O-GLYCOSYLATION OF AN SP1-DERIVED PEPTIDE BLACKS KNOWN SP1 PROTEIN INTERACTIONS

The O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins i s dynamic and abundant in the nucleus and cytosol. Several transcripti on factors, including Sp1, have been shown to contain this modificatio n; however, the functional role of O-GlcNAc in these proteins has not been determined. In this paper we describe the use of the previously c haracterized glutamine-rich transactivation domain of Sp1 (B-c) as a m odel to investigate the role of O-GlcNAc in Sp1's transcriptionally re levant protein-to-protein interactions with tile TATA-binding-protein- associated factor (TAF110) and holo-Sp1. When the model Sp1 peptide wa s overexpressed in primate cells, this 97-amino-acid domain of Sp1 was found to contain a dominant O-GlcNAc residue at high stoichiometry, w hich allowed the mapping and mutagenesis of this glycosylation site, I n vitro interaction studies between this segment of Sp1 and Drosophila TAF110 or holo-Sp1 indicate that the O-GlcNAc modification functions to inhibit the largely hydrophobic interactions between these proteins . In HeLa cells, the mutation at the mapped glycosylation site was per missive far transcriptional activation. We propose the hypothesis that the removal of O-GlcNAc from an interaction domain cars be a signal f ur protein association, O-GlcNAc may thereby prevent untimely and ecto pic interactions.