Identification of a Novel Role of ZMIZ2 Protein in Regulating the Activity of the Wnt/β-Catenin Signaling Pathway

Suk Hyung Lee,Chunfang Zhu,Yue Peng,Daniel T Johnson,Lynn Lehmann,Zijie Sun

doi:10.1074/jbc.m113.529727

Abstract

ZMIZ2, also named ZIMP7, is a protein inhibitor of activated STAT (PIAS)-like protein and a transcriptional coactivator. In this study, we investigated the interaction between ZMIZ2 and β-catenin, a key regulator of the Wnt signaling pathway. We demonstrated that the expression of exogenous ZMIZ2 augments TCF (T cell factor) and β-catenin-mediated transcription. In contrast, shRNA knockdown of ZMIZ2 expression specifically represses the enhancement of TCF/β-catenin-mediated transcription by ZMIZ2. Using Wnt3a-conditioned medium, we demonstrated that ZMIZ2 can enhance Wnt ligand-induced TCF/β-catenin-mediated transcription. We also showed a promotional role of ZMIZ2 in enhancing β-catenin downstream target gene expression in human cells and in Zmiz2 null (Zmiz2(-/-)) mouse embryonic fibroblasts (MEFs). The regulatory role of Zmiz2 in Wnt-induced TCF/β-catenin-mediated transcription can be restored in Zmiz2(-/-) MEFs that were infected with adenoviral expression vectors for Zmiz2. Moreover, enhancement of Zmiz2 on TCF/β-catenin-mediated transcription was further demonstrated in Zmiz2 knockout and Axin2 reporter compound mice. Furthermore, the protein-protein interaction between ZMIZ2 and β-catenin was identified by co-immunoprecipitation and in vitro protein pulldown assays. We also observed recruitment of endogenous ZMIZ2 onto the promoter region of the Axin 2 gene, a β-catenin downstream target promoter, in a Wnt ligand-inducible manner. Finally, a promotional role of ZMIZ2 on cell growth was demonstrated in human cell lines and Zmiz2 knockout MEFs. Our findings demonstrate a novel interaction between ZMIZ2 and β-catenin and elucidate a novel mechanism for PIAS-like proteins in regulating Wnt signaling pathways.

Highlights

ZMIZ2 is a transcriptional coactivator, but its biological role has not been fully investigated
Plasmids expressing T cell factor 1 (TCF1), ZMIZ1, ZMIZ2, and either the Topflash reporter containing a multiple ␤-catenin/TCF binding motif or Fopflash containing a multiple-mutated motif were transfected into HEK293 cells
The greatest induction of the pGL3-OT reporter was observed in the samples cotransfected with TCF1, ␤-catenin, and ZMIZ2 proteins, which is significantly different from the samples transfected with TCF1 and ␤-catenin or ZMIZ2 proteins only (p Ͻ 0.05)

Summary

Background

ZMIZ2 is a transcriptional coactivator, but its biological role has not been fully investigated. A promotional role of ZMIZ2 on cell growth was demonstrated in human cell lines and Zmiz knockout MEFs. Our findings demonstrate a novel interaction between ZMIZ2 and ␤-catenin and elucidate a novel mechanism for PIAS-like proteins in regulating Wnt signaling pathways. These proteins promote the phosphorylation of serine and threonine residues in the N-terminal region of ␤-catenin and, thereby, target it for degradation by the ubiquitin proteasome pathway [27] Wnt signaling inhibits this process and leads to the accumulation of ␤-catenin in the nucleus, in which ␤-catenin forms transcriptionally active complexes with members of the Lef/Tcf family of transcription factors [28]. Down-regulation of other ␤-catenin downstream targets, such as CD44 and c-Jun, were observed in Zmiz null mice These data provide the first line of evidence demonstrating an interaction between the Wnt/␤catenin and ZMIZ signaling pathways

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION