Cross-talk between Two Essential Nutrient-sensitive Enzymes: O-GlcNAc TRANSFERASE (OGT) AND AMP-ACTIVATED PROTEIN KINASE (AMPK)*

Nutrient-sensitive pathways regulate both O-GlcNAc transferase (OGT) and AMP-activated protein kinase (AMPK), cooperatively connecting metabolic homeostasis to regulation of numerous intracellular processes essential for life. Similar to phosphorylation, catalyzed by kinases such as AMPK, O-GlcNAcylation is a highly dynamic Ser/Thr-specific post-translational modification of nuclear, cytoplasmic, and mitochondrial proteins catalyzed exclusively by OGT. OGT and AMPK target a multitude of intracellular proteins, with the net effect to protect cells from the damaging effects of metabolic stress. Despite hundreds of studies demonstrating significant overlap in upstream and downstream signaling processes, no study has investigated if OGT and AMPK can directly regulate each other. We show acute activation of AMPK alters the substrate selectivity of OGT in several cell lines and nuclear localization of OGT in C2C12 skeletal muscle myotubes. Nuclear localization of OGT affects O-GlcNAcylation of numerous nuclear proteins and acetylation of Lys-9 on histone 3 in myotubes. AMPK phosphorylates Thr-444 on OGT in vitro; phosphorylation of Thr-444 is tightly associated with AMPK activity and nuclear localization of OGT in myotubes, and phospho-mimetic T444E-OGT exhibits altered substrate selectivity. Conversely, the α- and γ-subunits of AMPK are O-GlcNAcylated, O-GlcNAcylation of the γ1-subunit increases with AMPK activity, and acute inhibition of O-GlcNAc cycling disrupts activation of AMPK. We have demonstrated significant cross-talk between the O-GlcNAc and AMPK systems, suggesting OGT and AMPK may cooperatively regulate nutrient-sensitive intracellular processes that mediate cellular metabolism, growth, proliferation, and/or tissue function.     

Differential Regulation of the Ten-Eleven Translocation (TET) Family of Dioxygenases by O-Linked β-N-Acetylglucosamine Transferase (OGT)

The ten-eleven translocation (TET) family of dioxygenases (TET1/2/3) converts 5-methylcytosine to 5-hydroxymethylcytosine and provides a vital mechanism for DNA demethylation. However, how TET proteins are regulated is largely unknown. Here we report that the O-linked β-GlcNAc (O-GlcNAc) transferase (OGT) is not only a major TET3-interacting protein but also regulates TET3 subcellular localization and enzymatic activity. OGT catalyzes the O-GlcNAcylation of TET3, promotes TET3 nuclear export, and, consequently, inhibits the formation of 5-hydroxymethylcytosine catalyzed by TET3. Although TET1 and TET2 also interact with and can be O-GlcNAcylated by OGT, neither their subcellular localization nor their enzymatic activity are affected by OGT. Furthermore, we show that the nuclear localization and O-GlcNAcylation of TET3 are regulated by glucose metabolism. Our study reveals the differential regulation of TET family proteins by OGT and a novel link between glucose metabolism and DNA epigenetic modification.     

Essential role of O-GlcNAcylation in stabilization of oncogenic factors

A reversible post-translational protein modification which involves addition of N-acetylglucosamine (GlcNAc) onto hydroxyl groups of serine and/or threonine residues which is known as O-GlcNAcylation, has emerged as a potent competitor of phosphorylation. This glycosyltransfer reaction is catalyzed by the enzyme O-linked β-N-acetylglucosamine transferase (OGT). This enzyme uses uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the end product of hexosamine biosynthetic pathway, to modify numerous nuclear and cytosolic proteins. O-GlcNAcylation influences cancer cell metabolism in such a way that hyper-O-GlcNAcylation is considered as a prominent trait of many cancers, and is proposed as a major factor enabling cancer cell proliferation and progression. Growing evidence supports a connection between O-GlcNAcylation and major oncogenic factors, including for example, c-MYC, HIF-1α, and NF-κB. A comprehensive study of the roles of O-GlcNAc modification of oncogenic factors is warranted as a thorough understanding may help drive advances in cancer diagnosis and therapy. The focus of this article is to highlight the interplay between oncogenic factors and O-GlcNAcylation along with OGT in cancer cell proliferation and survival. The prospects for OGT inhibitors will also be discussed.     

