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O glycosylation of an Sp1-derived peptide blocks known Sp1 protein interactions
1Department of Cell Biology, University of Alabama at Birmingham, 35294, USA.
Abstract:
The O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins is dynamic and abundant in the nucleus and cytosol. Several transcription factors, including Sp1, have been shown to contain this modification; however, the functional role of O-GlcNAc in these proteins has not been determined. In this paper we describe the use of the previously characterized glutamine-rich transactivation domain of Sp1 (B-c) as a model to investigate the role of O-GlcNAc in Sp1's transcriptionally relevant protein-to-protein interactions with the TATA-binding-protein-associated factor (TAF110) and holo-Sp1. When the model Sp1 peptide was overexpressed in primate cells, this 97-amino-acid domain of Sp1 was found to contain a dominant O-GlcNAc residue at high stoichiometry, which allowed the mapping and mutagenesis of this glycosylation site. In 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 permissive for transcriptional activation. We propose the hypothesis that the removal of O-GlcNAc from an interaction domain can be a signal for protein association. O-GlcNAc may thereby prevent untimely and ectopic interactions.
Insights
O-linked N-acetylglucosamine (O-GlcNAc) protein modification inhibits Sp1 interactions. Removing O-GlcNAc from Sp1 domains can signal protein association, preventing unwanted interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Post-translational Modifications
Background:
- O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic protein modification found in the nucleus and cytosol.
- The functional role of O-GlcNAc in transcription factors like Sp1 remains largely undetermined.
- Sp1's glutamine-rich transactivation domain (B-c) is a model for studying O-GlcNAc's role in protein interactions.
Purpose of the Study:
- To investigate the functional role of O-GlcNAc in Sp1's protein-to-protein interactions.
- To determine how O-GlcNAc modification affects Sp1 interactions with TAF110 and holo-Sp1.
- To explore the hypothesis that O-GlcNAc removal signals protein association.
Main Methods:
- Overexpression of a model Sp1 peptide (B-c domain) in primate cells.
- Mapping and mutagenesis of the dominant O-GlcNAc glycosylation site.
- In vitro interaction studies using Sp1 peptide segments and Drosophila TAF110 or holo-Sp1.
- Transcriptional activation assays in HeLa cells.
Main Results:
- A dominant O-GlcNAc residue was identified at high stoichiometry in the model Sp1 peptide.
- O-GlcNAc modification was found to inhibit hydrophobic interactions between Sp1 and TAF110/holo-Sp1.
- Mutation of the glycosylation site in HeLa cells permitted transcriptional activation.
Conclusions:
- O-GlcNAc modification of Sp1 inhibits its protein-to-protein interactions.
- Removal of O-GlcNAc from interaction domains may act as a signal for protein association.
- O-GlcNAc may prevent untimely and ectopic protein interactions, regulating transcription.