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. 2005 Feb 14;144(7):982–993. doi: 10.1038/sj.bjp.0706127

Figure 2.

Figure 2

Effect of deletions or a mutation within the CREB transactivation domain on the inhibition by cyclosporin A of membrane depolarization-induced CREB transcriptional activity. (a) C-terminal deletion analysis. The expression vectors for the indicated GAL4-CREB fusion proteins were transfected into HIT cells together with the luciferase reporter gene controlled by five copies of the GAL4-binding site (5xGal4E1BLuc). The white box represents the kinase-inducible domain of CREB (KID), the numbers indicate the aa present (referring to CREB-327). Cells were treated as indicated with KCl (45 mM) and cyclosporin A 5 μM (CsA). Luciferase activity is expressed relative to the mean value in each experiment of the activity measured in the respective control. Values are means±s.e.m. (n=6). (b) N-terminal deletion analysis. The expression vectors for the indicated GAL4-CREB fusion proteins were transfected together with the luciferase reporter gene controlled by five copies of the GAL4-binding site (5xGal4E1BLuc). The white box represents the KID, the numbers indicate the aa present (referring to CREB-327). Cells were treated as indicated with KCl (45 mM) and cyclosporin A 5 μM (CsA). Luciferase activity is expressed relative to the mean value in each experiment of the activity measured in the respective control. Values are means±s.e.m. (n=6). (c) Analysis of internal deletions within the KID. The expression vectors for the indicated GAL4-CREB fusion proteins were transfected together with the luciferase reporter gene controlled by five copies of the GAL4-binding site (5xGal4E1BLuc). The white box represents the KID, the numbers indicate the aa present (referring to CREB-327). Δ indicates the amino acids internally deleted. Cells were treated as indicated with KCl (45 mM) and cyclosporin A 5 μM (CsA). Luciferase activity is expressed relative to the mean value in each experiment of the activity measured in the respective control. Values are means±s.e.m. (n=6). (d) Ser142 of CREB-341 is not required for cyclosporin A sensitivity of the CREB transactivation domain. Expression vectors for the GAL4 DNA-binding domain fused to the transactivation domain of CREB-341 either wild type (CREB wild-type) or bearing a Ser133Ala mutation (CREB S133A) or a Ser142Ala mutation (CREB S142A) were transfected together with the luciferase reporter gene under control of five copies of the GAL4-binding site (5xGal4E1BLuc). Ser133 of CREB-341 corresponds to Ser119 of CREB-327. Cells were treated as indicated with KCl (45 mM), forskolin 10 μM (cAMP), and cyclosporin A 5 μM (CsA). Luciferase activity is expressed relative to the mean value in each experiment of the activity measured in the respective control. Values are means±s.e.m. (n=6).