The Glyco-Switch of Life: O-GlcNAcylation in Cell Fate Decision

Abstract

O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a unique type of protein glycosylation that intricately links cellular metabolism to various signaling pathways. This reversible, nutrient-sensitive modification dynamically regulates a wide range of biological processes, including apoptosis, cell proliferation, and differentiation. Recent studies have made substantial progress in elucidating the pivotal roles of O-GlcNAcylation in modulating key oncogenes and signaling cascades. Aberrant O-GlcNAc cycling has been associated with a variety of pathological conditions, including cancer, metabolic disorders, and neurodegenerative diseases, underscoring its critical influence on cell fate decisions. In this review, we will highlight recent advances in understanding how O-GlcNAcylation modulates major cell fate regulating pathways, including nuclear factor kappaB (NF-κB), Notch, G protein–coupled receptor (GPCR) signaling, and transforming growth factor beta (TGF-β). We propose that O-GlcNAcylation integrates extracellular signals with intracellular metabolic states, functioning as an essential “glyco-switch” sensor that modulates cell fate decisions in both physiological and pathological contexts.

Introduction

Mammalian cells constantly integrate diverse signals from their environments with intracellular conditions to make critical decisions that determine their fate—whether to survive, proliferate, differentiate, or undergo programmed cell death. This process, broadly referred to as cell fate decision, is fundamental to development, homeostasis, and disease progression (Moris et al. 2016). Among the numerous molecular mechanisms orchestrating cell fate, protein post-translational modifications (PTMs) have emerged as pivotal regulatory switches. One such dynamic and nutrient-sensitive modification is O-linked β-N-acetylglucosaminylation (O-GlcNAcylation).

O-GlcNAcylation typically refers to the attachment of a single N-acetylglucosamine (GlcNAc) moiety to serine and threonine residues of intracellular proteins (Torres and Hart 1984). A single pair of human enzymes catalyze this modification: O-GlcNAc transferase (OGT), which installs the modification using UDP-GlcNAc as the sugar donor, and O-GlcNAcase (OGA), which hydrolyzes the modification (Haltiwanger et al. 1992; Lubas et al. 1997; Heckel et al. 1998; Comtesse et al. 2001). Both OGT and OGA play indispensable roles in cell fate decisions. Genetic deletion of OGT results in embryonic lethality, whereas deletion of OGA leads to perinatal lethality (Shafi et al. 2000; Muha et al. 2021; Formichetti et al. 2025). Unlike classical glycosylation, which occurs in the endoplasmic reticulum (ER) and Golgi apparatus, O-GlcNAcylation is predominantly found in the nucleocytoplasmic compartment (Torres and Hart 1984; Holt and Hart 1986). This modification acts like a “Glyco-Switch” sensing the cellular nutrient status, and integrates the inputs from glucose, amino acid, and lipid metabolism through the hexosamine biosynthetic pathway (HBP) (Paneque et al. 2023). Here, the “Glyco-Switch” denotes a reversible and dynamic tuner instead of a binary on/off switch. An exception is epidermal growth factor (EGF) domain-specific OGT (EOGT), an ER-resident enzyme that utilizes the same sugar donor (UDP-GlcNAc) to modify a distinct set of secreted and membrane proteins at EGF-like repeats, including Notch1 (Sakaidani et al. 2011; Yang et al. 2021; Shu et al. 2022). This EOGT-catalyzed modification is referred to as extracellular O-GlcNAcylation.

Recent studies have unveiled a growing body of evidence supporting O-GlcNAcylation as a key regulator of cell fate decisions. The connection of O-GlcNAcylation with cellular metabolism originates from its dependency on the HBP, positioning this unique type of glycosylation as a direct transducer of nutrient status and stress response (Liu et al. 2021; Wells and Hart 2024). This regulatory capacity is particularly evident in stem cell systems, where proteomic analyses have identified nearly 1,000 O-GlcNAc-modified proteins in mouse embryonic stem cells (mESCs), including core pluripotency transcription factors NANOG, SOX2, and OCT4 (Hao et al. 2023). Further studies demonstrate that OGT inhibition influences the expression of ~2,500 differentiation-associated genes (Cheng et al. 2020; Chen et al. 2025). Similarly, in neural systems, O-GlcNAcylation precisely regulates the balance between stem cell maintenance and differentiation. OGT deficiency induces neuronal apoptosis, impaired corticogenesis, and reduced neural stem cell proliferation, whereas acute OGT knockdown promotes neuronal differentiation (Cheng et al. 2020; Chen et al. 2025). These findings highlighted the context-dependent effects of OGT on cell survival versus differentiation.

O-GlcNAcylation often exerts its regulatory roles through crosstalk with major signaling pathways. In this review, we will highlight recent discoveries linking O-GlcNAcylation to key regulatory cascades, including survival regulators such as nuclear factor kappaB (NF-κB) (Sen and Baltimore 1986; Guo et al. 2024), developmental pathways such as Notch (Bridges 1916; Metz and Bridges 1917; Yochem et al. 1988; Zhou et al. 2022) and G-protein coupled receptor (GPCR) signaling (Callihan et al. 2011; Pedro et al. 2020), as well as differentiation mediators like transforming growth factor beta (TGF-β) (de Larco and Todaro 1978; Moses et al. 1981; Roberts et al. 1981; Deng et al. 2024), all of which play well-established roles in cell fate determination. Our goal is to provide a coherent framework for understanding how O-GlcNAcylation functions as a molecular switch that integrates extracellular cues and metabolic states to govern cell fate decisions and contribute to health and disease.