O-GlcNAc modification affects the ATM-mediated DNA damage response

Background

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. The role of O-GlcNAcylation in the ataxia-telangiectasia mutated (ATM)-mediated DNA damage response is unknown. It is unclear whether ATM, which is an early acting and central component of the signal transduction system activated by DNA double strand breaks, is an O-GlcNAc-modified protein.

Methods

The effect of O-GlcNAc modification on ATM activation was examined using two inhibitors, PUGNAc and DON that increase and decrease, respectively, levels of protein O-GlcNAcylation. To assess O-GlcNAcylation of ATM, immunoprecipitation and immunoblot analyses using anti-ATM or anti-O-GlcNAc antibody were performed in HeLa cells and primary cultured neurons. Interaction of ATM with O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc to target proteins, was examined by immunoprecipitation and immunoblot analyses using anti-ATM.

Results

Enhancement of protein O-GlcNAcylation increased levels of X-irradiation-induced ATM activation. However, decreases in protein O-GlcNAcylation did not affect levels of ATM activation, but these decreases did delay ATM activation and ATM recovery processes based on assessment of de-phosphorylation of phospho-ATM. Thus, activation and recovery of ATM were affected by O-GlcNAcylation. ATM was subjected to O-GlcNAcylation, and ATM interacted with OGT. The steady-state O-GlcNAc level of ATM was not significantly responsive to X-irradiation or oxidative stress.

General significance

ATM is an O-GlcNAc modified protein, and dynamic O-GlcNAc modification affects the ATM-mediated DNA damage response.     

Cancer Metabolism and Elevated O-GlcNAc in Oncogenic Signaling

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a carbohydrate post-translational modification on hydroxyl groups of serine and/or threonine residues of cytosolic and nuclear proteins. Analogous to phosphorylation, O-GlcNAcylation plays crucial regulatory roles in cellular signaling. Recent work indicates that increased O-GlcNAcylation is a general feature of cancer and contributes to transformed phenotypes. In this minireview, we discuss how hyper-O-GlcNAcylation may be linked to various hallmarks of cancer, including cancer cell proliferation, survival, invasion, and metastasis; energy metabolism; and epigenetics. We also discuss potential therapeutic modulation of O-GlcNAc levels in cancer treatment.     

Dual functionality of O-GlcNAc transferase is required for Drosophila development

Post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc) catalysed by O-GlcNAc transferase (OGT) has been linked to regulation of diverse cellular functions. OGT possesses a C-terminal glycosyltransferase catalytic domain and N-terminal tetratricopeptide repeats that are implicated in protein–protein interactions. Drosophila OGT (DmOGT) is encoded by super sex combs (sxc), mutants of which are pupal lethal. However, it is not clear if this phenotype is caused by reduction of O-GlcNAcylation. Here we use a genetic approach to demonstrate that post-pupal Drosophila development can proceed with negligible OGT catalysis, while early embryonic development is OGT activity-dependent. Structural and enzymatic comparison between human OGT (hOGT) and DmOGT informed the rational design of DmOGT point mutants with a range of reduced catalytic activities. Strikingly, a severely hypomorphic OGT mutant complements sxc pupal lethality. However, the hypomorphic OGT mutant-rescued progeny do not produce F2 adults, because a set of Hox genes is de-repressed in F2 embryos, resulting in homeotic phenotypes. Thus, OGT catalytic activity is required up to late pupal stages, while further development proceeds with severely reduced OGT activity.     

