O-GlcNAcylation and Phosphorylation Crosstalk in Vascular Smooth Muscle Cells: Cellular and Therapeutic Significance in Cardiac and Vascular Pathologies

Abstract

More than 400 different types of post-translational modifications (PTMs), including O-GlcNAcylation and phosphorylation, combine to co-ordinate almost all aspects of protein function. Often, these PTMs overlap and the specific relationship between O-GlcNAcylation and phosphorylation has drawn much attention. In the last decade, the significance of this dynamic crosstalk has been linked to several chronic pathologies of cardiovascular origin. However, very little is known about the pathophysiological significance of this crosstalk for vascular smooth muscle cell dysfunction in cardiovascular disease. O-GlcNAcylation occurs on serine and threonine residues which are also targets for phosphorylation. A growing body of research has now emerged linking altered vascular integrity and homeostasis with highly regulated crosstalk between these PTMs. Additionally, a significant body of evidence indicates that O-GlcNAcylation is an important contributor to the pathogenesis of neointimal hyperplasia and vascular restenosis responsible for long-term vein graft failure. In this review, we evaluate the significance of this dynamic crosstalk and its role in cardiovascular pathologies, and the prospects of identifying possible targets for more effective therapeutic interventions.

Keywords: O-GlcNAcylation; crosstalk; phosphorylation; vascular smooth muscle cells.

Figure 1

Schematic of the hexosamine biosynthetic pathway. Glucose is converted to fructose-6P (fructose-6-phosphate) once it enters the HBP. Next, glutamine-fructose-6P amidotransferase 1 (GFAT), the HBP’s rate-limiting enzyme, adds an amino group from glutamine to fructose-6-phosphate to create glucosamine-6-phosphate (GlcN-6-P). Following this, in the presence of acetyl-CoA, glucosamine-phosphate N-acetyltransferase (GNPNAT, EMeg32) quickly acetylates GlcN-6P to form N-acetylglucosamine-6-phosphate (GlcNAc-6P). GlcNAc phosphomutase (PGM3/AGM1) then isomerises this compound to produce N-acetylglucosamine-1-phosphate (GlcNAc-1P). This is then added to the sugar by UDP-N-acetylhexosamine pyrophosphorylase 1 (UAP/AGX1) to yield UDP-GlcNAc which is the amino sugar substrate for protein O-GlcNAcylation. Protein O-GlcNAcylation is a nutrient- and stress-responsive PTM controlled by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). While OGT catalyses the attachment of the O-GlcNAc moiety from a UDP-GlcNAc substrate to target proteins, OGA reverses this process [47]. O-GlcNAcylation occurs on serine and threonine residues of target proteins, sites that may also be targets for phosphorylation by Ser/Thr-directed protein kinases. Glc-6P (Glucose-6-phosphate), Fruc-6P (fructose-6-phosphate), GFAT (glutamine:fructose-6-phosphate amidotransferase), GNA1/GNPNAT1 (glucosamine-6-phosphate N-acetyltransferase), GlcNAc-1P (N-acetylglucosamine-1-phosphate), PGM3/AGM1 (phosphoglucomutase), UDP-GlcNAc (uridine diphosphate-N-acetylglucosamine), S (serine), and T (threonine).

Figure 2

A schematic of how O-GlcNAcylation and phosphorylation crosstalk regulates calmodulin kinase IV (CaMKIV) activity. O-GlcNAcylated and -phosphorylated sites are demonstrated using the CaMKIV structure. In the inactive state, O-GlcNAcylation of the naked protein prevents proximal activating phosphorylation due to O-GlcNAc occupancy of residue sites, which makes the protein dormant. Upon activation, OGA hydrolyses the O-GlcNAc residue, making the phosphorylation sites available for kinase activation. However, there is usually reduced activity when there is shared occupancy of the sites or when phosphorylation of the sites is incomplete. OGT (O-GlcNAc transferase) and OGA (O-GlcNAcase).

Figure 3

Schematic of key mechanisms by which O-GlcNAcylation and phosphorylation crosstalk mediates cardiac and vascular pathologies. A: O-GlcNAcylation inhibits eNOS activity and promotes VSMC migration and proliferation resulting in vascular restenosis and formation of neointimal hyperplasia [7]. O-GlcNAcylation also causes excessive generation of reactive oxygen species (ROS) through NADPH oxidase activation [133]. ROS modulates the activities of miR-200 family of microRNAs [134]. microRNAs play essential role in modulating vascular reactivity [135]. B: Acute increases in O-GlcNAcylation in response to stress have been shown to enhance cell survival and cardio protection [136]. Conversely, in chronic conditions, it inhibits the activity of acetaldehyde dehydrogenase 2 (ALDH2), an important cardioprotective enzyme, which results in exacerbation of myocardial injury [137]. C: O-GlcNAcylation upregulates the expression of extracellular signal-regulated kinase 1/2 (ERK1/2), known mediators of cardiac overload causing cardiac hypertrophy [138]. D: The IL-10 signalling pathway is inhibited by increased O-GlcNAcylation, causing increase in vascular resistance resulting in hypertension. IL-10 is a pleiotropic cytokine that reduces vascular responses to the potent vasoconstrictor endothelin-1 (ET-1) by downregulating the activity of ERK1/2 [139]. E: O-GlcNAcylation promotes proatherogenic genes like thrombospondin and reduces the action of atheroprotective proteins like eNOS, which in turn promotes atherosclerosis and coronary artery disease [140]. F: In response to cellular signal(s), phosphorylation modifies the serine and threonine residues of target proteins to largely limit the effect(s) of O-GlcNAcylation.