The Glyco-Switch in Metabolism, Stress Responses, and Diseases

Glucose is a crucial energy source for many cellular processes. Cancer cells, in particular, favor glycolysis even in the presence of oxygen, so called the Warburg effect (Warburg 1925; Liberti and Locasale 2016). This phenomenon underscores the importance of glucose metabolism in health and disease. The HBP is prominent in that it converts glucose to UDP-GlcNAc, the sugar donor for protein glycosylation, including O-GlcNAcylation. The rate-limiting step of HBP is the conversion of glucosamine-6-phosphate, catalyzed by glutamine-fructose 6-phosphosphate amidotransferase (GFAT) (Marshall et al. 1991). This process is tightly controlled by nutrient availability and feedback regulation, as glucose utilization through the HBP varies across cell types and is influenced by stress and disease conditions (Mergenthaler et al. 2013; Tran and Wang 2019; Nakrani et al. 2025). High glucose level increases cellular UDP-GlcNAc through HBP flux, thereby elevating protein O-GlcNAcylation (Wang et al. 2023). Acute stress induces a rapid, transient upregulation of O-GlcNAcylation, closely associated with enhanced stress tolerance, cell survival, cardioprotection, and neuroprotection (Xue et al. 2024). Such transient O-GlcNAc activation represents a pro-survival mechanism and holds promise as a therapeutic strategy for stress-related disorders. O-GlcNAcylation modulates countless cellular processes, and disrupted O-GlcNAc homeostasis alters cell survival (Ferrer et al. 2014), proliferation (Levine et al. 2021), and epithelial to mesenchymal transition (EMT) (Minh and Reginato 2023). When dysregulated, these changes contribute to pathogenesis. For example, in Ishikawa endometrial cancer cells, hyper-O-GlcNAcylation drives upregulation of EMT-related genes, resulting in increased cellular proliferation and migration (Jaskiewicz and Townson 2019). Inversely, hypo-O-GlcNAcylation downregulates the expression of pro-EMT genes and reduces cell proliferation and migration (Jaskiewicz and Townson 2019; Queiroz et al. 2022). O-GlcNAcylation also regulates pluripotency in human embryonic stem cells (hESCs). For instance, OGA contributes to maintaining naïve pluripotency, regulating the balance between naïve and primed hESCs (Liu et al. 2024). In addition, the OGT mutant (C921Y), clinically associated with intellectual disability, exhibits impaired catalytic activity that slows down the cell self-renewal, as evidenced by decreased expression of pluripotency markers OCT4, SOX2, and alkaline phosphatase (Omelková et al. 2023). Moreover, O-GlcNAcylation can exert both pro- and anti-apoptotic effects. For example, O-GlcNAcylation is upregulated in pancreatic cancer cells, where it exerts an anti-apoptotic effect. This effect can be reversed by inhibiting O-GlcNAc cycling enzymes (OGT and OGA), highlighting their potential as therapeutic targets (Ma et al. 2013). O-GlcNAcylation can also promote apoptosis through the attenuation of AKT phosphorylation (Shi et al. 2015). Taken together, these findings highlight the multifaceted roles of O-GlcNAcylation in regulating key cell fate processes, including proliferation, differentiation, and apoptosis.

O-GlcNAcylation Regulates Signaling Pathways in Cell Fate Decision

Cell fate determination events, such as differentiation, proliferation, and EMT, are tightly controlled by a number of signaling pathways that can integrate environmental cues with cellular conditions to fine-tune their responses. Below, we highlight key pathways in cell fate decisions, including NF-κB, Notch, GPCR, and TGF-β, and discuss how they are regulated by O-GlcNAcylation.

NF-κB Signaling

Nuclear factor kappaB (NF-κB) is a family of evolutionarily conserved master transcription factors that play indispensable roles in regulating critical cellular processes, including proliferation, differentiation, and cell death (Guo et al. 2024). In resting cells, NF-κB remains inactive in the cytoplasm as pre-formed complexes bound to inhibitory proteins, IκBs. Upon stimulation, IκB proteins undergo phosphorylation and ubiquitination, resulting in their proteasomal degradation and subsequent activation of NF-κB (Figure 1) (Sakurai et al. 1999; Chen and Greene 2004). More recent studies have revealed that the activity of NF-κB is modulated by PTMs such as O-GlcNAcylation, phosphorylation, ubiquitination, and acetylation, as well as their intricate crosstalk (Figure 1) (Hou et al. 2012; Ma et al. 2013; Christian et al. 2016).

Figure 1. O-GlcNAcylation regulates NF-κB signaling network.

In resting cells, NF-κB (p50 in complex with p65 or c-Rel subunits) remains inactive in the cytoplasm by forming a complex with IκB. Upon stimulation, O-GlcNAcylation of upstream IKKβ inhibits its negative regulatory phosphorylation, thereby promoting IKKβ activation. Activated IKKβ phosphorylates IκB, leading to its ubiquitination and proteasomal degradation. This process releases the NF-κB complex, allowing it to translocate into the nucleus. O-GlcNAcylation of NF-κB subunit p65 facilitates its acetylation, enhancing its nuclear translocation and DNA binding, thereby promoting transcriptional activation of target genes. Similarly, O-GlcNAcylation of c-Rel subunit significantly amplifies its activity as a transcription factor, promoting cytokine gene expression while repressing FOXP3 expression. G: O-GlcNAcylation; Ub: Ubiquitination; P: Phosphorylation; Ac: Acetylation.

Created in BioRender. Young, M. (2025) https://BioRender.com/57nxma7

O-GlcNAcylation modulates NF-κB activation by modifying upstream regulatory kinases and adaptor proteins. For example, the IKKβ kinase, a central NF-κB activator, requires O-GlcNAcylation of S733 to counteract the inactivating phosphorylation at the same site for full catalytic activity, positioning it as a nutrient-sensitive gatekeeper for pathway initiation (Figure 1, Table 1) (May et al. 2002; Schomer-Miller et al. 2006). This regulation propagates through the signaling cascade, with transforming growth factor beta-activated kinase 1 (TAK1) activation being coordinated by O-GlcNAcylation of its adaptors TAB1 (S395) and TAB3 (S408) (Pathak et al. 2012; Authier et al. 2020). Clinically, TAB3 O-GlcNAcylation correlates with aggressive breast cancer (Tao et al. 2016), highlighting its potential as both a prognostic biomarker and therapeutic target.

Table 1.

Identified O-GlcNAcylation sites and their functional roles in NF-κB, Notch, GPCR, and TGF-β signaling pathways.

