Interaction between O-GlcNAc Modification and Tyrosine Phosphorylation of Prohibitin: Implication for a Novel Binary Switch

Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.
PLoS ONE (Impact Factor: 3.23). 02/2009; 4(2):e4586. DOI: 10.1371/journal.pone.0004586
Source: PubMed


Prohibitin (PHB or PHB1) is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with O-linked beta-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114) and tyrosine 259 (Tyr259) in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121) and threonine 258 (Thr258) respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s) or known tyrosine phosphorylation site(s) revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.

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Available from: Sudharsana Rao Ande,
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    • "In many ways, O-GlcNAcylation is similar to O-phosphorylation: for instance, like phosphate, N-acetylglucosamine moiety can be attached and removed rapidly in response to internal or environmental changes [4,20,21]; and both O-GlcNAcylation and O-phosphorylation occur on Ser and/or Thr residues, which hints O-GlcNAcylation has a direct competition with O-phosphorylation [1]. Furthermore recent studies have revealed that besides phosphorylation on serine/threonine, also about 68.02% of the O-GlcNAcylated proteins are known to be tyrosine phosphorylated [22-24]. Thus, an increasing number of phosphorylated proteins have been found in mitochondria, and the site-specific interplay between O-phosphorylation and O-GlcNAcylation has been widely recognized. "
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    ABSTRACT: O-linked β-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification (PTM) consisting of a single N-acetylglucosamine moiety attached via an O-β-glycosidic linkage to serine and threonine residues. Glycosylation with O-GlcNAc occurs on myriad nuclear and cytosolic proteins from almost all functional classes. However, with respect to O-GlcNAcylated proteins special in mitochondria, little attention has been paid. In this study, we combined mass spectrometry and immunological methods to perform global exploration of O-GlcNAcylated proteins specific in mitochondria of rat liver. First, highly purified mitochondrial proteins were obviously shown to be O-GlcNAcylated by immunoblot profiling. Then, β-elimination followed by Michael Addition with Dithiothreitol (BEMAD) treatment and LC-MS/MS were performed to enrich and identify O-GlcNAcylated mitochondrial proteins, resulting in an unambiguous assignment of 14 O-GlcNAcylation sites, mapping to 11 O-GlcNAcylated proteins. Furthermore, the identified O-GlcNAcylated mitochondrial proteins were fully validated by both electron transfer dissociation tandem mass spectrometry (ETD/MS/MS) and western blot. Thus, for the first time, our study definitely not only identified but also validated that some mitochondrial proteins in rat liver are O-GlcNAcylated. Interestingly, all of these O-GlcNAcylated mitochondrial proteins are enzymes, the majority of which are involved in a wide variety of biological processes, such as urea cycle, tricarboxylic acid cycle and lipid metabolism, indicating a role for protein O-GlcNAcylation in mitochondrial function.
    PLoS ONE 10/2013; 8(10):e76399. DOI:10.1371/journal.pone.0076399 · 3.23 Impact Factor
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    • "mSin3A and HDAC1 are O-GlcNAcylated in HepG2 liver carcinoma cells, and are recruited to gene loci by the OGT enzyme [35], which also O-GlcNAcylates itself [30]. The activity of OGT is regulated by cellular concentrations of UDP-GlcNAc substrate [36,37], which is increased in the diabetic heart [38]. "
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    ABSTRACT: Exercise causes physiological cardiac hypertrophy and benefits the diabetic heart. Mammalian switch-independent 3A (mSin3A) and histone deacetylases (HDACs) 1 and 2 regulate hypertrophic genes through associations with the DNA binding proteins repressor element-1 silencing transcription factor (REST) and O-linked beta-N-acetylglucosamine transferase (OGT). O-linked beta-N-acetylglucosamine (O-GlcNAc) is a glucose derivative that is chronically elevated in diabetic hearts, and a previous study showed that exercise reduces cardiac O-GlcNAc. We hypothesized that O-GlcNAc and OGT would physically associate with mSin3A/HDAC1/2 in the heart, and that this interaction would be altered by diabetes and exercise. 8-week-old type 2 diabetic db/db (db) and non-diabetic C57 mice were randomized to treadmill exercise or sedentary groups for 1 or 4 weeks. O-GlcNAc was significantly higher in db hearts and increased with exercise. Db hearts showed lower levels of mSin3A, HDAC1, and HDAC2 protein, but higher levels of HDAC2 mRNA and HDAC1/2 deacetylase activity. Elevated HDAC activity was associated with significantly blunted expression of alpha-actin and brain natriuretic peptide in db hearts. In sedentary db hearts, co-immunoprecipitation assays showed that mSin3A and OGT were less associated with HDAC1 and HDAC2, respectively, compared to sedentary C57 controls; however, exercise removed these differences. These data indicate that diabetes and exercise oppositely affect interactions between pro-hypertrophic transcription factors, and suggest that an increase in total cardiac O-GlcNAc is a mechanism by which exercise benefits type 2 diabetic hearts.
    Cardiovascular Diabetology 07/2013; 12(1):101. DOI:10.1186/1475-2840-12-101 · 4.02 Impact Factor
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    • "However, emerging evidence suggests that the relationship between O-GlcNAc modification and serine/threonine phosphorylation is more extensive than initially thought [7,8]. Recently we have reported that tyrosine phosphorylation interacts with O-GlcNAc modification, a phenomenon which was previously not known [9]. Subsequently, two more articles were published showing that O-GlcNAc modification of insulin receptor substrate 1 (IRS1) occurs in close proximity of tyrosine phosphorylation sites and affects the tyrosine phosphorylation dependent function of IRS1 [10,11]. "
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    ABSTRACT: Post-translational modification of proteins at serine and threonine side chains by β-N-acetylglucosamine (O-GlcNAc) mediated by the enzyme β-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68%) than its normal occurrence (~2%) alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.
    Cell Communication and Signaling 01/2011; 9(1):1. DOI:10.1186/1478-811X-9-1 · 3.38 Impact Factor
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