Lymphocyte activation induces rapid changes in nuclear and cytoplasmic glycoproteins.

Abstract

A unique form of nucleoplasmic and cytoplasmic protein glycosylation, 0-linked GlcNAc, (O-GlcNAc) is present on proteins ranging from those of yeast to man, including many chromatin proteins, transcription factors, nuclear pore proteins, and certain types of cytoskeletal proteins.In this report we have studied the effects of cellular activation on O-GlcNAc-modified proteins, using T lymphocytes as a model system. Results indicate that the apparent levels of O-GlcNAc on many nuclear proteins increases rapidly after lymphocyte activation, returning to control levels after a few hours. In contrast, the apparent levels of O-GkcNAc on a distinct population of cytosolic proteins decreases rapidly after cellular activation and also returns to control levels after a few hours. These data are consistent with the hypothesis that O-GlcNAc is a regulatory modification and suggest that O-GlcNAc modification may play an important role in the early stages of T-lymphocyte activation.A unique form of nucleoplasmic and cytoplasmic protein glycosylation in which N-acetylglucosamine monosaccharides are O-glycosidically linked to serine or threonine residues (O-GlcNAc) has been described in organisms including viruses, yeast, protozoa, insects, frogs, rodents, and man but not bacteria (for reviews, see refs. 1 and 2). O-GlcNAcmodified proteins are enriched in nuclei (3) and nuclear pores and are particularly dense in chromatin (4). Subsets of the family of proteins compromising RNA polymerase II transcription factors, but not RNA polymerase I or III transcription factors, appear to be glycosylated by O-GlcNAc (5, 6). One such glycosylated transcription factor, c-fos, is important for the activation of the interleukin 2 gene, which encodes a lymphokine critical to T-cell activation and commitment (7).Aside from the apparent role of O-GlcNAc-bearing nuclear pore glycoproteins in nuclear transport (8-11) and the possible involvement of O-GlcNAc in enhancing the activities of transcription factors (5), virtually nothing is known concerning the functions of these monosaccharide moieties. It has been suggested that O-GlcNAc might be required for the proper assembly of multimeric protein complexes (12) or that it might serve as a nuclear targeting signal (13) analogous to the mannose 6-phosphate-mediated targeting of proteins into lysosomes (14). Based upon the sequences surrounding sites of attachment of O-GlcNAc on three proteins (15), it also has been suggested that O-GlcNAc might reversibly block potential phosphorylation sites on proteins (1, 12). The sites of attachment of O-GlcNAc are similar to the phosphorylation sites of several known serine/threonine kinases (for a review, see ref. 16) and a recently described growth factor-sensitive proline-directed serine/threonine kinase (17, 18). In addition, the sites of attachment of O-GlcNAc are strikingly homologous to previously defined proline-acidic residue-(serine or threonine) regions, which are believed to regulate proteolysis of rapidly turning-over regulatory proteins (19...

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

“…Additionally, we conducted nearly all of our experiments with primary human T cells to complement prior work that relied mostly on immortalized cell lines and, thus, may not constitute an accurate representation of normal cell physiology. We found that the activation of human T cells through the TCR induces a gradual increase in overall O-GlcNAc levels over the course of several hours, similar to previous work characterizing a T cell hybridoma activated with PMA and ionomycin (37). However, in that study, cytosolic O-GlcNAc levels underwent a transient decrease with a corresponding increase in nuclear O-GlcNAc levels.…”

“…Ultimately, these signal-transduction cascades dictate cellular behavior, a prime example of which is the activation of T cells during an adaptive immune response to a pathogen. Compared with other types of posttranslational modifications, the glycosylation of intracellular proteins by O-GlcNAc in T cells remains largely unexplored, despite several studies indicating that it possesses functional significance (23,25,(37)(38)(39).…”

Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance.

“…The products of this pathway are used for protein glycosylation so that alterations in regulated N-or O-linked glycosylation could play a role. In particular, the widespread occurrence of cytosolic O-linked N-acetyl glucosamine on transcription factors, RNA polymerase (32), and nuclear pore proteins (33) and the fact that at least some of the effects of GFA overexpression are transcriptional (34) suggest one potential area for further study.…”

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

“…Like phosphorylation, the levels of O-GlcNAc respond to both intracellular and extracellular stimuli including insulin, nutrient levels, and cellular stress (Whelan et al 2008(Whelan et al , 2010Walgren et al 2003;Kearse and Hart 1991;Zachara et al 2004b;Song et al 2008;Hart et al 2011), with the latter being the main focus of this review. In response to diverse forms of cellular stress, O-GlcNAc levels are elevated on numerous proteins (Zachara et al 2004b).…”