Abstract 1019: Localization and activity of β-catenin is regulated by O-GlcNAc modification at Serine 23

Abstract INTRODUCTION: β-catenin (β-cat) is a essential regulator of the Wnt signalling pathway that serves as a structural anchor at the adherens junctions linking E-cadherin to the actin cytoskeleton and as a transcriptional co-activator of the LEF/TCF family of transcription factors. Cytoplasmic β-cat levels are stringently regulated by the Wnt pathway through a complex comprising axin, APC, GSK3-β and CK-1, which tags β-cat for degradation by phosphorylation at Serine (S) 33, 37, 45 and Threonine (T) 41 residues. We previously reported that endogenous β-cat is post-translationally modified with O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc inversely regulated β-cat nuclear localization and transcriptional activity (TA). We propose to determine the mechanism(s) by which O-GlcNAc of β-cat regulates its nuclear localization and TA. METHODS & RESULTS: We determined O-GlcNAc sites at the N-Terminal (NT) of β-cat using pEGFP-NT β-cat constructs. pEGFP tagged NT S33Alanine (A), 37A, 45A and T41A mutant constructs were generated using site directed mutagenesis to target the phosphorylation sites of β-cat. Fusion constructs were expressed in prostate cancer (CaP; DU145) cell line and treated with PUGNAc, a drug that increases global levels of O-GlcNAc. β-cat fusion proteins (FP) were immunoprecipitated (IP) with anti-GFP antibody. Wheat Germ Agglutinin (WGA)-HRP was utilized to identify O-GlcNAc by Western Blot analysis. Interestingly, we observed that mutation of the phosphorylations sites did not alter the O-GlcNAc of β-cat. We also generated Alanine mutations of T40, T42, and T40/42 of the NT of β-cat. These represented mutations of sites adjacent T41, a site that was highly predicted to be O-GlcNAc modified. Mutation of these sites did not alter the O-GlcNAc of β-cat. Recently, Plakoglobin, a closely related protein of β-cat, was found to be O-GlcNAcylated at T14, which shares homology to S23 of β-cat. We developed an NT and full length pEGFP tagged S23Glycine mutant (S23G). S23G or wild type (WT) NT and full length FPs were expressed in DU145 cell lines. Transfected cells were treated with PUGNAc and IPed with anti-GFP. WGA-HRP was used to identify levels of O-GlcNAc. NT S23G levels of O-GlcNAc were decreased compared to WT with PUGNAc treatment. Nuclear localization of full length GFP-β-cat (WT and S23G) constructs were visualized using confocal microscopy. We confirmed subcellular localization of β-cat by nuclear/cytosolic fractionation. Both methods indicated that while endogenous and GFP-WT β-cat localized to the plasma membrane upon PUGNAc treatment there was no change in localization of S23G β-cat. Soft Agar Assays confirmed that while WT cells exhibited significant reduction in anchorage independent growth potential upon PUGNAc treatment there was no change in the S23G β-cat. CONCLUSIONS: O-GlcNAc of S23 regulates β-cat nuclear localization and TA. This is a novel regulatory mechanism for β-cat function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1019. doi:10.1158/1538-7445.AM2011-1019

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