O-GlcNAc in cancer biology

O-linked β-N-actylglucosamine (O-GlcNAc) is a carbohydrate post-translational modification on hydroxyl groups of serine and/or threonine residues of cytosolic and nuclear proteins. Analogous to phosphorylation, O-GlcNAcylation plays crucial regulatory roles in a variety of cellular processes. O-GlcNAc was termed a nutritional sensor, as global levels of the modification are elevated in response to increased glucose and glutamine flux into the hexosamine biosynthetic pathway. A unique feature of cancer cell energy metabolism is a shift from oxidative phosphorylation to the less efficient glycolytic pathway (Warburg effect), necessitating greatly increased glucose uptake. Additionally, to help meet increased biosynthetic demands, cancer cells also up-regulate glutamine uptake. This led us to hypothesize that the universal feature of increased glucose and glutamine uptake by cancer cells might be linked to increased O-GlcNAc levels. Indeed, recent work in many different cancer types now indicates that hyper-O-GlcNAcylation is a general feature of cancer and contributes to transformed phenotypes. In this review, we describe known/potential links between hyper-O-GlcNAcylation and specific hallmarks of cancer, including cancer cell proliferation, survival, cell stresses, invasion and metastasis, aneuploidy, and energy metabolism. We also discuss inhibition of hyper-O-GlcNAcylation as a potential novel therapeutic target for cancer treatment.     

Inhibition of mTOR affects protein stability of OGT

Autophagy regulates cellular homeostasis through degradation of aged or damaged subcellular organelles and components. Interestingly, autophagy-deficient beta cells, for example Atg7-mutant mice, exhibited hypoinsulinemia and hyperglycemia. Also, autophagy response is diminished in heart of diabetic mice. These results implied that autophagy and diabetes are closely connected and affect each other. Although protein O-GlcNAcylation is up-regulated in hyperglycemia and diabetes, and O-GlcNAcylated proteins play an important role in metabolism and nutrient sensing, little is known whether autophagy affects O-GlcNAc modification and vice versa. In this study, we suppressed the action of mTOR by treatment of mTOR catalytic inhibitors (PP242 and Torin1) to induce autophagic flux. Results showed a decrease in global O-GlcNAcylation, which is due to decreased OGT protein and increased OGA protein. Interestingly, knockdown of ATG genes or blocking of lysosomal degradation enhanced protein stability of OGT. In addition, when proteasomal inhibitor was treated together with mTOR inhibitor, protein level of OGT almost recovered to control level. These data suggest that mTOR inhibition is a more efficient way to reduce protein level of OGT rather than that of CHX treatment. We also showed that not only proteasomal degradation regulated OGT stability but autophagic degradation also affected OGT stability in part. We concluded that mTOR signaling regulates protein O-GlcNAc modification through adjustment of OGT stability.     

Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition

Background

DNA replication represents a critical step of the cell cycle which requires highly controlled and ordered regulatory mechanisms to ensure the integrity of genome duplication. Among a plethora of elements, post-translational modifications (PTMs) ensure the spatiotemporal regulation of pivotal proteins orchestrating cell division. Despite increasing evidences showing that O-GlcNAcylation regulates mitotic events, the impact of this PTM in the early steps of the cell cycle remains poorly understood.

Methods and results

Quiescent MCF7 cells were stimulated by serum mitogens and cell cycle progression was determined by flow cytometry. The levels of O-GlcNAc modified proteins, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) were examined by Western blotting and OGA activity was measured during the progression of cells towards S phase. A global decrease in O-GlcNAcylation was observed at S phase entry, concomitantly to an increase in the activity of OGA. A combination of two-dimensional electrophoresis, Western blotting and mass spectrometry was then used to detect and identify cell cycle-dependent putative O-GlcNAcylated proteins. 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated through G1/S transition were identified and the O-GlcNAc variations of Cytokeratin 8, hnRNP K, Caprin-1, Minichromosome Maintenance proteins MCM3, MCM6 and MCM7 were validated by immunoprecipitation.

Conclusions

The dynamics of O-GlcNAc is regulated during G1/S transition and observed on key proteins involved in the cytoskeleton networks, mRNA processing, translation, protein folding and DNA replication.

General significance

Our results led us to propose that O-GlcNAcylation joins the PTMs that take part in the regulation of DNA replication initiation.     

