journal homepage: www.elsevier.com/locate/yexcr Available online at www.sciencedirect.com Research Article β-Catenin is O-GlcNAc glycosylated at Serine 23: Implications for β-catenin's subcellular localization and transactivator function Jacqueline R. Ha, Li Hao, Geetha Venkateswaran, Yu Hao Huang, Elizabeth Garcia, Sujata Persad n Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2E1 articleinformation Article Chronology: Received 10 July 2013 Received in revised form 26 November 2013 Accepted 28 November 2013 Available online 14 December 2013 Keywords: β-Catenin O-GlcNAcylation Subcellular localization Transcriptional function abstract Background: We have previously reported that β-catenin is post-translationally modified with a single O-linked attachment of β-N-acetyl-glucosamine (O-GlcNAc). We showed that O-GlcNAc regulated β-catenin's subcellular localization and transcriptional activity. Objective: The objectives of this investigation were to identify the putative O-GlcNAc sites of β-catenin and the relevance of identified sites in the regulation of β-catenin's localization and transcriptional activity. Method: Missense mutations were introduced to potential O-GlcNAc sites of pEGFP-C2-N- Terminal- or pEGFP-C2-Wild Type-β-catenin by site-directed mutagenesis. We determined the levels of O-GlcNAc-β-catenin, subcellular localization, interaction with binding partners and transcriptional activity of the various constructs. Results: Serine 23 of β-catenin was determined as a site for O-GlcNAc modification which regulated its subcellular distribution, its interactions with cellular partners and consequently its transcriptional activity. Significance: O-GlcNAcylation of Serine 23 is a novel regulatory modification for β-catenin's subcellular localization and transcriptional activity. This study is the first report to characterize site specific regulation of β-catenin by the O-GlcNAc modification. & 2013 Elsevier Inc. All rights reserved. Introduction Beta (β)-catenin is a multifunctional protein that serves a struc- tural role at the adherens junctions [1] and a regulatory function as a transcriptional co-activator of the wnt/wingless canonical signal transduction pathway. Activation of this pathway results in stabilization and accumulation of β-catenin in the cytosol, and subsequent localization of the protein into the nucleus. Deregulated Wnt signaling results in increased levels of β- catenin in the nucleus and constitutive transcription of Wnt target genes [2–5], some of which are involved in cell proliferation, invasion and metastasis. Under normal conditions, cytosolic levels of β-catenin are tightly regulated by the ubiquitin-proteasome system, which requires the targeted phosphorylation of highly conserved serine (Ser) and threonine (Thr) residues (Ser 33, Ser 37, Thr 41, and Ser 45) at the 0014-4827/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2013.11.021 n Corresponding author. Fax: þ1 780 492 0723. E-mail addresses: sujata.persad@ualberta.ca, sujata.persad@med.ualberta.ca (S. Persad). EXPERIMENTAL CELL RESEARCH 321 (2014) 153 – 166