Biomedical subjects

Stephen A Whelan

Publications and source records attributed to Stephen A Whelan.

4 recordsLinked to original sources

PubMed2006

Reciprocal keratin 18 Ser48 O-GlcNAcylation and Ser52 phosphorylation using peptide analysis.

Phosphorylation and O-GlcNAcylation of keratin 18 (K18) are highly dynamic and involve primarily independent K18 populations. We used in vitro phosphorylation and O-GlcNAcylation of wild-type, phospho-Ser52, glyco-Ser48, and Ser-to-Ala mutant 17mer peptides (K18 amino acids 40-56), which include the major K18 glycosylation (Ser48) and phosphorylation (Ser52) sites, to address whether each modification blocks the other. The glyco-K18 peptide blocks Ser52 phosphorylation by protein kinase C, an in vivo K18 kinase, while the phospho-K18 peptide blocks its O-GlcNAcylation. Our findings support the reciprocity of these two post-translational modifications. Therefore, regulation of protein Ser/Thr phosphorylation and glycosylation at proximal sites can be interdependent and provides a potential mechanism of counter regulation.

Acylation↗

PubMed2006

Identification of O-GlcNAc sites on proteins.

O-linked N-acetylglucosamine (O-GlcNAc) is a monosaccharide posttranslational modification that modifies serine/threonine residues of nucleocytoplasmic proteins in metazoans. O-GlcNAc, like phosphorylation, is dynamic and responsive to numerous stimuli in diverse regulatory pathways. O-GlcNAc may also be found adjacent to or at the same sites as phosphorylation, demonstrating the potential for a reciprocal function on some of these proteins. Like most posttranslational modifications, O-GlcNAc is substoichiometric and may be found at multiple sites with other posttranslational modifications present. Additionally, there is no consensus sequence defining the addition of O-GlcNAc to the peptide backbone, further complicating identification and site mapping. This chapter describes several strategies to confirm that proteins are O-GlcNAc modified and provide subsequent determination of O-GlcNAc attachment sites. We have listed the strengths and limitations of each protocol to allow readers to decide which suits their system and availability of resources. These protocols include galactosyltransferase labeling, immunoblotting, using mass spectrometry based on beta-elimination followed by Michael addition with dithiothreitol, and chemoenzymatic labeling, enrichment, and detection.

Acetylglucosamine↗

PubMed2003

Proteomic approaches to analyze the dynamic relationships between nucleocytoplasmic protein glycosylation and phosphorylation.

O-linked beta-N-acetylglucosamine (O-GlcNAc) is both an abundant and dynamic posttranslational modification similar to phosphorylation that occurs on serine and threonine residues of cytosolic and nuclear proteins in all metazoans and cell types examined, including cardiovascular tissue. Since the discovery of O-GlcNAc more than 20 years ago, the elucidation of O-GlcNAc as a posttranslational modification has been slow, albeit similar to the rate of acceptance of phosphorylation, because of the lack of tools available for its study. Identifying O-GlcNAc posttranslational modifications on proteins is a major challenge to proteomics. The recent development of mild beta-elimination followed by Michael addition with dithiothreitol has significantly improved the site mapping of both O-GlcNAc and O-phosphate in functional proteomics. beta-Elimination followed by Michael addition with dithiothreitol facilitates the study of the labile O-GlcNAc modification in the etiology of disease states. We discuss how recent technological innovations will expand our present understanding of O-GlcNAc and what the implications are for diabetes and cardiovascular complications.

Acetylglucosamine↗

PubMed2003

O-GlcNAc: a regulatory post-translational modification.

Beta-N-acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification of nuclear and cytosolic proteins. The enzymes for its addition and removal have recently been cloned and partially characterized. While only about 80 mammalian proteins have been identified to date that carry this modification, it is clear that this represents just a small percentage of the modified proteins. O-GlcNAc has all the properties of a regulatory modification including being dynamic and inducible. The modification appears to modulate transcriptional and signal transduction events. There are also accruing data that O-GlcNAc plays a role in apoptosis and neurodegeneration. A working model is emerging that O-GlcNAc serves as a metabolic sensor that attenuates a cell's response to extracellular stimuli based on the energy state of the cell. In this review, we will focus on the enzymes that add/remove O-GlcNAc, the functional impact of O-GlcNAc modification, and the current working model for O-GlcNAc as a nutrient sensor.

Acetylglucosamine↗