Perturbations in O-linked β-N-Acetylglucosamine Protein Modification Cause Severe Defects in Mitotic Progression and Cytokinesis

Abstract

The dynamic modification of nuclear and cytoplasmic proteins with O-linked ␤-N-acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification that is rapidly responsive to morphogens, hormones, nutrients, and cellular stress. Here we show that O-GlcNAc is an important regulator of the cell cycle. Increased O-GlcNAc (pharmacologically or genetically) results in growth defects linked to delays in G 2 /M progression, altered mitotic phosphorylation, and cyclin expression. Overexpression of O-GlcNAcase, the enzyme that removes O-GlcNAc, induces a mitotic exit phenotype accompanied by a delay in mitotic phosphorylation, altered cyclin expression, and pronounced disruption in nuclear organization. Overexpression of the O-GlcNAc transferase, the enzyme that adds O-GlcNAc, results in a polyploid phenotype with faulty cytokinesis. Notably, O-GlcNAc transferase is concentrated at the mitotic spindle and midbody at M phase. These data suggest that dynamic O-GlcNAc processing is a pivotal regulatory component of the cell cycle, controlling cell cycle progression by regulating mitotic phosphorylation, cyclin expression, and cell division.Because of the discovery of cyclins 22 years ago (1), a working model of the cell cycle has slowly been constructed. The cell cycle oscillator is composed of protein phosphorylation, timed expression of cyclins, and well orchestrated cell division (2). Nevertheless, a detailed mechanism of the cell cycle is still incomplete. Knockouts of proteins thought critical for proper cell cycle function such as cyclin D only partially disrupt the cell cycle, and function can be completely restored by cyclin E knock-in (3). However, cyclin E knockouts are viable (4), and ablation of cyclin-dependent kinases (CDK2 and CDK4) do not cause cell death or cell cycle defects (5, 6). Recently, RNA-mediated interference was used to knockdown the Drosophila kinome (7). One-third of all kinases knocked down in this study caused cell cycle defects. Clearly, many pathways in the cell cycle have redundant features, and other mechanisms of growth control exist. Despite the publication of more than 7000 papers within the last 5 years on the roles of phosphorylation in the cell cycle, the mechanisms regulating cell cycle progression are still not well understood. Although phosphorylation is the molecular mechanism generally associated with the regulation of cell cycle proteins, another potential regulator that has not been studied in this context is the abundant post-translational modification O-GlcNAc 2 (8).O-GlcNAc is a ubiquitous post-translational modification in which a single ␤-N-acetylglucosamine molecule is O-linked to serine or threonine residues on cytoplasmic and nuclear proteins (9). O-GlcNAc is thought to act as a modulator of protein function in a manner analogous to protein phosphorylation; the addition of O-GlcNAc to the protein backbone is dynamic and responds to morphogens (10), cellular stress (11), and changes in glucose metabolism (12, 13). O-GlcNAc transferase (OGT) (14 -16) adds and...

Drosophila O -GlcNAc transferase (OGT) is encoded by the Polycomb group (PcG) gene, super sex combs ( sxc )

“…Six different sxc alleles were used in the current study. The origins of the EMS-induced alleles, sxc 1 , sxc 3 , sxc 4 , and sxc 5 , are described in Ingham (8); sxc 1 was induced on a second chromosome bearing NC130 , an EMS-induced allele obtained from the Bloomington Stock Center, was generated more recently using a second chromosome bearing the markers cn and bw (32); its status as an sxc allele was confirmed by complementation (D. Sinclair, unpublished results). sxc 2637 , also obtained from the Bloomington Stock Center, was generated in an extensive P-element-based gene disruption study (33) and was originally designated l (2)02637.…”

“…S2 and S3). The missense allele sxc 4 does show an immunoreactive protein of the expected size for OGT, yet enzyme activity is nearly completely absent (Figs. S2 and S3).…”

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

“…PUGNAc is suitable for use in cells (10-100 μM, 4-18 h) and in animals (50 mg/kg, 4-12 h) ; however, prolonged use (>36 h) can lead to cell cycle defects (Slawson et al 2005). While widely used, recent evidence demonstrates that PUGNAc can also inhibit other lysosomal glycosidases including HexA and HexB (Macauley et al 2005;Ficko-Blean et al 2008), suggesting that PUGNAc may have effects on other cellular pathways aside from O-GlcNAcylation.…”

“…Techniques such as over expression of OGT (Zachara et al 2004b), OGlcNAcase (Slawson et al 2005), and GFAT (Chen et al 1997;Marshall et al 2004;James et al 2000) by both transient transfection and viral transduction have been successful, as has reducing the expression of these enzymes using RNA interference (Zachara et al 2004b;Ngoh et al 2009a;Hsieh et al 2012). OGT has also been overexpressed using a tetracycline-inducible OGT stably expressed in HeLa cells, although for effective overexpression a histone deacetylase inhibitor was included with the tetracycline for appropriate regulation of OGT (Marshall et al 2003).…”

Caenorhabditis elegans ortholog of a diabetes susceptibility locus: oga-1 ( O -GlcNAcase) knockout impacts O -GlcNAc cycling, metabolism, and dauer

“…Knockouts of OGT are embryonic and stem cell lethal (44), and conditional knockouts of OGT in various tissues have not been fully completed (45). Overexpression of OGA induces a mitotic exit phenotype (46), suggesting that it too may perform essential functions. We originally identified C. elegans OGT (25) and have shown that knockouts of ogt-1 in the nematode, although viable and fertile, have effects on insulin-like signaling (25,26).…”