Discovered in 1984, intracellular O-linked β-N-acetylglucosamine (O-GlcNAc) has been shown to occur on thousands of proteins within the nucleus, cytoplasm and mitochondria. O-GlcNAc modifications can have significant impact on various cellular processes controlled by the cycling addition and hydrolysis of GlcNAc on key proteins catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The biosynthesis of UDP-GlcNAc, the substrate of OGT, involves the metabolism of several biomolecules, making the amount of O-GlcNAc sensitive to the nutrient levels present in the cell. Discussed herein is the biosynthesis of UDP-GlcNAc, structural and mechanistic properties of the cycling enzymes, the various biological functions of O-GlcNAc in both healthy and pathological systems, and how GlcNAc is being targeted in a therapeutic sense.

Mutations and dysregulation of kinases play causal roles in human disease development, signaling, and metabolism. Understanding the function of kinases is of an outstanding interest for biomarker discovery and also necessary for the development of agonists and antagonists for the use in disease therapy. O‐linked beta‐N‐acetylglucosamine (O‐GlcNAcylation) is a post‐translational modification known to regulate different aspects of a proteins function including localization, activity, and stability. Like phosphorylation, O‐GlcNAcylation, which modifies serine and threonine residues on nuclear and cytoplasmic proteins, is a ubiquitous, reversible process that regulates numerous cellular processes. Recent evidence indicates that site‐specific crosstalk between O‐GlcNAcylation and phosphorylation and the O‐GlcNAcylation of kinases play an important role in regulating cell signaling. Hence, it is very important to investigate the O‐GlcNAcylation of the kinome. Previously, we identified 42 kinases that are substrates for O‐GlcNAcylation using an in vitro OGT assay with [H3] radiolabel on a functional kinase array. However, using [H3] has serve senstitive limiations suggesting a more sensitive approach is needed to probe large libraries of proteins. Herein, using a novel kinase microarray coupled with an in vitro OGT labeling assay with immuno fluorophore detection technique, we report a simple, yet sensitive, strategy by which this method can be used to profile the entire kinome for O‐GlcNAcylated proteins.Grant Funding Source: NIH R01CA42486, R01DK61671; N01‐HV‐00240; P01HL107153, R24DK084949

Like phosphorylation, the addition of O-linked beta-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.

Mitosis must faithfully divide the genome such that each progeny inherits the same genetic material. DNA condensation is crucial in ensuring that chromosomes are correctly attached to the mitotic spindle for segregation, preventing DNA breaks or constrictions from the contractile ring. Histones form an octameric complex of basic proteins important in regulating DNA organization and accessibility. Histone post-translational modifications are altered during mitosis, although the roles of these post-translational modifications remain poorly characterized. Here, we report that N-acetylglucosamine (O-GlcNAc) transferase (OGT), the enzyme catalyzing the addition of O-GlcNAc moieties to nuclear and cytoplasmic proteins at serine and threonine residues, regulates some aspects of mitotic chromatin dynamics. OGT protein amounts decrease during M phase. Modest overexpression of OGT alters mitotic histone post-translational modifications at Lys-9, Ser-10, Arg-17, and Lys-27 of histone H3. Overexpression of OGT also prevents mitotic phosphorylation of coactivator-associated arginine methyltransferase 1 (CARM1) and prevents its correct cellular localization during mitosis. Moreover, OGT overexpression results in an increase in abnormal chromosomal bridge formation. Together, these results show that regulating the amount of OGT during mitosis is important in ensuring correct chromosomal segregation during mitosis.

Dynamic posttranslational modification of serine and threonine residues of nucleocytoplasmic proteins by β-N-acetylglucosamine (O-GlcNAc) is a regulator of cellular processes such as transcription, signaling, and protein–protein interactions. Like phosphorylation, O-GlcNAc cycles in response to a wide variety of stimuli. Although cycling of O-GlcNAc is catalyzed by only two highly conserved enzymes, O-GlcNAc transferase (OGT), which adds the sugar, and β-N-acetylglucosaminidase (O-GlcNAcase), which hydrolyzes it, the targeting of these enzymes is highly specific and is controlled by myriad interacting subunits. Here, we demonstrate by multiple specific immunological and enzymatic approaches that histones, the proteins that package DNA within the nucleus, are O-GlcNAcylated in vivo. Histones also are substrates for OGT in vitro. We identify O-GlcNAc sites on histones H2A, H2B, and H4 using mass spectrometry. Finally, we show that histone O-GlcNAcylation changes during mitosis and with heat shock. Taken together, these data show that O-GlcNAc cycles dynamically on histones and can be considered part of the histone code.