Pathway	Subunit	O-GlcNAcylation site	Function	References
NF-κB	p65	T305	Antagonize p65’s phosphorylation at T308.	(Ma et al. 2017)
		T305, S319	Facilitate CBP/P300-dependent acetylation of p56 at K310. Promote nuclear translocation and transcriptional output.	(Allison et al. 2012)
		T352	Critical for transcriptional activation.	(Yang et al. 2008)
		T322, T352	Enhance p65’s DNA binding affinity to promoters of pro-inflammatory genes. Enhance NF-κB-dependent anti-apoptotic gene expression, promoting pathologies such as colitis and colitis-associated cancer.Required for the anchorage-independent growth of pancreatic ductal adenocarcinoma cells.	(Ma et al. 2013; Phoomak et al. 2016)
	cRel	S350	Contribute to hyperglycemic-induced inflammation via the expression of TCR-induced, CD28RE-dependent genes such as IL-2, CSF2, and IFNG. Suppress FOXP3 expression needed for the development of immunosuppressive Treg cells.	(Ramakrishnan et al. 2013)
Notch	TM/ICD	S2264, S2271	Inhibit Itch binding and Notch ubiquitination.	(Chen et al. 2021)
	EGF2	S94	These O-GlcNAcylation modifications share the same function: required for Delta-like ligands binding to Notch.	(Sawaguchi et al. 2017)
	EGF10	T405
	EGF17	T671
	EGF20	S784
GPCR	β1AR	Unmapped	Decrease cAMP and PKA signal. Increase cardiomyocyte cell death.	(Cao et al. 2020)
	RAC	Unmapped	Increase GTP binding/activation. Increase activation of p42/44 and p38 MAPK.	(Kneass and Marchase 2005)
	DRP1	T585, T586	Modulate phosphorylation at S637. Promote translocation to mitochondria. Contribute to increased mitochondrial fragmentation and decreased membrane potential.	(Gawlowski et al. 2012)
	RAB3a	Unmapped	Decrease binding to GTP and function. Attenuate tumor suppressive effects in HCC.	(Wu et al. 2018).
	Gα12	Unmapped	Stabilize binding to Arhgef12. Positive regulation of RhoA/ROCK pathway.	(Zhang et al. 2023)
	MYPT1	S379, T381, S566, T570, T577, S585, S589, T590, T592, T594 & T637	Activation of MYPT1 leading to inactive MLC.Decrease actin contraction in response to S1P and LPA treatment.	(Morales et al. 202; Tóth et al. 2021)
	PKA	Unmapped	Positive regulation of PKA with increased phosphorylation of CREB and Tau. Alter subcellular localization of PKA catalytic subunits.	(Xie et al. 2016)
	CREB	S40	Disrupt binding between CREB and CRTC. Negatively regulate neuronal growth.	(Rexach et al. 2012)
TGF-β	MORC2	T556	Transcriptional activation of CTGF and Snail.	(Liu et al. 2022)
	SP1	26 reported sites	Increase TGF-β1 expression. Decrease SMAD7 expression.	(Aguilar et al. 2014)
	SMAD3	Unmapped	Increase SMAD3 phosphorylation and activity.	(Vang et al. 2024)
	SMAD4	T63	Increase stability through inhibition of proteasomal degradation.	(Kim et al. 2020)
	Snail	S112	Reduction of degradation and E-Cadherin repression to promote EMT.	(Park et al. 2010)
	TWIST1	S31	Inhibit protein degradation.	(Li et al. 2023)

Beyond its role in kinase activation, O-GlcNAcylation exhibits context-dependent suppression of NF-κB signaling via different mechanisms. In mesenchymal stem cells, O-GlcNAcylation of leptin inhibits p65 nuclear translocation, thereby promoting cell senescence and osteogenic differentiation (Zhang et al. 2024). Similarly, elevated global O-GlcNAcylation blocks RANKL-induced p65 phosphorylation, resulting in suppressed osteoclastogenesis (Li et al. 2022). The pathway is further modulated through RIPK1 O-GlcNAcylation, which disrupts formation of the RIPK1/FADD/Caspase-8 complex and NF-κB activation, thereby attenuating sunitinib-induced RIPK1-dependent apoptosis (Zeng et al. 2024). Notably, the relationship between O-GlcNAcylation and NF-κB extends to cell cycle regulation, where NF-κB-mediated G1 to S phase transition, achieved through p53 antagonism and cyclin D1 upregulation, demonstrates an inverse correlation with O-GlcNAcylation levels (Chen et al. 2001). This suggests the existence of a nutrient-sensitive checkpoint controlling cellular proliferation, highlighting the complex, context-dependent interplay between O-GlcNAcylation and NF-κB signaling.

In addition to modulating upstream kinases and adaptor proteins, O-GlcNAcylation directly modifies NF-κB subunits. NF-κB consists of subunits: p65 (RelA), RelB, c-Rel, p50/p105 (NF-κB1), and p52/p100 (NF-κB2). Among these, the p65 and c-Rel subunits are known to be O-GlcNAcylated. O-GlcNAcylation regulates NF-κB (p65 subunit) through a multi-layered control system, influencing its transcriptional activity, DNA binding, and interaction with co-regulators. Exposure to high glucose or glucosamine induces NF-κB O-GlcNAcylation and increases NF-κB-dependent gene expression (Ramakrishnan et al. 2013). O-GlcNAcylation of NF-κB was first reported in 2002, when it was shown to enhance NF-κB-dependent promoter activity through modification of the p65 subunit in rat mesangial cells (James et al. 2002). Subsequent studies confirmed this modification in human cells, where it was found to be essential for lymphocyte activation (Golks et al. 2007). Prior research has identified specific O-GlcNAcylation sites on NF-κB subunits, each contributing distinct functional roles to the regulation of NF-κB signaling. A number of O-GlcNAcylation sites have been mapped on p65, including residues T305, S319, T322, S337, T352, and S374, with T352 being critical for transcriptional activation (Table 1) (Yang et al. 2008). Notably, O-GlcNAcylation of T322 and T352 enhances p65’s DNA binding affinity to promoters of pro-inflammatory genes (e.g., IL-6, TNF-α, and MCP-1), which in turn enhances NF-κB-dependent anti-apoptotic gene expression in a feed-forward loop, promoting pathologies such as colitis and colitis-associated cancer (Figure 1) (Phoomak et al. 2016). Furthermore, O-GlcNAcylation of p65 at T305 and S319 residues facilitates CBP/p300-dependent acetylation of p65 at K310, forming a coordinated PTM code that amplifies nuclear translocation and transcriptional output (Figure 1) (Allison et al. 2012). Additionally, OGT overexpression elevates the expression of p300, IKKα, and IKKβ and further enhances IKK-mediated phosphorylation of p65 at S536, further stabilizing its activation (Ma et al. 2017). Conversely, phosphorylation at p65 T308 residue may antagonize O-GlcNAcylation at T305 of the same protein, suggesting dynamic crosstalk between these PTMs. Importantly, O-GlcNAcylation of p65 at T322 and T352 residues is also required for the anchorage-independent growth of pancreatic ductal adenocarcinoma cells, underscoring the functional significance of this modification in disease (Table 1) (Ma et al. 2013). By enhancing anti-apoptotic signaling and promoting gene transcriptions that favor survival and proliferation, O-GlcNAcylation of the p65 subunit of NF-κB directly contributes to cell fate decisions under both physiological and pathological conditions.

