Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease

Abstract

O-GlcNAcylation is the addition of β-D-N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. O-linked N-acetylglucosamine (O-GlcNAc) was not discovered until the early 1980s and still remains difficult to detect and quantify. Nonetheless, O-GlcNAc is highly abundant and cycles on proteins with a timescale similar to protein phosphorylation. O-GlcNAc occurs in organisms ranging from some bacteria to protozoans and metazoans, including plants and nematodes up the evolutionary tree to man. O-GlcNAcylation is mostly on nuclear proteins, but it occurs in all intracellular compartments, including mitochondria. Recent glycomic analyses have shown that O-GlcNAcylation has surprisingly extensive cross talk with phosphorylation, where it serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amounts of O-GlcNAcylation underlie the etiology of insulin resistance and glucose toxicity in diabetes, and this type of modification plays a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation. Current data justify extensive efforts toward a better understanding of this invisible, yet abundant, modification. As tools for the study of O-GlcNAc become more facile and available, exponential growth in this area of research will eventually take place.

Figure 1

The hexosamine biosynthetic pathway provides the sugar substrate for O-GlcNAcylation. When glucose enters into the cell, a small percentage is funneled directly into the hexosamine biosynthetic pathway, where it is converted into UDP-N-acetylglucosamine (UDP-GlcNAc). The enzyme O-GlcNAc transferase (OGT) catalyzes the addition of the amino sugar to nuclear and cytoplasmic proteins, whereas the enzyme O-GlcNAcase catalyzes the removal of the sugar. Modified proteins are involved in multiple cellular processes, such as transcription and translation, nutrient sensing, neuronal function, cell cycle, and stress. Acetyl-CoA, acetyl coenzyme A; Fru, fructose; GFAT, glucose:fructose 6-phosphate amidotransferase; Glc, glucose; NAc, N-acetyl; O-GlcNAc, O-linked N-acetylglucosamine.

Figure 2

O-GlcNAcylation and phosphorylation cross talk to regulate protein function. (a) O-linked N-acetylglucosamine (O-GlcNAc) and phosphate can modify the same amino acids reciprocally or amino acids proximal to each other. For example, serine 15 of myosin can be phosphorylated or O-GlcNAcylated. Additionally, serine 19, which is proximal to serine 15, can also be phosphorylated. Each of the modifications can potentially influence each other and alter the rate of addition and turnover of the other modification. (b) Kinases are targets for O-GlcNAcylation. The calcium calmodulin kinase IV (CaMKIV) structure is used to illustrate functions of corresponding CaMKIV-O-GlcNAcylated and -phosphorylated sites. In the inactive state, O-GlcNAc blocks a proximal activating phosphorylation. Upon activation, the O-GlcNAc residue is removed by O-GlcNAcase (OGA), and the phosphorylation site is accessible for kinase activation. (c) O-GlcNAc transferase (OGT) is targeted to substrates by interacting proteins. For this example, the MYPT1-PP1δhuman complex structure is used to illustrate a composite complex with structures of bacterial homologs of OGT and OGA. MYPT1 interacts with OGT and thus potentially targets the latter to myosin light chain for O-GlcNAcylation. MLCK, myosin light chain kinase; VSMC, vascular smooth muscle cell.

Figure 3

Identification of O-linked N-acetylglucosamine (O-GlcNAc) sites is facilitated by photocleavable biotin tagging, combined with collision-assisted dissociation (CAD) and electron transfer dissociation (ETD) mass spectrometry. O-GlcNAc peptides are enriched by first labeling O-GlcNAc groups with N-azidoacetylgalactosamine (GalNAz) using a mutant galactosyltransferase (GalT1), followed by click chemistry addition of a photocleavable biotin tag. Tagged peptides are purified over an avidin column and subsequently are released from the beads by photochemical cleavage. CAD of tagged peptides generates diagnostic ions at mass/charge (m/z) 503.1 and 300.2. ETD enables peptide sequencing and O-GlcNAc site localization. An ETD spectrum of a sample peptide (SISQSISGQK) indicates that Ser1 is modified with a tagged GlcNAc-GalNAz moiety.

Figure 4

O-linked N-acetylglucosamine (O-GlcNAc) is a nutrient sensor and underlies glucose toxicity. Chronic elevation in cellular nutrients (e.g., glucose) increases the glucose shunt into the hexosamine biosynthetic pathway, which elevates the availability of the metabolite substrate UDP-GlcNAc for O-GlcNAc transferase (OGT). Increased flux through this pathway is associated with altered O-GlcNAcylation of key proteins for cellular homeostasis and survival (IRS1, PI3K, Akt, and eNOS), resulting in alterations in multiple cellular pathways. Increased levels of O-GlcNAcylation, particularly on the effectors of the insulin/phosphoinositide signaling pathway, disrupt regulation of their activity by OGT, which results in key pathogenic contributions to glucose toxicity and insulin resistance, the two major hallmarks of diabetes mellitus. Note that proteins are shown as light red squares, and the cellular metabolites are light purple squares. OGT is shown as a green circle and O-GlcNAcase (OGA) as a blue circle. Arrows denote activation, whereas blunt ends denote inhibition.

Figure 5

O-linked N-acetylglucosamine (O-GlcNAc) signaling is crucial for proper neuronal function. (a) O-GlcNAcylation of tubulin is important in maintaining the soluble pool of the protein during cellular rearrangement. (b) O-GlcNAc transferase (OGT) is targeted to kinesin by the vesicle trafficking protein Milton, which regulates axonal transport. (c) Neuronal filament solubility is partially controlled by O-GlcNAcylation. (d ) Long-term potentiation is enhanced by increases in cellular O-GlcNAcylation. (e) At the synapse, many proteins are heavily modified by O-GlcNAc. The tau protein is O-GlcNAcylated in the normal physiological state, but in tau paired-helical tangles, associated with neurodegenerative disease, the tau is no longer O-GlcNAcylated but is heavily phosphorylated. Miro, Bassoon, and Piccolo are proteins.