Identification of IFITM3 and MGAT1 as novel interaction partners of BRI3 by yeast two-hybrid screening

Abstract

BRI3 (brain protein I3) is one of the Wnt/β-catenin pathway target genes as indicated by the results of serial analysis of geneexpression (SAGE) and microarray analyses performed in our laboratory. The Wnt/β-catenin signaling pathway is an evolutionarilyconserved pathway, which has important functions in early vertebrate development, axis formation, cellular proliferation, andmorphogenesis. Previous studies showed that BRI3 expression is upregulated at both mRNA and protein levels upon β-cateninactivation by various approaches, such as lithium treatment and overexpression of Wnt ligands in Huh7 (hepatocellular carcinoma) celllines. Moreover, with regard to the previous literature, BRI3 was found to have a very important role in the TNFα-mediated cell deathpathway. In this study, we screened a human liver cDNA library by yeast two-hybrid assay using BRI3 protein as bait, with the aim offinding novel interaction partners of BRI3. Library screening by yeast mating resulted in the identification of three candidate positiveclones. Among these, IFITM3 and MGAT1 proteins were confirmed as interaction partners by using cotransformation in yeast cells andcoimmunoprecipitation from mammalian cell lines. Considering the poor functional characterization of BRI3 to date, identification ofnovel BRI3-interacting proteins is an essential first step in determining the action mechanism of BRI3 with respect to the Wnt/β-cateninpathway.