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Beta-catenin plays a pivotal role in the transcriptional activation of Wnt-responsive genes by binding to TCF/LEF transcription factors. Although it has been suggested that the COOH-terminal region of beta-catenin functions as an activation domain, the mechanisms of activation remain unclear. To screen for potential transcriptional coactivators that bind to the COOH-terminal region of beta-catenin, we used a novel yeast two-hybrid system, the Ras recruitment system (RRS) that detects protein-protein interactions at the inner surface of the plasma membrane. Using this system, we isolated the CREB-binding protein (CBP). Armadillo (Arm) repeat 10 to the COOH terminus of beta-catenin is involved in binding to CBP, whereas beta-catenin interacts directly with the CREB-binding domain of CBP. Beta-catenin synergizes with CBP to stimulate the activity of a synthetic reporter in vivo. Conversely, beta-catenin-dependent transcriptional activation is repressed by E1A, an antagonist of CBP function, but not by an E1A mutant that does not bind to CBP. The activation of Wnt target genes such as siamois and Xnr3 in Xenopus embryos is also sensitive to E1A. These findings suggest that CBP provides a link between beta-catenin and the transcriptional machinery, and possibly mediates the oncogenic function of beta-catenin.
Figure 1. Interaction between β-catenin and CBP. A, Complementation of the cdc25-2 mutation through the interaction of β-catenin COOH-terminal region with CBP. The temperature-sensitive cdc25-2 yeast cells were cotransformed with the indicated plasmids and the CBP expression plasmid isolated in the screening, and incubated on galactose containing plates either at 25 (left) or 37°C (right). B, Fine mapping of the β-catenin domain that binds to CBP in yeast. Cells were cotransformed with the indicated plasmids together with the CBP expression plasmid, and tested for the growth at 37°C as described for A. The numbers indicate the locations in the amino acid sequence of β-catenin. ++, Efficient growth; +, poor growth; −, no growth. C, β-Catenin directly binds to CREB-binding domain of CBP. Bacterially expressed and purified His-tagged βcatR10-C or myc-tagged β-catenin was incubated with buffer (lane 5), GST (lanes 2 and 6), or GST-CBP 451–682 (lanes 3 and 7). After extensive washing, bound proteins were eluted and resolved by SDS-PAGE. βCatR10-C and β-catenin were detected by an anti-His antibody and an anti-myc antibody, respectively. Approximately 10% of the input is shown (lanes 1 and 4). D, Coimmunoprecipitation of CBP and β-catenin. COS7 cells were transfected with expression plasmids for CBP and/or myc-tagged ptβ-catenin as indicated. Cell lysates were immunoprecipitated with anti-CBP polyclonal antibodies 2 d after transfection. The immunoprecipitates were separated by SDS-PAGE and subjected to Western blotting with anti-myc antibodies.
Figure 2. CBP synergizes with β-catenin to activate transcription. HeLa cells were transiently transfected with 1 μg of pTOPFLASH or pFOPFLASH reporter, indicated amounts of CBP expression plasmid, either with or without 1 μg of pt β-catenin–encoding plasmid. Transfections were carried out in triplicate and the means ± SD are shown. Where they are not evident, this is due to minimal variability between transfections.
Figure 3. E1A represses β-cateninâmediated transcriptional activation. A, The reporter plasmid pTOPFLASH (1 μg) was transfected into HeLa cells with or without 1 μg of pt β-cateninâencoding plasmid. The expression vector encoding E1A or E1A mutCBP (100 or 500 ng) were cotransfected as indicated. B, SW480 cells were cotransfected with 100 ng of the pTOPFLASH reporter, and increasing amounts (100 or 400 ng) of CBP expression plasmid, 5 ng each of the plasmids encoding E1A WT, E1A mutRB, or E1A mutCBP, or 50 ng of dnLEF-1 expression plasmid as indicated. The activity of the pTOPFLASH reporter in the absence of exogenous CBP, E1A, or dnLEF-1 was set at 100%.
Figure 4. Endogenous CBP is required for transactivation by GAL4-βcatR10-C fusion protein. HeLa cells were transfected with 1 μg of GAL4 reporter construct, pFR-Luc, 50 ng of GAL4 1â147 or GAL4-βcatR10-C, and increasing amounts of E1A or E1A mutCBP expression plasmid (25 or 500 ng) in combinations as indicated.
Figure 5. The activation of endogenous Wnt-responsive genes is sensitive to E1A. Both blastomeres of two-cell stage Xenopus embryos were injected with 20 pg of Xwnt-8, 0.5 ng of E1A mutCBP, or E1A, and 3.2 ng of CBP RNAs as indicated. Animal cap explants were dissected at stage 8 to 9 and analyzed for gene expression by RT-PCR.
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