Mediator Is a Transducer of Wnt/β-Catenin Signaling

Seokjoong Kim,Thomas G Boyer,Xuan Xu,Andreas Hecht

doi:10.1074/jbc.m602696200

Abstract

Signal transduction within the canonical Wnt/beta-catenin pathway drives development and carcinogenesis through programmed or unprogrammed changes in gene transcription. Although the upstream events linked to signal-induced activation of beta-catenin in the cytoplasm have been deciphered in considerable detail, much less is known regarding the mechanism by which beta-catenin stimulates target gene transcription in the nucleus. Here, we show that beta-catenin physically and functionally targets the MED12 subunit in Mediator to activate transcription. The beta-catenin transactivation domain bound directly to isolated MED12 and intact Mediator both in vitro and in vivo, and Mediator was recruited to Wnt-responsive genes in a beta-catenin-dependent manner. Disruption of the beta-catenin/MED12 interaction through dominant-negative interference- or RNA interference-mediated MED12 suppression inhibited beta-catenin transactivation in response to Wnt signaling. This study thus identifies the MED12 interface within Mediator as a new component and a potential therapeutic target in the Wnt/beta-catenin pathway.

Highlights

Promoters of Wnt target genes that function in developmental regulation, cell proliferation, and cell/cell as well as cell/matrix interactions [11,12,13,14,15,16,17,18]
The ␤-Catenin Transactivation Domain Binds Directly to Mediator— To investigate a possible physical interaction between ␤-catenin and Mediator, we initially examined the abilities of several Glutathione S-Transferase (GST)-␤-catenin derivatives to bind Mediator present in HeLa nuclear extract using a GST pulldown assay and immunoblot analysis using antibodies specific for Mediator subunits MED1, MED6 (ARC/DRIP33), MED12 (TRAP230, ARC/DRIP240), MED16 (TRAP95, DRIP92), MED17 (TRAP80, ARC/DRIP/CRSP77), MED23, and CDK8
To determine whether ␤-catenin binds intact Mediator directly, we examined the ability of GST-␤cat-FL and GST-␤cat-C to bind Mediator purified from a HeLa/S3-derived cell line expressing a hemagglutinin epitope-tagged Mediator MED6 subunit

Summary

Introduction

Promoters of Wnt target genes that function in developmental regulation, cell proliferation, and cell/cell as well as cell/matrix interactions [11,12,13,14,15,16,17,18]. The ␤-Catenin Transactivation Domain Binds Directly to Mediator— To investigate a possible physical interaction between ␤-catenin and Mediator, we initially examined the abilities of several GST-␤-catenin derivatives to bind Mediator present in HeLa nuclear extract using a GST pulldown assay and immunoblot analysis using antibodies specific for Mediator subunits MED1, MED6 (ARC/DRIP33), MED12 (TRAP230, ARC/DRIP240), MED16 (TRAP95, DRIP92), MED17 (TRAP80, ARC/DRIP/CRSP77), MED23, and CDK8 (human SRB10).

Results

Conclusion