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. Author manuscript; available in PMC: 2010 Feb 18.
Published in final edited form as: J Biol Chem. 2007 Jan 9;282(11):8380–8392. doi: 10.1074/jbc.M611322200

Figure 5. NLS1, the Ser/Pro repeat, and the polyproline regions spanning aa 261–365 play a key role in the nuclear localization and activation of NFAT3.

Figure 5

A, NFAT3-(261–365) is a physiological target of RSK2. The pGFP-NFAT3-(261–365) expression vector was transfected into RSK2+/+ and RSK2−/− MEF cells. Cells were cultured for 36 h in a 5% CO2 incubator, and then proteins were extracted with Nonidet P-40 cell lysis buffer. GFP-NFAT3-(261−365) was immunoprecipitated (IP) with anti-GFP antibody (GFP-Ab). The proteins were resolved by SDS-PAGE, transferred onto PVDF membranes, hybridized with an HRP-conjugated phosphoserine (p-serine)-specific antibody, and visualized using the ECL detection kit. WB, Western blot; IgG H.C., IgG heavy chain. B, RSK2 deficiency suppresses the N-terminal transactivation activity of NFAT3. pBIND-wtNFAT3-(1–365) was transfected with the luciferase reporter plasmid (containing five Gal4-binding sites located upstream of the minimal TATA box of the major late promoter in adenovirus) into RSK2+/+ and RSK2−/− MEF cells. Luciferase activity was analyzed at 36 h. A pBIND mock expression vector was transfected as a control. C, differential binding affinity of wild-type and mutant NFAT3 for RSK2.The binding affinity of wild-type and mutant NFAT3 for RSK2 was analyzed by mammalian two-hybrid assay. D, serine phosphorylation is required for NFAT3 activity. The 3× NFAT-luciferase (Luc) reporter plasmid was cotransfected with pGFP-wtNFAT3FL or pGFP-mtNFAT3FL into RSK2+/+ and RSK2−/− MEF cells, and 36 h after transfection, luciferase activity was measured. E, effect of CsA (a calcineurin inhibitor) on NFAT3 activity. The 3× NFAT-luciferase reporter plasmid was transfected with pcDNA3-FLAG-wtNFAT3FL Cells were treated with CsA and/or A23187for 24 h as indicated, and then firefly luciferase activity was analyzed. F, effect of the MEK1 inhibitor PD98059 on NFAT3activity.The3× NFAT-luciferase reporter plasmid was transfected; cells were treated for 24 h with A23187 (1 μm) and/or PD98059 (10 μm); and then firefly luciferase activity was analyzed. For B and F, firefly luciferase activity was normalized against Renilla luciferase activity (pRL-SV40). Each bar indicates the mean ± S.D. of values obtained from triplicate experiments, and significant differences were evaluated using Student's t test (*, p < 0.05;**, p < 0.001).