Unlike p65, c-Rel contains a single identified O-GlcNAcylation site at S350, which is essential for its DNA-binding and transactivation capacity (Table 1) (Ramakrishnan et al. 2013). O-GlcNAcylation regulates the c-Rel subunit of NF-κB through parallel mechanisms, though with distinct functional outcomes. Under hyperglycemic conditions (30 mM glucose), O-GlcNAcylation occupancy at the S350 residue of c-Rel increases from a basal 5% to 25%, significantly amplifying its activity (de Jesus et al. 2021). Upon T-cell receptor (TCR) activation, O-GlcNAcylation of c-Rel at S350 residue enhances the CD28RE-dependent expression of key cytokine genes (IL2, IFNG, and CSF2), amplifying inflammatory responses (Figure 1). Conversely, elevated O-GlcNAcylation reduces c-Rel binding at the forkhead box P3 (FOXP3) promoter, suppressing the transcription of this immunosuppressive regulatory gene (Figure 1). This mechanism contributes to the dysregulation of immune tolerance, particularly in autoimmune diabetes, where impaired FOXP3 expression coincides with raised proinflammatory gene activation.

The O-GlcNAcylation of NF-κB drives distinct pathological outcomes across diseases. In cancer, this modification promotes tumor progression by facilitating EMT through increased matrix metalloproteinase activity while simultaneously blocking cell death pathways via enhanced Bcl2 family expression (Ferrer et al. 2014; Lu et al. 2022). Metabolic diseases exhibit tissue-specific dysregulation, with O-GlcNAcylated NF-κB triggering retinal ganglion cell death in diabetic retinopathy yet driving liver inflammation in metabolic syndrome (Kim et al. 2016; Xie et al. 2023). Within the immune system, O-GlcNAc modifications favor pro-inflammatory macrophage states and disrupt regulatory T cell development, contributing to autoimmune pathogenesis (Liu et al. 2019; Machacek et al. 2019). These observations underscore how O-GlcNAc-dependent NF-κB regulation diversely influences disease progression through cell fate determination.

Notch Signaling

Notch signaling is a highly conserved pathway in metazoans and plays critical roles in cell fate determination, differentiation induction, and stem cell maintenance (Lasky and Wu 2005; Zhou et al. 2022). Notch signaling can serve either as a tumor suppressor or an oncogenic factor, depending on the context (Radtke and Raj 2003; Dontu et al. 2004). For instance, Notch exerts tumor suppressor functions in solid tumors such as liver cancer, small-cell lung cancer, and a few subtypes of brain cancers (Shi et al. 2024). Conversely, Notch can act as an oncogene. Aberrant activation of Notch in hematopoietic cells leads to T-cell leukemia in both mice and humans (Wang et al. 2016). Furthermore, Notch mutation has been proposed as a predictive biomarker for immune checkpoint blockade therapy in many cancers (Zhou et al. 2022). Interestingly, the Notch receptor undergoes various types of glycosylation in the ER, including O-fucosylation, O-glucosylation, and O-GlcNAcylation, contributing to receptor folding, ligand engagement, and downstream functions such as hematopoietic differentiation (Stanley and Tanwar 2022). Moreover, EOGT-catalyzed O-GlcNAcylation and POFUT1-catalyzed O-fucosylation on Notch EGF repeats synergize at distinct consensus sites to support ligand binding and signal transduction (Nauman et al. 2023). In this review, we mainly focus on O-GlcNAc-mediated regulation of Notch.

Notch signaling is activated through direct cell–cell interaction, where a membrane-bound ligand binds to the Notch receptor on the same or adjacent cell. The interaction between Notch ligand and receptor triggers proteolytic cleavage and releases the Notch intracellular domain (NICD), which transfers to the nucleus to regulate target gene expression (Figure 2) (Zhou et al. 2022). Different species encode different sets of Notch receptors and ligands. For instance, Drosophila has a single Notch receptor and two ligands, Delta (Dl) and Serrate (Ser). Mammals have four Notch paralogs (Notch1, Notch2, Notch3, and Notch4) and five ligands (delta-like ligand 1 (DLL1), delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), Jagged-1 (JAG1), and Jagged-2 (JAG2)) with both overlapping and distinct functions. For example, DLL1 regulates cell differentiation and intercellular communication (Zhang et al. 2021). DLL3 promotes apoptosis and suppresses cell growth (Owen et al. 2019). DLL4 activates NF-κB signaling to enhance tumor metastasis (Pitulescu et al. 2017; Zhou et al. 2022). JAG1 supports angiogenesis, and JAG2 promotes cell survival and proliferation (Houde et al. 2004; Li et al. 2014).

Figure 2. Schematic illustration of Notch protein O-GlcNAcylation in normal and OGT/EOGT-deficient cells.

In normal cells (blue background, left), the Notch protein undergoes O-GlcNAcylation by EOGT at its EGF repeat domain in the endoplasmic reticulum (ER) following translation. After S1 cleavage in the Golgi apparatus, Notch is transported to the cell membrane. O-GlcNAcylation at the EGF domain enhances Notch’s binding to Delta-like ligands, while exerting minimal effect on the interactions with Jagged ligands. Concurrently, OGT catalyzes O-GlcNAcylation at the Notch’s transmembrane/intracellular domain (TM/ICD), protecting Notch from ubiquitination by the E3 ligase Itch and preventing proteasomal degradation. In OGT- or EOGT-deficient cells (yellow background, right), the lack of EOGT-mediated glycosylation weakens Notch binding with Delta-like ligands, and the absence of OGT catalyzed O-GlcNAcylation allows Itch-mediated ubiquitination, leading to enhanced degradation of Notch and suppression of Notch signaling activity. NICD: Notch Intracellular Domain; G: O-GlcNAcylation; Ub: Ubiquitination.

