Nuclear factor of activated T cells (NFAT)-dependent transactivation regulated by the coactivators p300/CREB-binding protein (CBP) - PubMed (original) (raw)

Nuclear factor of activated T cells (NFAT)-dependent transactivation regulated by the coactivators p300/CREB-binding protein (CBP)

C García-Rodríguez et al. J Exp Med. 1998.

Abstract

p300 and cAMP response element-binding protein (CREB)-binding protein (CBP) are members of a family of coactivators involved in the regulation of transcription and chromatin. We show that transcription factors of the nuclear factor of activated T cells (NFAT) family bind p300/CBP and recruit histone acetyltransferase activity from T cell nuclear extracts. The NH2-terminal transactivation domain of NFAT1 and the phospho-CREB- and E1A-binding sites of p300/CBP are involved in the interaction. The viral oncoprotein E1A inhibits NFAT-dependent transactivation in a p300-dependent manner. Recruitment of the coactivators p300/CBP by the transactivation domains of NFAT proteins is likely to play a critical role in NFAT-dependent gene expression during the immune response.

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Figures

Figure 1

Figure 1

NFAT1 associates with HATs in nuclear extracts. Cl.7W2 T cells were stimulated with ionomycin and PMA for 25 min at 37°C, and nuclear extracts were immunoprecipitated (i.p.) with antibodies against the DBD (α_DBD_) or a COOH-terminal peptide (α_Ct_) of NFAT1. An irrelevant antibody was used as a control. The immune complexes were tested for HAT activity as described in Materials and Methods. The results are representative of three independent experiments. Bars, The mean and range of duplicates in one such experiment.

Figure 2

Figure 2

Interaction of p300 and NFAT1 in cells. (A) 293T cells expressing NFAT1 and HA-p300 were stimulated with ionomycin and PMA for 25 min at 37°C. Whole-cell extracts were immunoprecipitated (i.p.) with antibodies against an NH2-terminal peptide (α_Nt_), the DBD (α_DBD_), and a COOH-terminal peptide (α_Ct_) of NFAT1. An irrelevant antibody was used as a control. The immune complexes were analyzed by Western blotting for HA-p300 (top) and NFAT1 (bottom, blot with α_Nt_). The expressed HA-p300, detected with anti-HA (α_HA_), appeared as several bands (lanes 5 and 6), and the bottom band seemed to be preferentially coimmunoprecipitated with NFAT1 (lanes 3 and 4). (B) Lysates from 293T cells expressing NFAT1 alone, HA-p300 alone, or both were immunoprecipitated (i.p.) with anti-HA (α_HA_), and immune complexes were analyzed by Western blotting for HA-p300 (top) and NFAT1 (bottom). Input, 1% of the total amount of cell extract used for immunoprecipitations. Arrows, The different forms detected by immunoblotting.

Figure 2

Figure 2

Interaction of p300 and NFAT1 in cells. (A) 293T cells expressing NFAT1 and HA-p300 were stimulated with ionomycin and PMA for 25 min at 37°C. Whole-cell extracts were immunoprecipitated (i.p.) with antibodies against an NH2-terminal peptide (α_Nt_), the DBD (α_DBD_), and a COOH-terminal peptide (α_Ct_) of NFAT1. An irrelevant antibody was used as a control. The immune complexes were analyzed by Western blotting for HA-p300 (top) and NFAT1 (bottom, blot with α_Nt_). The expressed HA-p300, detected with anti-HA (α_HA_), appeared as several bands (lanes 5 and 6), and the bottom band seemed to be preferentially coimmunoprecipitated with NFAT1 (lanes 3 and 4). (B) Lysates from 293T cells expressing NFAT1 alone, HA-p300 alone, or both were immunoprecipitated (i.p.) with anti-HA (α_HA_), and immune complexes were analyzed by Western blotting for HA-p300 (top) and NFAT1 (bottom). Input, 1% of the total amount of cell extract used for immunoprecipitations. Arrows, The different forms detected by immunoblotting.

Figure 3

Figure 3

Regions of NFAT1 and CBP involved in the interaction. (A) In vitro binding experiments were performed using 6xHisNFAT1(1–415) and GST-CBP fusion proteins immobilized on glutathione-Sepharose beads. After six washes, the samples were analyzed by Western blotting analysis for NFAT1. (B) 293T cells coexpressing HA-p300 and either full-length NFAT1 (NFAT1-FL) or NFAT1ΔReg were stimulated with ionomycin and PMA for 25 min at 37°C. Whole-cell extracts were immunoprecipitated with anti-HA (α_HA_). Immune complexes were analyzed by Western blotting for HA-p300 (top) and NFAT1 (bottom). Input, 1% of the total amount of cell extract used for immunoprecipitations. Arrows, The different forms detected by immunoblotting.

Figure 3

Figure 3

Regions of NFAT1 and CBP involved in the interaction. (A) In vitro binding experiments were performed using 6xHisNFAT1(1–415) and GST-CBP fusion proteins immobilized on glutathione-Sepharose beads. After six washes, the samples were analyzed by Western blotting analysis for NFAT1. (B) 293T cells coexpressing HA-p300 and either full-length NFAT1 (NFAT1-FL) or NFAT1ΔReg were stimulated with ionomycin and PMA for 25 min at 37°C. Whole-cell extracts were immunoprecipitated with anti-HA (α_HA_). Immune complexes were analyzed by Western blotting for HA-p300 (top) and NFAT1 (bottom). Input, 1% of the total amount of cell extract used for immunoprecipitations. Arrows, The different forms detected by immunoblotting.

Figure 4

Figure 4

E1A inhibits NFAT-dependent transactivation in a p300-dependent manner. (A) The NFAT3x-Luc reporter plasmid and expression plasmids encoding full-length NFAT1 (NFAT1-FL) or NFAT1ΔReg were cotransfected into Jurkat cells along with expression plasmids encoding wild-type E1A (WT) or the E1A mutant defective for p300 binding (Mut). (B) The GAL4-Luc reporter and expression plasmid encoding GAL4-NFAT1(1–415), GAL4-NFAT2(1–418), or GAL4-NFAT1ΔSP2 were cotransfected into Jurkat cells along with expression plasmids encoding wild-type E1A (WT) or the E1A mutant defective in p300 binding (Mut). After 36 h of transfection, cells were left unstimulated or stimulated overnight with ionomycin and PMA and assayed for luciferase assay. Results are representative of several experiments. Transfection efficiencies were normalized by measuring hGH expression from a cotransfected RSV-hGH plasmid.

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