O-Glycosylation of B-Catenin Regulates its Nuclear Localization and Transcriptional Activity

Abstract

B-catenin is a transcriptional co-activator in the Wnt signaling pathway. Upon Wnt stimulation, cytosolic B-catenin translocates into the nucleus where it forms complexes with members of the TCF /LEF family of transcription factors to activate gene transcription. Translocation into the nucleus is followed by transcriptional activation of B-catenin's target genes, which are involved in proliferation, angiogenesis and oncogenesis, is a crucial step in the progression of a subset of cancers. The cellular expression of B-catenin is known to be regulated by phosphorylation. However, the mechanisms(s) responsible for B-catenin nuclear entry is not well understood. Recently, B-catenin was reported to be post-translationally modified by O-glycosylation in breast cancer cells. We investigated whether O-glycosylation regulates the signal transduction properties of the protein. Our results indicated that while there are higher levels of total B-catenin in the nucleus of two prostate cancer cell lines (DU-145 and LNCaP) compared to that in a normal prostate epithelial cell line (PNT1A), most of it was in the unglycosylated form. Also, the normal prostate cell line exhibited higher levels of O-glycosylated B-catenin in both the nucleus and cytosol than what was seen in the two prostate cancer cell lines. We carried out further experiments using PUGNAc, a non-cytotoxic reversible inhibitor of O-GlcNAcase, which causes a time dependent increase in cellular levels of O-glycosylated B-catenin. Treatment of prostate cancer cells with PUGNAc caused a decrease in the expression of B-catenin in the nucleus with increasing cellular O-glycosylation of the protein suggesting that O-glycosylation was hindering B-catenin nuclear translocation. Additional studies showed that O-glycosylation of B-catenin decreased that transcriptional activity of a TopFlash reporter plasmid and the protein expression of two B-catenin target genes. Our results suggest that O-glycosylation of B-catenin may represent a novel mechanism important in the regulation of the nuclear localization and transcriptional activity of B-catenin.