PMC

Phosphorylation increases NFAT binding to 14-3-3. (A) [³⁵S]methionine-labeled NFAT3 was prepared by in vitro translation in the presence of various concentrations of the protein kinase inhibitor H7 (0, 1, 10, 100, and 1,000 μM). The amount of NFAT3 in the lysate was detected after SDS-PAGE by autoradiography (middle panel). The binding of NFAT3 to immobilized GST–14-3-3τ was examined by autoradiography (upper panel; sizes are indicated in kilodalton) and was quantitated by PhosphorImager analysis (lower panel). (B) Calcineurin inhibits NFAT3 binding to 14-3-3. [³⁵S]methionine-labeled NFAT3 prepared by in vitro translation was incubated with calcineurin (2.5 and 5 U) and calcineurin inhibitor peptide (10 μg). The amount of NFAT3 in the lysate was detected after SDS-PAGE by autoradiography (middle panel). The binding of NFAT3 to immobilized GST–14-3-3τ was examined by autoradiography (upper panel) and was quantitated by PhosphorImager analysis (lower panel).

Calcium and cAMP regulate NFAT binding to 14-3-3. (A) Jurkat T cells were incubated without and with dibutyryl cAMP (500 μM) plus IBMX (50 μM) for 20 min prior to treatment with ionomycin (2 μM, 20 min) or with ionomycin plus PMA (100 nM, 20 min). NFATp proteins were immunoprecipitated, and 14-3-3 present in the immunoprecipitates (IP) was detected by immunoblot analysis (IB). (B) Jurkat T cells were incubated without and with CsA (100 ng/ml, 20 min) prior to treatment with ionomycin (2 μM, 20 min). Extracts prepared from the Jurkat T cells were incubated with immobilized GST–14-3-3τ. Bound NFATp and Raf-1 were detected by immunoblot analysis. The presence of equal amounts of NFATp in the lysates was confirmed by immunoblot analysis. The amount of GST–14-3-3τ was confirmed by Coomassie blue staining. (C) Jurkat T cells were incubated without and with dibutyryl cAMP (500 μM) plus IBMX (50 μM) for 20 min prior to treatment with ionomycin (2 μM, 20 min). Extracts prepared from the Jurkat T cells were incubated with immobilized GST–14-3-3τ. Bound NFATp and Raf-1 were detected by immunoblot analysis. The presence of equal amounts of NFATp in the lysates was confirmed by immunoblot analysis.

NFAT interacts with 14-3-3. (A) Immobilized GST and GST-NFAT3 (residues 1 to 308) were incubated with extracts prepared from [³⁵S]methionine-labeled BHK cells. Bound proteins were separated by SDS-PAGE and detected by autoradiography. (B) Recombinant GST and GST-NFAT3 (residues 1 to 308) were phosphorylated by incubation with 1 mM ATP and purified PKA catalytic subunit. Immobilized GST and GST-NFAT3 were incubated with extracts prepared from Jurkat T cells. Bound 14-3-3 was detected by immunoblot (IB) analysis. The GST and GST-NFAT3 fusion proteins were detected by staining with Coomassie blue. (C) NFATp interacts with 14-3-3 in vivo. NFATp was immunoprecipitated from Jurkat T cell extracts by using a rabbit antibody to NFATp. Preimmune antibody was used as a control. 14-3-3 in the immunoprecipitates (IP) was detected by immunoblot (IB) analysis. (D) NFAT proteins bind 14-3-3. NFAT3, NFAT4, NFATp and NFATc were expressed in COS cells. NFATp was immunoprecipitated with an antibody to NFATp. Epitope-tagged NFAT3, NFAT4, and NFATc were immunoprecipitated with the anti-Flag monoclonal antibody (Ab) M2. 14-3-3 in the cells lysate and in the immunoprecipitates (IP) was detected by immunoblot (IB) analysis. Extracts prepared from mock-transfected cells were used as a control. IgG, immunoglobulin G.

Identification of the 14-3-3 binding site on NFAT. (A) Deletion analysis of NFAT3. The structure of NFAT3 is illustrated schematically. Flag-tagged NFAT3 proteins corresponding to residues 1 to 902, 1 to 580, 1 to 450, 1 to 365, 1 to 308, 1 to 260, 1 to 160, and 1 to 112 were expressed in COS cells and detected by immunoblot analysis of cell lysates with monoclonal antibody M2. The binding of NFAT3 and Raf-1 to immobilized GST–14-3-3τ was examined. Bound NFAT3 and Raf-1 were detected by immunoblot analysis. (B) Replacement of Ser-272, Ser-273, Ser-274, and Ser-289 with Ala decreases NFAT3 binding to 14-3-3. Epitope-tagged wild-type and mutated NFAT3 were expressed in COS cells and detected by immunoblot (IB) analysis of cell lysates with monoclonal antibody M2 (lower panel). The NFAT3 proteins were immunoprecipitated, and 14-3-3 present in the immunoprecipitates (IP) was detected by immunoblot analysis (upper panel; sizes are indicated in kilodaltons. (C) Immobilized GST–14-3-3τ was incubated with [³⁵S]methionine-labeled wild-type and mutated [Ala^{272,273,274,289}] NFAT3 prepared by in vitro translation. Control experiments were performed with in vitro-translated luciferase. Proteins in the lysate and bound to the immobilized GST–14-3-3τ were detected by autoradiography and quantitated by PhosphorImager analysis. (D) Epitope-tagged wild-type and mutated [Ala^{272,273,274,289}] NFAT3 were expressed in COS cells without (Control) and with an expression vector for the PKA catalytic subunit (PKA). NFAT3 proteins were immunoprecipitated, and 14-3-3 present in the immunoprecipitates (IP) was detected by immunoblot analysis (IB).

