Abstract
Hyperactivated beta-catenin is a commonly found molecular abnormality in colon cancer, and its nuclear accumulation is thought to promote the expression of genes associated with cellular proliferation and transformation. The p300 transcriptional co-activator binds to beta-catenin and facilitates transcription by recruiting chromatin remodeling complexes and general transcriptional apparatus. We have found that beta-TrCp1/Fbw1a, a member of the Skp1/Cullin/Rbx1/F-box E3 ubiquitin ligase complex, binds directly to p300 and co-localizes with it to beta-catenin target gene promoters. Our data show that Fbw1a, which normally targets beta-catenin for degradation, works together with p300 to enhance the transcriptional activity of beta-catenin, whereas other F-box/WD40 proteins do not. Fbw1a also cooperates with p300 to co-activate transcription by SMAD3, another Fbw1a ubiquitylation target, but not p53 or HIF-1alpha, which are substrates for other ubiquitin ligase complexes. These results suggest that, although Fbw1a is part of a negative feedback loop for controlling beta-catenin levels in normal cells, its overexpression and binding to p300 may contribute to hyperactivated beta-catenin transcriptional activity in colon cancer cells.
Highlights
CREB3-binding protein (CBP) and p300 are large multidomain proteins that can bind to and activate over 80 different transcription factors thereby affecting a wide spectrum of cellular growth, development, and differentiation pathways [8, 9]
Several lines of evidence show that ubiquitylation machinery may cooperate with co-activator complexes to enhance transcription, and, in some cases, their recruitment to target gene promoters facilitates transcription factor degradation (18 –20)
We found that p300 and CBP bind to -TrCp1/ Fbw1a, the substrate recognition partner associated with the Skp1/Cullin/Rbx1/F-box (SCF) E3 ubiquitin ligase complex that normally targets -catenin for proteasome-mediated degradation [26]
Summary
DNA Plasmids—The vector, pGEX6P.1 (Amersham Biosciences) was used to express p300 fragments fused to GST. Co-immunoprecipitation and Western blotting verified that the WD40 domain, which contains the K(V/L)WXL motif, confers binding to p300, whereas other fragments of Fbw1a (F-box, mid) did not (Fig. 4B). This result, along with the in vitro interaction studies -catenin immunoprecipitate and further enriched at the axin promoter as compared with negative control regions of DNA (Fig. 7H) These results confirm that both p300 and Fbw1a are co-localized with -catenin transcriptional complexes at the axin target gene promoter
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