Article Figures & Data
Figures
- FIGURE 1.
Activation of the CD28RE/AP-1 luciferase reporter by DA-MEKK1 in parallel with induced c-Jun and c-Rel binding to this element. A, CD28RE/AP-1 Luc assay showing the effects of different stimuli. Jurkat-tTA cells were transfected with 10 μg of 4xCD28RE/AP-1 Luc, rested for 24 h, and then stimulated with the range of stimuli for 6 h as described in Materials and Methods. Cells were lysed and assayed for luciferase activity. The fold increase (top of each row) was calculated against luciferase values obtained in untreated cells. B, DA-MEKK1 expression by a tetracycline-repressible system. Jurkat-tTA cells were transfected with 20 μg of pTPH into which we had subcloned MEKKΔ (DA-MEKK1). After incubating these cells for 24 h in the presence (+) or the absence (−) of 0.1 μg/ml tetracycline, cell lysates were analyzed by Western blotting, using the anti-MEKK1 Ab at a dilution of 1/1000. C, Reporter gene assay showing that both CD28RE and AP-1 binding sites are required for activation of CD28RE/AP-1 by DA-MEKK1. Jurkat-tTA cells were cotransfected with the reporter gene constructs and DA-MEKK1. Luciferase assays were performed 24 h later in cells grown in the presence or the absence of tetracycline. The fold increase was calculated against the luciferase value for unstimulated, tetracycline-positive (+) cells. D, EMSA showing c-Rel and c-Jun binding to the CD28RE/AP-1. Cells were transfected with DA-MEKK1 as in B and selected in hygromycin for 4 wk to obtain a relatively homogeneous MEKKΔ-expressing population. Twenty-four hours after removal of tetracycline, nuclear extracts were prepared from DA-MEKK1-expressing cells as well as untransfected cells stimulated with P+I or OKT3 plus 9.3 mAb. Gel shift assays were performed as previously described (33). Supershift analysis was performed by adding 1 μg of anti-c-Jun, anti-c-Rel, or nonimmune serum (NIS) to nuclear extracts from DA-MEKK1-expressing cells.
- FIGURE 2.
Kinase-inactive SEK1/MKK4 and mutant c-Jun (TAM67) interfere in the activation of CD28RE/AP-1. A, DN-SEK1 decreases CD28RE/AP-1 Luc activity. Cells were transfected with 20 μg of an empty vector (pCDNA1.1) or with 20 μg of DN-SEK1 plus 10 μg of Luc vector and 6 μg of pCMV-βGal. After resting for 24 h, cells were stimulated with anti-CD3 plus anti-CD28 or P+I for 6 h as described in Fig. 1A. β-Galactosidase values were used to adjust for differences in transfection efficiency. B, Wild-type (wt-) c-Jun enhances, while DN-c-Jun decreases CD28RE/AP-1 activity. Cells were transfected with 20 μg of pCDNA1.1, 20 μg of wt-c-Jun, or 20 μg of DN-c-Jun together with 10 μg of the Luc vector and 6 μg of the β-Gal vector.
- FIGURE 3.
PD098059, a specific MEK1 inhibitor, inhibits the expression of IL-2 message as well as activation of the distal NF-AT/AP-1, but does not interfere with activation of the CD28RE/AP-1 response element. A, RT-PCR assay showing absent IL-2 mRNA expression during cellular stimulation in the presence of PD098059. Jurkat-tTA cells were preincubated in 20 μM PD098059 (lanes 1–3) or 0.2% DMSO (lanes 4–6) for 30 min. Cells were stimulated for 8 h as described in Fig. 1A. RT-PCR analysis was performed as previously described (18). B, PD098059 fails to inhibit CD28RE/AP-1 reporter activity. Cells were transfected with the Luc vector, rested, and stimulated with anti-CD3 plus anti-CD28 or P+I in the presence or the absence of the drug for 6 h. C, PD098059 interferes in the activation of the distal NF-AT/AP-1 element. Cells were transfected with 10 μg of 4xNFAT/AP-1 Luc vector (pΔODLO), rested, and stimulated in the absence or the presence of 20 μM PD098059 for 6 h.
- FIGURE 4.
