. 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, the cell cycle, and changes in glucose metabolism (1). The mechanisms by which O-GlcNAc act are complex, and changes in O-GlcNAc levels have been shown to alter the behavior of specific proteins by modulating the following: 1) the half-life and proteolytic processing of proteins (2-7); 2) subcellular localization (8 -14); 3) protein-protein interactions (6, 15, 16); 4) DNA binding (17); and 5) enzyme activity or regulation (18 -20). One mechanism by which O-GlcNAc may mediate these events is by altering protein phosphorylation. Notably, phosphorylation and O-GlcNAc are reciprocal on some well studied proteins, which include the C-terminal domain of the large subunit of RNA polymerase (21, 22), the c-myc protooncogene (23-25), SV40 large T-antigen (26), estrogen receptor- (7), and endothelial nitric-oxide synthase (18). These observations suggest that O-GlcNAc and phosphorylation may modulate each other (27-29).Increasing extracellular glucose concentrations affects the functioning of key cellular proteins in an O-GlcNAc-dependent manner, including endothelial nitric-oxide synthase (18), mSin3a (30), the transcription factors, YY1 (31), Sp1 (5, 32-34), CREB (35), and the 26 S proteosomal complex (36, 37). UDPGlcNAc:polypeptide O--N-acetylglucosaminyltransferase (OGT; EC 2.4.1.94), the enzyme that adds O-GlcNAc, is responsive across the physiological range of UDP-GlcNAc. Moreover, the substrate specificity of OGT changes at different UDPGlcNAc concentrations (38). Both in vitro and in vivo data support a model where increased UDP-GlcNAc levels, due to hyperglycemia, result in increased O-GlcNAc levels, leading to insulin resistance, a hallmark of type II diabetes (1, 39). These data and others have led researchers to propose that O-GlcNAc is a nutritional sensor (1, 39 -41).In response to multiple forms of stress, cells rapidly increase glucose uptake. The ability of cells to transport glucose has been linked to the capacity of cells to respond and survive deleterious cellular conditions (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56). In many studies, blocking both glycolysis (48,51,57) and the hexosamine biosynthetic pathway (58 -61) results in decreased survival of cells. In some instances, alternative energy sources have been provided suggesting that depletion of ATP levels does NOT explain the decrease in survival (48,51,57). Several insulin-resistant models, including the long lived Caenorhabditis elegans Daf-2 knockout, have an increased stress tolerance to a variety of agents (62)(63)(64). Based upon these data, and recent observations suggesting that heat shock protein (HSP) 70 may act as an O-GlcNAc lectin (65), we investigated the possible link between stress tolerance and O-GlcNAc. We demonstrate that in response to all forms of cellular stress tested, multiple cell lines
Like phosphorylation, the addition of O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways, but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division.
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...
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