The intersections between O-GlcNAcylation and phosphorylation: implications for multiple signaling pathways

Abstract

A paradigm-changing discovery in biology came about when it was found that nuclear and cytosolic proteins could be dynamically glycosylated with a single O-linked beta-N-acetylglucosamine (O-GlcNAc) moiety. O-GlcNAcylation is akin to phosphorylation: it occurs on serine and/or threonine side chains of proteins, and cycles rapidly upon cellular activation. O-GlcNAc and phosphate show a complex interplay: they can either competitively occupy a single site or proximal sites, or noncompetitively occupy different sites on a substrate. Phosphorylation regulates O-GlcNAc-cycling enzymes and, conversely, O-GlcNAcylation controls phosphate-cycling enzymes. Such crosstalk is evident in all compartments of the cell, a finding that is congruent with the fundamental role of O-GlcNAc in regulating nutrient- and stress-induced signal transduction. O-GlcNAc transferase is recruited to the plasma membrane in response to insulin and is targeted to substrates by forming transient holoenzyme complexes that have different specificities. Cytosolic O-GlcNAcylation is important for the proper transduction of signaling cascades such as the NFkappaB pathway, whereas nuclear O-GlcNAc is crucial for regulating the activity of numerous transcription factors. This Commentary focuses on recent findings supporting an emerging concept that continuous crosstalk between phosphorylation and O-GlcNAcylation is essential for the control of vital cellular processes and for understanding the mechanisms that underlie certain neuropathologies.

Fig. 1.

The O-GlcNAcylation cycle. Glucose is metabolized through the hexosamine biosynthetic pathway to form the high-energy intermediate UDP-GlcNAc, which serves as the sugar donor for OGT. The O-GlcNAc cycle starts when OGT catalyzes the transfer of GlcNAc from UDP-GlcNAc to serine and/or threonine residues of a protein substrate through β-glycosidic attachment. Although no consensus sequence has been identified for OGT, proline and valine are the most common amino acids found prior to the serine or threonine target site. The O-GlcNAcylated protein can be a substrate for O-GlcNAcase, which hydrolyses the glycosidic linkage to generate free GlcNAc and naked protein.

Fig. 2.

O-GlcNAc and phosphate regulate the insulin signaling pathway at the plasma membrane. (A) In the basal state, OGT is concentrated in the nucleus of the insulin-responsive cell. (B) Upon insulin (I) binding to its receptor (IR), active PI3K catalyzes the phosphorylation of the plasma-membrane phospholipid phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P₂; PIP2] to PtdIns(3,4,5)P₃ (PIP3), driving the translocation of OGT from the nucleus to the plasma membrane by its binding to PtdIns(3,4,5)P₃. OGT is then tyrosine phosphorylated by active IR. This occurs simultaneously with the activation of other molecules of the pathway such as phosphoinositide-dependent kinase-1 (PDK1), IRS-1 and Akt, which is mediated in part by phosphorylation events. (C) Active OGT can now O-GlcNAcylate substrates of the pathway, including IR, IRS-1, Akt and OGT itself. O-GlcNAcylation of IRS-1 occurs in parallel with its phosphorylation at several serine residues that are known to inactivate IRS-1. Comparatively, O-GlcNAcylation of Akt inhibits its (activating) phosphorylation at a threonine residue, thereby inhibiting its kinase activity. Thus, the overall effect of O-GlcNAcylation of insulin signaling molecules leads to attenuation of the pathway.

Fig. 3.

Multifunctional enzymatic complexes regulate the interplay between O-GlcNAc and phosphate in many cellular processes, including the cell cycle. (A) A supra-macromolecular complex formed of OGT, O-GlcNAcase, kinase(s) and phosphatase(s) (PP) regulates the crosstalk between O-GlcNAcylation and phosphorylation of a naked protein, allowing its rapid and dynamic modification by O-GlcNAc, phosphate or both. The possibility of such molecular diversity within the same substrate allows the fine-tuning of protein activity in response to cellular changes. Note that dephosphorylation and removal of O-GlcNAc are catalyzed by the same complex; these events must be tightly regulated to avoid futile cycles. (B) Perturbations of a mitotic complex formed by OGT, O-GlcNAcase, Aurora kinase B and PP1 by adenoviral overexpression of OGT (vOGT) or O-GlcNAcase (vO-GlcNAcase) in HeLa cells results in disruption of cellular and nuclear structure, and in aneuploidy (upper panel), as well as in defective cytokinesis with a mitotic-exit phenotype (lower panel). White arrows point at midbody. This figure has been modified from Slawson et al. (Slawson et al., 2005).

Fig. 4.

Four different types of O-GlcNAc–phosphate crosstalk on protein substrates. (A) Alternative and competitive occupancy at the same amino acid residue, such as in the transactivation domain (TAD) of the transcription factor Myc, which can be either O-GlcNAcylated or phosphorylated at threonine 58. (B) Alternative and reciprocal occupancy at different sites, such as in the regulatory domain (RD) of the transcription factor C/EBPβ, which is either phosphorylated at threonine 179, serine 184 and threonine 188, or O-GlcNAcylated at serines 180 and 181. (C) Simultaneous occupancy at different sites, such as in IRS-1, which is both O-GlcNAcylated at multiple residues in the C-terminal domain (which contains several docking sites for SH2-domain-containing proteins) and phosphorylated at serines 307, 632 and 635 (which are known to attenuate the insulin signaling pathway). (D) Site-dependent reciprocal or simultaneous occupancy, such as in CaMKIV, which exists either as an O-GlcNAc–phospho active protein (at residues threonine 57/serine 58 for O-GlcNAc and threonine 200 for phosphate) or as an O-GlcNAcylated inactive protein (at serine 189). AD, activation domain; CaMBD, calmodulin-binding domain; DBD, DNA-binding domain; HLH, helix-loop-helix domain; PHD, pleckstrin-homology domain; PKD, protein kinase domain; PRD, proline-rich domain; PTBD, phospho-tyrosine-binding domain; SRD, serine-rich domain.