg of various NFATp deletion mutants. Cell lysates were immunoprecipitated with anti-MEF2D antibody and probed with anti-flag antibody.

Fig. 5. An NFATp mutant defective in DNA binding remains competent for enhancement of MEF2D-dependent Nur77 reporter gene activity. (A) DNA binding-deficient NFAT mutant NFATp(K522A/R524A) is incapable of activating an NFAT-luciferase reporter gene. (B) NFATp(K522A/R524A) is as active in enhancing MEF2D activity as the wild-type protein. Jurkat T cells were treated with PMA (40 nM) and ionomycin (1 µM) for 12 h and the luciferase activity was normalized to β-gal activity.

Fig. 2. NFATp interacts with MEF2D and enhances MEF2D-dependent reporter gene activation. (A) NFATp and MEF2D form a complex. Jurkat T cells (4 × 10⁷/lane) were transfected with pSG-flag-NFATp or with pSG-c-myc-MEF2D, respectively. NFAT-overexpressing cell lysates were prepared and immunoprecipitated with anti-Flag antibodies and the bound endogenous MEF2D was detected by western blotting using anti-MEF2D antibodies. A similar procedure was used to detect the presence of endogenous NFAT in c-Myc-MEF2D immuno precipitate. (B) Association of endogenous MEF2D, NFAT and p300 with the Nur77 promoter. Various endogenous proteins were immuno precipitated from nuclear lysates prepared from Jurkat T cells transfected with pGLNur77(–307 to –242)-luc with specific antibodies as indicated, and the co-precipitated Nur77 promoter DNA was amplified using primers specific to the promoter region of Nur77 encoded in pGLNur77(–307 to –242)-luc. (C) NFATp enhances the activation of the MEF2D-dependent Gal4 reporter gene.

Fig. 4. DNA binding is not required for NFAT-mediated MEF2 transcriptional activation. (A) DNA sequences of the minimal Nur77 promoter. Putative NFAT- and MEF2-binding sites are overlined and underlined, respectively. Nucleotides that were mutated are indicated by lower case letters. (B) NFATp enhancement of the p300-mediated activation of the Nur77-luciferase reporter gene is dependent on the MEF2D-binding site, but independent of the NFAT-binding sites. (C) NFATp enhances p300 binding to the minimal MEF2-responsive element on the Nur77 promoter. DO11.10 cells were transfected with 2.5 µg of MEF2D, 10 µg of p300, 5 µg of pNur77(–347 to –242)-luciferase reporter gene and varying amounts of NFATp (0–10 µg). Transfected cells were stimulated with 40 nM PMA and 1 µM ionomycin for 1.5 h. Upon treatment with 1% formaldehyde, cell lysates were immunoprecipitated with anti-p300 polyclonal antibodies. CHIP assays were carried out as described in Materials and methods.

Fig. 1. Calcineurin is necessary for TCR-mediated Nur77 expression and activates an MEF2D-dependent reporter gene. (A) DO11.10 T-hybridoma cells (1 × 10⁷/lane) were pre-treated with the indicated concentrations of FK506, CsA or rapamycin for 30 min prior to a 3 h treatment with PMA (40 nM) and ionomycin (1 µM). Cells were lysed with a lysis buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40 and 1 mM PMSF) and incubated with anti-Nur77 antibody–protein A/G beads for 2 h. Nur77 protein present in the immunoprecipitate was detected with anti-Nur77 antibody. (B) DO11.10 cells were transfected with pSGMEF2D, pGLNur77(–307 to –242) luciferase reporter plasmid along with either pSGCNβ2(1–401/WT) or pSGCNβ2(1–401/H160N), and reporter gene activity was determined as previously described (). (C) DO11.10 cells were transfected with Gal4-MEF2D, pG5-luciferase reporter plasmid along with either pSGCNβ2(1–401/WT) or pSGCNβ2(1–401/H160N). Luciferase activities were normalized to β-galactosidase activity.