Determination of O-glycosylation sites of β-Cantenin

Abstract

Cells respond to their environment through dynamic posttranslational modification of their existing proteins. There are more than 20 posttranslational modifications that occur on eukaryotic proteins. Several of these modifications, with phosphorylation being the hallmark, participate in signal transduction. Generally, glycosylation is not thought to participate directly in signaling. Complex N-and 0-linked glycosylation occurs on membrane-bound or secreted proteins that are synthesized in the endoplasmic reticulum and Golgi apparatus. The lumenal or extracellular localization ofthese glycans restricts their potential for dynamic responsiveness to signals. In contrast, 0-GlcNAc is a simple monosaccharide modification that is abundant on serine or threonine residues ofnucleocytoplasmic proteins. An 0-GlcNAc site consensus motif has not yet been identified. However, many attachment sites are identical to those used by serine/threonine) kinases, and a neural network program has been developed to predict 0GlcNAc sites. The dynamic glycosylation of serine or threonine residues on nuclear and cytosolic proteins by 0-linked beta-N-acetylglucosamine (0-GlcNAc) is abundant in all multicellular eukaryotes. On several proteins, 0-GlcNAc and 0-phosphate alternatively occupy the same or adjacent sites, leading to the hypothesis that one function of this saccharide is to transiently block phosphorylation. Many proteins have been identified that carry this modification, including transcription factors, cytoskeletal proteins, nuclear pore proteins, oncogene products, and tumor suppressors. 0-GlcNAc appears to modify a large number of nucleocytoplasmic proteins· One of important regulatory proteins on which this project concentrates is β-catenin. Here, we examined where does this type ofposttranslational modification takes place on the protein. Our results indicated that P-catenin is 0-glycosylated on both the N-terminus and Cterminus, but not at the ARMADILLO segment. Further, we show that the known phosphorylation sites located at theN-terminal "destruction box" of this protein are not involved in 0-glycosylation. Furthermore, we demonstrated that the threonines adjacent to phosphorylation-site Threonin41 are not essential in 0-glycosylation process. In addition, treatment ofprostate cancer lines with PUGNAc, a non-cytotoxic reversible inhibitor ofOGlcNAcase, caused a decrease in the expression oftransfected P-catenin in the nucleus with increasing cellular 0-glycosylation ofthe protein suggesting that 0-glycosylation was hindering P-catenin's nuclear translocation. Additional studies showed that 0-glycosylation of P-catenin decreased transcriptional activity of a TopFlash reporter plasmid. In summary, our results show that P-catenin is 0-glycosylated on theN-and C-terminus, but not on ARMADILLO segment, and that phosphorylation sites are not the critical for 0-glycosylation. Furthermore, our data show that 0-glycosylation of P-catenin may represent a novel mechanism important in the regulation of the nuclear localization and transcriptional activity of P-catenin.