CD28RE/AP-1 Luc reporter assay showing that expression of DA-MEKK1 leads to stronger stimulation than DA-MEK1. A, Western blot showing levels of DA-MEKK1 and DA-MEK1 expression. Cells were transfected with 20 μg of DA-MEK1 or 5 μg of DA-MEKK1 (supplemented with 15 μg of pCDNA1.1). DA-MEK1 was detected using the hemagglutinin tag, while DA-MEKK1 was visualized with an anti-MEKK Ab. B, Luciferase assay comparing the effects of DA-MEK1 and DA-MEKK1 on activation of CD28RE/AP-1. Cells were transfected with 20 μg of pCDNA1.1, 20 μg of DA-MEK1, or 5 μg of DA-MEKK1 (with 15 μg of pCDNA1.1) plus 10 μg of 4xCD28RE/AP-1 and 6 μg of pCMV-βGal. Cells were rested for 24 h and stimulated with anti-CD3 plus anti-CD28 or P+I for 6 h.
- FIGURE 5.
IκB degradation and phosphorylation are regulated by CD28, which also induces IKKβ activation. A, Western blot showing IκBα and IκBβ degradation in response to CD3 plus CD28 coligation. Cells were stimulated with OKT3 or OKT3 plus 9.3 mAb for the indicated time periods. Cell lysates were analyzed by Western blotting using anti-IκBα or anti-IκBβ serum as previously described (34). B, Western blot showing the expression of transfected IKKα and IKKβ. Cells were transfected with 20 μg of tagged IKKα or 20 μg of tagged IKKβ, then rested for 24 h. Cell lysates were precipitated with anti-FLAG (M2) Ab, and immunoprecipitates were subjected to immunoblotting. C, Immune complex kinase assay showing activation of IKKβ during CD28 costimulation. Cells were transfected as described in B. After 24 h, cells were stimulated with the indicated stimuli for 10 min. Cell lysates were immunoprecipitated with 1 μg of M2, and these precipitates were incubated together with GST-IκBα1–100 and [32P]ATP. The increase in IKKβ activity by CD28 costimulation was fourfold. D, DN-IKKs decrease CD28RE/AP-1 Luc activity. Cells were transfected with kinase-inactive IKK constructs or an empty vector together with 10 μg of Luc plus 6 μg of β-Gal vector. Luciferase assays were performed after cellular stimulation with the indicated stimuli for 8 h. E, Western blot showing equivalent levels of DN-IKKα and DN-IKKβ expression. Anti-FLAG immunoprecipitates, obtained as described in D, were analyzed by anti-FLAG immunoblotting.
- FIGURE 6.
While wild-type NIK (wt-NIK) increases CD28RE/AP-1 Luc activity, DN-NIK does not interfere in the activation of this response element. A, The wt-NIK enhances CD28RE/AP-1 Luc activity. Cells were transfected with 20 μg of pCDNA1.1 or 20 μg of wt-NIK together with 10 μg of the Luc vector and 6 μg of pCMV-βGal. After resting for 24 h, cells were stimulated for 8 h. B, Western blot showing DN-NIK and wt-NIK expression. Cells were transfected with 20 μg of DN-NIK or 20 μg of wt-NIK and then rested for 24 h. Cell lysates were analyzed with a 1/1000 dilution of M2 Ab. C, DN-NIK does not interfere in activation of the CD28RE/AP-1 Luc reporter. Cells were transfected with 20 μg of pCDNA1.1 or 20 μg of DN-NIK together with 10 μg of the Luc vector and 6 μg of pCMV-βGal. After resting for 24 h, cells were stimulated as indicated for 8 h.
- FIGURE 7.
DA-MEKK1 preferentially induces IKKβ kinase activity, while kinase-inactive IKKα and IKKβ constructs inhibit CD28RE/AP-1 Luc activation. A, In vitro kinase assay looking at the activation of IKKs by DA-MEKK1. Jurkat-tTA cells were transfected with either wt-IKKα or wt-IKKβ together with an empty vector or pCMV-DA-MEKK1. After resting for 24 h, cells were lysed, and in vitro kinase assays were performed as described in Fig. 5C. The bottom panel is an anti-FLAG immunoblot to demonstrate that equivalent amounts of IKKα or IKKβ protein were expressed. B, DA-MEKK1-induced CD28RE/AP-1 Luc activity is negatively regulated by DN-IKKα. Cells were transfected with 20 μg of pTPH(DA-MEKK1), 10 μg of the Luc vector, 6 μg of pCMV-βGal, and either 20 μg of pCDNA1.1 or 20 μg of DN-IKKα. Transfected cell populations were divided into aliquots receiving 0.1 μg/ml tetracycline (gene off) or no tetracycline (gene on) for 24 h. Cells were left unstimulated or were stimulated with OKT3 plus 9.3 mAb for an additional 6 h. C, DA-MEKK1-induced CD28RE/AP-1 Luc activity is negatively regulated by DN-IKKβ. The assay was performed as described in B, except for the use of a DN-IKKβ in place of a DN-IKKα expression vector.
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