Created in BioRender. Young, M. (2025) https://BioRender.com/ljbsqay

The O-GlcNAc modification of Notch receptor was first identified in Drosophila Schneider 2 (S2) cells in 2008, where O-GlcNAc was detected on epidermal growth factor-like 20 (EGF20) of Notch using the CTD110.6 antibody (Matsuura et al. 2008). This modification was also found on the EGF repeats of Drosophila Notch ligands, Delta and Serrate. Subsequent mass spectrometry analysis of Drosophila Notch and mouse Notch1 further confirmed that the O-GlcNAc modifications occur at the serine/threonine residues within the consensus sequence C5-X-X-G-X-(T/S)-G-X-X-C6 (Alfaro et al. 2012; Harvey et al. 2016; Kakuda and Haltiwanger 2017). While the Drosophila Notch protein has 18 potential sites for O-GlcNAc modifications, only five of these sites have been observed to be robustly modified in Drosophila S2 cells and embryos (Pandey et al. 2020). In contrast, mouse Notch1 has 17 EGF repeats with consensus sites, most of which are O-GlcNAcylated (Pandey et al. 2020). Notably, Notch O-GlcNAcylation on EGF repeats is catalyzed by EOGT within the ER (Varshney and Stanley 2017). Although EOGT likely operates through a mechanism distinct from that of OGT, we briefly discuss EOGT-mediated O-GlcNAcylation here to illustrate how O-GlcNAcylation can also influence cell fate via membrane-bound signaling pathways such as Notch.

Recent research has shown that the O-GlcNAcylation on Notch1 EGF repeats plays a critical role in ligand selectivity and signaling activation. Studies using EOGT knockdown/knockout models demonstrate that O-GlcNAcylation promotes Delta-like ligand (DLL1/DLL4)-mediated Notch signaling but has no significant effect on JAG1-induced activation (Figure 2, Table 1). Loss of EOGT impairs DLL1/DLL4 binding to Notch1, while JAG1 binding remains unaffected. This ligand-specific regulation is essential for vascular development, as endothelial EOGT deletion disrupts retinal angiogenesis, phenocopying mild Notch loss-of-function defects (Sawaguchi et al. 2017). Genetic interactions further confirm this, as EOGT deficiency exacerbates vascular abnormalities in Notch1 mutant mice and reduces Notch target gene expression in endothelial cells.

Besides the EOGT-catalyzed O-GlcNAcylation, Notch function is also regulated by O-GlcNAc transferase (OGT). OGT modifies Notch at its transmembrane/intracellular domain (TM/ICD) fragment, thus inhibiting its ubiquitination and stabilizing Notch by disrupting the association with E3 ligase Itch (Figure 2, Table 1) (Chen et al. 2021). OGT-catalyzed O-GlcNAcylation of Notch is essential for maintaining the pool of adult neural stem/progenitor cells and neurogenesis in mice. Furthermore, elevated cellular O-GlcNAc levels, induced by OGA inhibition, have been associated with decreased expression of Notch genes. This downregulation appears to trigger premature activation of neurogenic transcription factors, potentially through modulation of histone H3K27 methylation mediated by the histone methyltransferase EZH2 (Parween et al. 2022). Taken together, these findings support a conserved mechanism in which O-GlcNAcylation fine-tunes Notch signaling output to balance stem cell maintenance with lineage-specific differentiation.

G-Protein Coupled Receptor (GPCR) Signaling

G-protein coupled receptors (GPCRs) are a diverse class of integral membrane receptors that regulate numerous cellular processes in response to ligands, including hormones, neurotransmitters, photons, and other stimuli (Hilger et al. 2018). Upon ligand binding, GPCRs initiate signaling cascades through both G-protein-dependent and -independent mechanisms (Liccardo et al. 2022). GPCRs influence cell fate through various mechanisms, promoting survival or cell death depending on the transmitted signal (New et al. 2007; Li et al. 2024). G-proteins are usually heterotrimeric complexes composed of Gα and Gβγ subunits, activated by ligand-bound GPCRs. Upon activation, Gα exchanges guanosine diphosphate (GDP) for guanosine triphosphate (GTP) and dissociates from Gβγ, allowing both G-protein subunits to signal independently to downstream effectors. Monomeric small GTPases also mediate GPCR signal transduction and modulate key cellular processes, including cell death, proliferation, and migration (Yin et al. 2023). Both heterotrimeric and monomeric forms of G-proteins are regulated by guanine nucleotide exchange factors (GEFs), which activate signaling by promoting GTP binding, and GTPase-activating proteins (GAPs), which terminate signaling by accelerating GTP hydrolysis. While GPCRs act as GEFs for heterotrimeric G-proteins, monomeric GTPases rely on distinct cytosolic GEFs for activation (Bos et al. 2007; Ghosh et al. 2017). The vast diversity of GPCRs and G-proteins facilitates remarkable signaling promiscuity, enabling the modulation of key effectors involved in critical cell fate decisions − including apoptosis, proliferation, and differentiation − processes in which GPCR signaling is deeply implicated (New and Wong 2007; Callihan et al. 2011; Alhosaini et al. 2021; Li et al. 2024). GPCR signaling acts as a molecular hub that translates environmental stimuli into context-specific cellular responses, and this diverse signaling network is subject to O-GlcNAc regulation at multiple levels, warranting further characterization. Investigating how O-GlcNAcylation modulates GPCR signaling provides critical insights into the regulation of downstream effectors and the molecular mechanisms that govern cell fate decisions.