O-GlcNAc-transferase (OGT) substrate specificity is regulated by transiently interacting proteins. To further examine the regulation of OGT, we have identified 27 putative OGT-interacting proteins through a yeast two-hybrid screen. Two of these proteins, Trak1 (OIP106) and O-GlcNAcase, have been shown previously to interact with and regulate OGT. We demonstrate here that MYPT1 and CARM1 also interact with and target OGT. MYPT1 and CARM1 are substrates of OGT in vitro and in vivo. MYPT1 and CARM1 also function to alter OGT substrate specificity in vitro. Furthermore depletion of MYPT1 in Neuro-2a neuroblastoma cells alters GlcNAcylation of several proteins under basal conditions, suggesting that MYPT1 regulates OGT substrate specificity in vivo.

Enzymatic modification of serine and threonine residues on nucleocytoplasmic proteins with single N‐acetylglucosamine moiety through a β‐glycosidic linkage (O‐GlcNAc) is a dynamic post‐translational modification. Much like phosphorylation, protein O‐GlcNAcylation is responsive to cell stimuli and serves to regulate protein function. However unlike phosphorylation, which is mediated by more than 500 kinases, GlcNAcylation is regulated by transient targeting of a single catalytic subunit, O‐GlcNAc Transferase (OGT), to a multitude of substrates by interactor proteins. Recently, through a yeast two‐hybrid approach, we identified Coactivator Associated Arginine Methyltransferase (CARM1) as a putative binding partner with OGT. CARM1 methylates Arg residues of proteins, such as p300 and histone H3. Here, we demonstrate that not only does CARM1 associate with OGT, it also is O‐GlcNAcylated. Additionally, we show that it is reciprocally regulated by phosphorylation and O‐GlcNAcylation. When CARM1 becomes hyper‐phosphorylated during M phase, O‐GlcNAc on CARM1 decreases. This reciprocal relationship regulates substrate specificity, since methylation on Arg 17 of histone H3, a CARM1 specific substrate, increases dramatically during M phase. Overexpression of OGT alters not only the modification status of CARM1, but also the methylation of histone H3 by decreasing mitotic phosphorylation of CARM1 and concomitantly decreasing histone H3 methylation. Aberrant methylation of histone H3 caused by OGT overexpression could explain the gross defects associated with altering O‐GlcNAc levels during cell cycle. Supported by NIH HD13563. Dr. Hart receives royalty received by the university on the sales of the 110.6 antibody. Terms of the arrangement are managed by JHU.

Enzymatic modification of serine and threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine moiety through a ?-glycosidic linkage (O-GlcNAc) is a dynamic post-translational modification. Much like phosphorylation, protein O-GlcNAcylation is responsive to cell stimuli and can serve to regulate protein function. However, while there are a large number of different kinases and phosphatases, only a single O-GlcNAc Transferase (OGT) and O-GlcNAcase have been identified in mammals. Recently, through a yeast two-hybrid approach, we have identified Coactivator Associated Arginine Methyltransferase (CARM1) as a putative binding partner with OGT. CARM1 has been shown by other groups to methylate Arg residues of proteins such as p300 and histone H3. Additionally, CARM1 is required for maximal transcriptional activation of nuclear hormone receptor mediated transcription. Furthermore, CARM1, as well as other components of the transcriptional machinery, associates cyclically with the estrogen responsive pS2 promoter. We have recently shown that not only does CARM1 bind OGT, but also it itself is an O-GlcNAc modified protein. Our data indicate that the O-GlcNAc processing enzymes also cycle on and off the pS2 promoter and are involved in the transcription regulation at the pS2 promoter. Supported by NIH grant CA42486. Dr. Hart receives a share of royalty received by the university on sales of the CTD110.6 antibody. Terms of this arrangement are managed by JHU.

Estrogen receptor β is differentially regulated by alternative O-GlcNAcylation/O-phosphorylation at Ser16. NMR, CD, and molecular dynamics analyses of model peptides [1] show that these alternative modifications induce different peptide conformations, providing a molecular basis for their differential regulation of protein function.