The Early Metazoan Trichoplax adhaerens Possesses a Functional O-GlcNAc System

Protein O-GlcNAcylation is a reversible post-translational signaling modification of nucleocytoplasmic proteins that is essential for embryonic development in bilateria. In a search for a reductionist model to study O-GlcNAc signaling, we discovered the presence of functional O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and nucleocytoplasmic protein O-GlcNAcylation in the most basal extant animal, the placozoan Trichoplax adhaerens. We show via enzymatic characterization of Trichoplax OGT/OGA and genetic rescue experiments in Drosophila melanogaster that these proteins possess activities/functions similar to their bilaterian counterparts. The acquisition of O-GlcNAc signaling by metazoa may have facilitated the rapid and complex signaling mechanisms required for the evolution of multicellular organisms.     

Murine platelets are not regulated by O-linked beta-N-acetylglucosamine

It is generally appreciated that platelets derived from diabetic patients display increased responsiveness to low levels of agonists. O-GlcNAcylation has been linked to hyperglycemia-related effects in other tissues; therefore we examined this modification in platelets to determine if O-GlcNAcylation affects platelet function. This post-translational modification consists of an N-acetylglucosamine attached to serine and/or threonine residues. We examined O-GlcNAc levels in platelets from a hyperglycemic murine model of Type I diabetes with known hypersensitivity to agonists and a Type II diabetes model (ob/ob) lacking detectable alterations in the aggregation profile. Neither model showed marked increases in protein O-GlcNAcylation. Treatment of platelets with multiple O-GlcNAcase inhibitors led to O-GlcNAc accumulation on multiple platelet proteins. However, the inhibitor-induced accumulation of this modification does not correlate with any gross alterations in platelet aggregation. These data suggest that while the modification occurs in platelets, their activity is not globally sensitive to O-GlcNAc levels.     

Cross regulation between mTOR signaling and <Emphasis Type="Italic">O</Emphasis>-GlcNAcylation

The hexosamine biosynthetic pathway (HBP) integrates glucose, amino acids, fatty acids and nucleotides metabolisms for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is the nucleotide sugar donor for O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) processes. O-GlcNAc transferase (OGT) is the enzyme which transfers the N-acetylglucosamine (O-GlcNAc) residue onto target proteins. Several studies previously showed that glucose metabolism dysregulations associated with obesity, diabetes or cancer correlated with an increase of OGT expression and global O-GlcNAcylation levels. Moreover, these diseases present an increased activation of the nutrient sensing mammalian target of rapamycin (mTOR) pathway. Other works demonstrate that mTOR regulates protein O-GlcNAcylation in cancer cells through stabilization of OGT. In this context, we studied the cross-talk between these two metabolic sensors in vivo in obese mice predisposed to diabetes and in vitro in normal and colon cancer cells. We report that levels of OGT and O-GlcNAcylation are increased in obese mice colon tissues and colon cancer cells and are associated with a higher activation of mTOR signaling. In parallel, treatments with mTOR regulators modulate OGT and O-GlcNAcylation levels in both normal and colon cancer cells. However, deregulation of O-GlcNAcylation affects mTOR signaling activation only in cancer cells. Thus, a crosstalk exists between O-GlcNAcylation and mTOR signaling in contexts of metabolism dysregulation associated to obesity or cancer.     

Tracking of fast moving neuronal vesicles with ageladine A

O-Linked β-N-acetylglucosaminylation (O-GlcNAcylation) of nucleocytoplasmic proteins is a ubiquitous post-translational modification in multicellular organisms studied so far. Since aberrant O-GlcNAcylation has a link with insulin resistance, it is important to establish the status of O-GlcNAcylation in differentiation of mesenchymal cells such as preadipocytes. In this study, we found a differentiation-dependent drastic increase in the level of O-GlcNAcylation in mouse 3T3-L1 preadipocytes. The occurrence of the increase in O-GlcNAcylation, which correlated with the expression of C/EBPα, was in part due to increased expression of O-GlcNAc transferase. In addition to the well-known O-GlcNAcylated proteins such as nucleoporins and vimentin, pyruvate carboxylase, long chain fatty acid-CoA ligase 1, and Ewing sarcoma protein were identified as the proteins which are heavily O-GlcNAcylated with the adipocyte differentiation. Both adipocyte differentiation and the differentiation-dependent increase in O-GlcNAcylation were blocked by 6-diazo-5-oxo-norleucine. These results suggest that O-GlcNAcylation particilates, at least in part, in adipogenesis.