While direct O-GlcNAc regulation of GPCRs remains underexplored, emerging evidence suggests a significant role of this modification in GPCR signaling networks (Figure 3, Table 1). For instance, the GPCR β1-adrenergic receptor (β1AR) transduces cell death or survival signals via cAMP in cardiomyocytes (Shin et al. 2014) and is found to be O-GlcNAcylated by OGT (Cao et al. 2020). Overexpression of OGT reduces cAMP production and suppresses Protein Kinase A (PKA) activity − a downstream effector of cAMP − via a β1AR-dependent mechanism (Figure 3). This OGT-catalyzed modification is associated with increased intracellular Ca²⁺ levels and elevated cardiomyocyte cell death, highlighting the important role of O-GlcNAcylation in β1AR signaling, which is implicated in diabetic cardiomyopathy (Cao et al. 2020). Additional evidence of direct regulation of GPCR canonical components includes the G-protein subunit α12 (Gα12). This subunit serves as an important upstream regulator of the RhoA/Rho-associated protein kinase (ROCK) pathway, which plays a key role in neuronal development (Lu et al. 2023). In mouse cerebellar granule cells, Gα12 was found to be O-GlcNAcylated, a modification lost with OGT conditional knockout. O-GlcNAcylation of Gα12 stabilized its binding to the RGS-like domain of the Rho-specific guanine nucleotide exchange factor 12 (Arhgef12) (Figure 3), which is essential for downstream RhoA/ROCK activation (Zhang et al. 2023). Supporting this, a separate study found that O-GlcNAcylation enhanced RhoA/ROCK/myosin light chain (MLC) signaling, promoting motility, invasion, and migration in SKOV3 and 59M ovarian cancer cell lines (Niu et al. 2017). These findings establish O-GlcNAcylation as a modulator of Gα12-dependent RhoA–ROCK signaling.

Figure 3. O-GlcNAcylation regulates the GPCR signaling network.

The β1 adrenergic receptor (β1AR) is O-GlcNAcylated, which decreases cAMP formation and PKA signaling, while increasing intracellular Ca²⁺ accumulation and cell death. Glucosamine treatment or OGA inhibition by PUGNAc increases RAC and MAPK activities. O-GlcNAcylation inhibits RAB3a activity, which increases the capacity for migratory invasion. DRP1 O-GlcNAcylation results in its translocation to the mitochondria. O-GlcNAcylation of MYPT1 blocks its phosphorylation, inhibiting MLC activation. O-GlcNAcylation alters PKAcα/β subcellular localization and increases phosphorylation of downstream targets CREB and Tau. CREB O-GlcNAcylation blocks its interaction with CRTC and alters the expression of genes important for neuronal development. Gα12 can be O-GlcNAc modified, which promotes its association with Arhgef12. G: O-GlcNAcylation; P: Phosphorylation; GTP: Guanosine Triphosphate; MLC: Myosin Light Chain. Blue colored proteins represent GPCR-associated effectors. Green colored proteins represent GPCR canonical effectors.

Created in BioRender. Young, M. (2025) https://BioRender.com/wxpcz9x

In addition to direct regulation of GPCR signaling, evidence demonstrates that O-GlcNAcylation modulates downstream effectors in GPCR-associated pathways, particularly small GTPases, kinases, and transcription factors that transduce GPCR signals into cellular responses. A notable example is the Ras homology (Rho) family, a subset of the RAS superfamily of small GTPases that control numerous cellular processes, including cell polarity, division, and gene expression (Mosaddeghzadeh and Ahmadian 2021). Evidence supports that O-GlcNAcylation alters Rho GTPase activity. For instance, in human neutrophils, O-GlcNAcylation modulates the activity of the Rho GTPase, RAC (Kneass and Marchase 2005). This study demonstrated that OGA inhibition or glucosamine treatment enhances GTP-bound RAC levels, promoting cell motility and MAPK signaling in a PI3K-dependent manner (Figure 3). This pathway is highly relevant to cell fate determination and exhibits significant cross-talk with GPCR signaling (Zhang and Liu 2002). The GTPase Dynamin-Related Protein-1 (DRP1), which is important in mitochondrial fission, is O-GlcNAcylated at its T585 and T586 residues. This modification alters DRP1 function by reducing its phosphorylation at S637, thereby promoting its translocation from the cytosol to the mitochondria (Figure 3) (Gawlowski et al. 2012). The same study showed that enhanced O-GlcNAcylation of DRP1 is associated with mitochondrial fragmentation and a decrease in membrane potential. Although DRP1 is not a direct effector of GPCRs, GPCR-mediated activation of RhoA regulates DRP1 activity through ROCK-dependent phosphorylation. (Brand et al. 2018). Given the role of DRP1 in RhoA-mediated cardioprotection against oxidative stress, its O-GlcNAcylation may act as a regulatory mechanism linking extracellular cues to mitochondrial remodeling and cell survival pathways. O-GlcNAcylation has also been identified on the GTPase RAB3a, which plays pivotal roles in vesicular trafficking. RAB3a functions downstream of Gαo signaling initiated by the KDEL receptor, a non-canonical GPCR (Solis et al. 2017). In HEP3b Hepatocellular Carcinoma (HCC) cells, O-GlcNAcylation of RAB3a reduced its binding affinity to GTP and its activity (Figure 3) (Wu et al. 2018). This study demonstrated that in HCC, RAB3a acts as a tumor suppressor, inhibiting cell migration and metastasis; however, O-GlcNAcylation attenuates these tumor-suppressive effects by impairing RAB3a’s function. OGT knockdown enhances RAB3a-mediated inhibition of metastasis, which is reversed by OGT overexpression in vitro and in vivo. Wu and colleagues also demonstrate through clinical data that high OGT levels impair RAB3a’s protective role, leading to a worse prognosis in HCC patients. In HeLa cells, the GTPase RAB7a, a regulator of autophagy, is O-GlcNAc modified (Liu et al. 2020). While the function of O-GlcNAcylated RAB7a is unclear, OGT inhibition in CCD841CoN cells decreased RAB7a binding to autophagosome membranes, resulting in autophagosome accumulation and blocking their fusion with lysosomes (Ben Ahmed et al. 2024). This data suggest that O-GlcNAc is important for RAB7a-regulated autophagy, a crucial process in cell fate determination.