Phosphorylation by PKA inhibits NFAT transcription activity. (A) Mutational analysis of NFAT3 phosphorylation by PKA. The primary sequence of the NFAT homology domain of NFAT3 is compared with sequences of NFATp (NFAT1), NFATc (NFAT2), and NFAT4 (upper panel). Potential PKA phosphorylation sites are highlighted with filled boxes. NLS-1 and the conserved SP box C are indicated. Recombinant NFAT3 (1 μg) was phosphorylated by purified PKA catalytic subunit in the presence of [γ-³²P]ATP (middle panel; sizes are indicated in kilodaltons). Phosphorylated NFAT3 was detected by autoradiography and quantitated by PhosphorImager analysis (lower panel). (B) Replacement of Ser²⁷², Ser²⁷³, Ser²⁷⁴, and Ser²⁸⁹ with Ala increases the electrophoretic mobility of NFAT3 during SDS-PAGE. Epitope-tagged wild-type and mutated [Ala^{272,273,274,289}] NFAT3 were expressed in COS cells without (Control) and with activated calcineurin (ΔCN). NFAT3 proteins were detected in immunoblot analysis by using monoclonal antibody M2. (C) NFAT3 is phosphorylated by PKA in vivo. Epitope-tagged wild-type and mutated [Ala^{272,273,274,289}] NFAT3 were expressed in COS cells without (Control) and with PKA. The cells were labeled with [³²P]phosphate, and the NFAT3 proteins were isolated by immunoprecipitation. The phosphorylation of NFAT3 was examined by tryptic phosphopeptide mapping. (D) PKA phosphorylates NFAT3 on Ser²⁷² and Ser²⁸⁹ in vivo and in vitro. Recombinant NFAT3 (1 μg) was phosphorylated by purified PKA in the presence of [γ-³²P]ATP (upper left panel). The effect of replacement of Ser²⁷² and Ser²⁸⁹ with Ala was examined. The in vitro phosphorylated wild-type NFAT3 was examined by tryptic phosphopeptide mapping (upper right panel). The phosphorylation of NFAT3 in vivo was also examined by phosphopeptide mapping of NFAT3 co-expressed with PKA in COS cells (middle and lower panels). The effect of replacement of Ser²⁷², Ser²⁷³, Ser²⁷⁴ or Ser²⁸⁹ with Ala was examined. Electrophoresis and chromatography are the horizontal and vertical dimensions of the phosphopeptide maps, respectively. (E) Measurement of NFAT3 transcription activity in BHK cells. The effect of NFAT3 expression was examined in cotransfection assays using an NFAT-luciferase reporter plasmid. The cells were treated without (Untreated) and with ionomycin (2 μM) and PMA (100 nM) (I+P) for 16 h. The data are presented as relative luciferase activity (see Materials and Methods). (F) Mutation of the PKA phosphorylation sites blocks PKA-mediated inhibition of NFAT3 transcription activity. Wild-type and mutated [Ala^{272,273,274,289}] NFAT3 were expressed together with an NFAT-luciferase reporter plasmid in BHK cells. The effect of treatment with dibutyryl cAMP (500 μM) and IBMX (50 μM) or coexpression without (Control) and with PKA on cells treated (24 h) with ionomycin (2 μM) and PMA (100 nM) is presented. (G) Effect of PKA phosphorylation on the nuclear accumulation of NFAT3. The structure of NFAT3 is illustrated schematically to show the serine-rich region (SRR), NLS-1 and NLS-2, NES, the conserved SP boxes (A, B, and C), and the Rel domain. Epitope-tagged NFAT3 proteins were expressed in BHK cells and detected by immunofluorescence microscopy using monoclonal antibody M2. The subcellular distribution of wild-type (WT) and truncated NFAT3 (residues 1 to 580) was examined. The effect of mutation of the PKA phosphorylation sites and coexpression with PKA catalytic subunit or ΔCn is presented. The percentage of cells with NFAT3 in nucleus (n = 100) is presented, and images of representative cells are illustrated. The nuclei (blue) of transfected cells expressing NFAT3 (red) are indicated with arrowheads.