Sphingosine 1-phosphate (S1P) and Lysophosphatidic Acid (LPA) are GPCR ligands that influence cell survival or death, depending on cell type and context (Kamps and Coffman 2005). Both GPCR ligands activate the Rho/ROCK pathway, driving cell contraction, which is linked to cell fate as ROCK-mediated phosphorylation of myosin light chain (MLC) induces early apoptotic blebbing in cells (Street and Bryan 2011). Recent studies highlight O-GlcNAc’s role in regulating cellular contraction. In NIH3T3 fibroblasts, O-GlcNAc levels modulated sensitivity to S1P-induced actin contraction via the Myosin Phosphatase Targeting Subunit (MYPT1) (Pedowitz et al. 2021). MYPT1 regulates procontractile signaling by dephosphorylating MLC. O-GlcNAc modification of MYPT1 inhibits its ROCK-mediated phosphorylation, leading to decreased actin contraction in response to S1P treatment (Figure 3). O-GlcNAcylation of MYPT1 also inhibits MLC activation and actin contraction with LPA treatment (Morales et al. 2021). In addition, MYPT1 is implicated in cardiomyogenesis by influencing transcriptional regulation downstream of the Rho/ROCK signaling pathway (Ryan et al. 2013). Given that MYPT1 undergoes O-GlcNAc modification, its function may be responsive to nutrient cues, positioning it as a potential molecular link between metabolic state and cardiac cell fate decisions.

Protein Kinase A (PKA) is a cyclic Adenosine Monophosphate (cAMP) dependent kinase and is involved in many cellular processes such as metabolism, gene expression, cell proliferation, and differentiation (Jin et al. 2018). GPCR signaling stimulates cAMP production, which transmits downstream signals through PKA, a central effector in this pathway (Rehman et al. 2025). O-GlcNAcylation has been identified on both PKAcα and PKAcβ catalytic subunits of this enzyme, resulting in the positive regulation of PKA, increasing the phosphorylation of downstream targets CREB and Tau (Figure 3) (Xie et al. 2016). This study also showed that O-GlcNAcylation alters the subcellular localization of PKA in mouse neuroblastoma N2A cells, where PKAcα was translocated to the periphery of the nucleus and PKAcβ to the nucleus with OGT co-expression (Figure 3). CREB, a transcription factor downstream of GPCR signaling, is O-GlcNAcylated at residue Ser40. This modification disrupts the interaction of CREB with transcriptional co-activator CRTC, thereby affecting gene expression related to cell survival (Figure 3) (Rexach et al. 2012). Mutation of the CREB O-GlcNAcylation site (S40A) increases the expression of genes essential in neuronal development via a CRTC-dependent mechanism. Additionally, neuron growth is modulated by O-GlcNAcylation of CREB, as seen by the increase in dendrite growth in O-GlcNAc-deficient CREB S40A mutant cells compared to wild type. While CREB is classically activated downstream of GPCR signaling via PKA, it is important to note that other signaling networks, including those mediated by receptor tyrosine kinases, also converge to activate CREB (Zhang et al. 2020).

Emerging evidence suggests that O-GlcNAcylation can regulate various components of GPCR signaling; however, our current understanding of this regulation is far from complete. The context-dependent nature of GPCR signaling, coupled with the dynamic and reversible interplay between O-GlcNAcylation and phosphorylation, adds further complexity. Importantly, the contribution of O-GlcNAc-modulated GPCR signaling to disease pathogenesis remains underexplored. Closing this knowledge gap could uncover new mechanisms driving disease and identify novel therapeutic targets.

TGF-β Signaling

Transforming growth factor beta (TGF-β) is a cytokine that plays a pivotal role in regulating cellular homeostasis, cell growth, differentiation, and extracellular matrix production (Zhang et al. 2020). In mammals, there are three different TGF-β isoforms (TGF-β1, -β2, and -β3). Each TGF-β isoform is synthesized as an inactive precursor and then cleaved by Furin-like proteases after homodimerization, resulting in the formation of the small latency complex (SLC) (Deng et al. 2024). The SLC binds to latent TGF-β binding proteins (LTBPs) to form the large latency complex, which facilitates proper secretion and deposition into the extracellular matrix, where it remains latent until it is activated. TGF-β can be activated by several mechanisms, including mechanical stress and protease cleavage, which leads to the release of the mature cytokine from the large latency complex. Once released, TGF-β binds to its receptor (TGF-βR) and can signal through both canonical SMAD signaling and non-canonical pathways, thereby modulating cell fate decisions (Figure 4) (Furler et al. 2018; Massagué and Sheppard 2023).

Figure 4. O-GlcNAcylation regulates the TGF-β signaling network.

Glucose increases bioactive TGF-β, USF-2, and the externalization of type 1 and type 2 TGF-β receptors (TGF-βR). TGF-β1 leads to GFAT stabilization by reducing its ubiquitination. O-GlcNAcylation stabilizes Snail, leading to repression of E-Cadherin and promoting EMT. TWIST1 is stabilized by O-GlcNAcylation, leading to upregulated OGT transcriptional activation, promoting EMT. MORC2 O-GlcNAcylation increases Snail and CTGF expression and promotes EMT. USF-2 expression increases TGF-β1 and Collagen expression. Hyperglycemia increases SMAD2/3 activation, which translocates to the nucleus in conjunction with SMAD4, a process that is stabilized by O-GlcNAcylation. SMAD2/3 and SMAD4 interact with SP1 to induce expression of TGF-β-associated genes and EMT. SP1 O-GlcNAcylation also inhibits SMAD7 expression. SMAD3 phosphorylation and activity are enhanced by O-GlcNAc modification. HBP: Hexosamine Biosynthetic Pathway; EMT: Epithelial to Mesenchymal Transition; G: O-GlcNAcylation; Ub: Ubiquitination; P: Phosphorylation. Blue colored proteins represent TGF-β-associated effectors. Yellow colored proteins represent TGF-β canonical effectors.

Created in BioRender. Young, M. (2025) https://BioRender.com/27j87f9

The nutrient-sensing hexosamine biosynthetic pathway (HBP) and O-GlcNAcylation regulate TGF-β signaling at multiple levels, ranging from ligand expression to the modulation of downstream effector activity. In mesangial cells, glucosamine treatment elevates TGF-β1 expression and bioactivity, yet reduces cell proliferation in a TGF-β1-dependent manner (Kolm-Litty et al. 1998). TGF-β1 is also transcriptionally regulated by O-GlcNAc modification of the transcription factor SP1 (Table 1). Hyperglycemia induces SP1 O-GlcNAcylation and activation, leading to increased TGF-β1 expression (Du et al. 2000). Other studies link HBP flux to elevated expression of upstream stimulatory factor 2 (USF-2) and its enhanced binding to the glucose response element of the TGF-β1 promoter, leading to enhanced promoter activity (Figure 4) (Weigert et al. 2004). Further exploration of the link between HBP and TGF-β revealed that GFAT is responsible for positively regulating the expression of TGF-β1 (Weigert et al. 2001; Weigert et al. 2003).

Cellular glucose concentration also regulates the surface expression of TGF-β type 1 and type 2 receptors. In mouse embryonic fibroblasts and NRK-52E cells, glucose treatment does not affect the relative mRNA or protein levels of TGF-β receptors but increases their externalization (Wu and Derynck 2009). The same study also found that glucose treatment upregulates the mRNA and protein levels of TGF-β1, -β2, and -β3, and increases the amount of bioactive extracellular TGF-β (Figure 4). Proteins downstream in the canonical TGF-β pathway exhibit more direct evidence of regulation by O-GlcNAcylation and have been studied in the context of cancer, as well as in fibrosis, which is an area of increasing interest. For instance, a study in rat cardiac fibroblasts found that hyperglycemia results in increased protein expression of SMAD2/3. These SMADs translocate to the nucleus in a complex with SMAD4, where they can interact with SP1 and enhance transcription of target genes, including collagen (Aguilar et al. 2014). The same study also demonstrated that the hyperglycemia-associated increase in O-GlcNAcylated SP1 reduces protein expression of SMAD7, an inhibitory factor of SMAD signaling (Figure 4). A recent study also identified O-GlcNAc modification of SMAD3, which surprisingly coincided with its elevated phosphorylation and activity (Figure 4) (Vang et al. 2024). Inhibition of OGT decreases SMAD3 O-GlcNAcylation and phosphorylation, which reduces TGF-β1-induced collagen expression and deposition into the ECM. These studies highlight the ability of O-GlcNAcylation to regulate TGF-β1 in the context of tissue remodeling and fibrosis. The TGF-β pathway is highly linked with cell invasion and EMT, and emerging evidence suggests that the pathway is regulated extensively by O-GlcNAcylation. In A549 lung cancer cells, it was found that SMAD4 is O-GlcNAcylated at multiple sites, and O-GlcNAcylation of the residue Thr63 stabilizes SMAD4 by hindering its proteasomal degradation (Figure 4, Table 1) (Kim et al. 2020). O-GlcNAc stabilized SMAD4 was found to promote EMT in BEAS-2B lung epithelial cells (Wang et al. 2024). Hypo-O-GlcNAcylation induced by OGT inhibition decreases cell proliferation and, interestingly, reduces the expression of pro-EMT factor, TGF-β2 (Jaskiewicz and Townson 2019).

A more nuanced role for O-GlcNAcylation includes direct and indirect modulation of TGF-β effectors, particularly transcription factors targeted by TGF-β and co-activated by pathways like Notch and GPCR signaling. Recently, it was discovered that TGF-β1 treatment enhances GFAT stability by reducing its ubiquitination, providing potential evidence of HBP–TGF-β positive feedback loop (Liu et al. 2022). This study demonstrated that GFAT stability enhances O-GlcNAcylation of MORC2 at Thr556 (Table 1), leading to the transcriptional activation of TGF-β1 target genes Snail and CTGF and promoting migratory invasion in breast cancer cells (Figure 4). These findings underscore the role of O-GlcNAcylation as a regulatory node in cell decisions, as Snail is critical in EMT, and CTGF regulates migratory invasion in breast cancer cells (Kaufhold and Bonavida 2014; Kim et al. 2021). O-GlcNAcylation also directly modifies transcription factors downstream of TGF-β, fine-tuning their activities. One such transcription factor, Snail, is O-GlcNAcylated at Ser112 by OGT (Table 1). This glycosylation prevents Snail degradation and promotes EMT by repressing E-Cadherin expression (Figure 4) (Park et al. 2010). Similarly, TWIST1, a key EMT driver downstream of TGF-β/SMAD signaling, is stabilized by the O-GlcNAcylation at its Ser31 residue (Table 1). Notably, TWIST1 was also identified at the promoter of OGT, enhancing OGT transcription and providing evidence of a positive feedback loop with OGT (Figure 4) (Fan et al. 2015; Li et al. 2023).

Emerging evidence supports the pivotal role of O-GlcNAcylation in modulating TGF-β signaling. Despite these advances, the precise mechanisms underlying this regulation remain incompletely understood and warrant further investigation. The studies discussed herein reveal a dual regulatory function of O-GlcNAcylation in TGF-β signaling: it directly modulates core signaling components including SMAD proteins and TGF-β receptors (TGF-βRs), while also fune-tuning the activity of downstream effectors such as the transcription factor Snail. TGF-β influences both the stimulation and inhibition of the cell cycle and serves as a key regulator of EMT processes that are intimately involved in disease when dysregulated. Understanding how O-GlcNAcylation modulates TGF-β signaling is critical for elucidating the molecular basis of TGF-β-driven pathologies.

Conclusions

Dissecting how O-GlcNAcylation regulates important pathways in cell maintenance and cell fate decision is critical, but it does not come without challenges. The dynamic nature of O-GlcNAcylation makes it challenging to detect modifications on low-abundant proteins within the cell. Advances in proteomic analysis are uncovering new O-GlcNAcylated proteins, helping to fill knowledge gaps in how this modification regulates signaling pathways. O-GlcNAc modification is complex, as its effects can vary significantly between cell types, highlighting the need for further research to elucidate its role in a context-dependent manner, especially in complex disease models. Chemical biology, genetic, biophysical, and engineering strategies, as well as the development of O-GlcNAc databases (Wulff-Fuentes et al. 2021; Hu et al. 2024; Hou et al. 2025), are enhancing our ability to decipher the regulatory roles of O-GlcNAcylation, offering a more systematic understanding of its functions across different cellular environments. The growing application of artificial intelligence in research offers significant potential for synergizing data across different studies to paint a more complete picture of O-GlcNAcylation in these signaling networks. Currently, there is also limited understanding of the mechanism and function of EOGT-catalyzed O-GlcNAcylation. The identification of O-GlcNAcylation on extracellular protein domains, particularly on Notch receptors and their ligands, raises the important question of whether extracellular O-GlcNAcylation is more prevalent and functionally significant than previously appreciated. All of these highlight the need for advanced tools and context-specific models to decipher the multifaceted roles of O-GlcNAcylation, both within and outside the cell.

Data availability

All data discussed are derived from previously published sources, which are cited in the article.