In silico modulation of apoptotic Bcl-2 proteins by mistletoe lectin-1: functional consequences of protein modifications

The mistletoe lectin-1 (ML-1) modulates tumor cell apoptosis by triggering signaling cascades through the complex interplay of phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modification in pro- and anti-apoptotic proteins. In particular, ML-1 is predicted to induce dephosphorylation of Bcl-2-family proteins and their alternative O-GlcNAc modification at specific, conserved Ser/Thr residues. The sites for phosphorylation and glycosylation were predicted and analyzed using Netphos 2.0 and YinOYang 1.2. The involvement of modified Ser/Thr, and among them the potential Yin Yang sites that may undergo both types of posttranslational modification, is proposed to mediate apoptosis modulation by ML-1.     

Epidermal growth factor receptors: function modulation by phosphorylation and glycosylation interplay

Post-translational modifications (PTMs) of proteins induce structural and functional changes that are most often transitory and difficult to follow and investigate in vivo. In silico prediction procedures for PTMs are very valuable to foresee and define such transitory changes responsible for the multifunctionality of proteins. Epidermal growth factor receptor (EGFR) is such a multifunctional transmembrane protein with intrinsic tyrosine kinase activity that is regulated primarily by ligand-stimulated transphosphorylation of dimerized receptors. In human EGFR, potential phosphorylation sites on Ser, Thr and Tyr residues including five autophosphorylation sites on Tyr were investigated using in silico procedures. In addition to phosphorylation, O-GlcNAc modifications and interplay between these two modifications was also predicted. The interplay of phosphorylation and O-GlcNAc modification on same or neighboring Ser/Thr residues is termed as Yin Yang hypothesis and the interplay sites are named as Yin Yang sites. Amongst these modification sites, one residue is localized in the juxtamembrane (Thr 654) and two are found in the catalytic domain (Ser 1046/1047) of the EGFR. We propose that, when EGFR is O-GlcNAc modified on Thr 654, EGFR may be transferred from early to late endosomes, whereas when EGFR is O-GlcNAc modified on Ser 1046/1047 desensitization of the receptor may be prevented. These findings suggest a complex interplay between phosphorylation and O-GlcNAc modification resulting in modulation of EGFR's functionality.     

In silico modulation of HMGN-1 binding to histones and gene expression by interplay of phosphorylation and O-GlcNAc modification

Utilizing different computational methods; phosphorylation, O-GlcNAc modification and Yin Yang sites are predicted in HMGN-1. Prediction results suggest that interplay of phosphorylation and O-GlcNAc modification regulates binding and removal of HMGN-1 with the nucleosome and its translocation from nucleus to cytoplasm and back to nucleus, consequently modulating gene expression.     

Glycosylation Sites Flank Phosphorylation Sites on Synapsin I

Synapsin I is concentrated in nerve terminals, where it appears to anchor synaptic vesicles to the cytoskeleton and thereby ensures a steady supply of fusion-competent synaptic vesicles. Although phosphorylation-dependent binding of synapsin I to cytoskeletal elements and synaptic vesicles is well characterized, little is known about synapsin I's O-linked N-acetylglucosamine (O-GlcNAc) modifications. Here, we identified seven in vivo O-GlcNAcylation sites on synapsin I by analysis of HPLC-purified digests of rat brain synapsin I. The seven O-GlcNAcylation sites (Ser55, Thr56, Thr87, Ser516, Thr524, Thr562, and Ser576) in synapsin I are clustered around its five phosphorylation sites in domains B and D. The proximity of phosphorylation sites to O-GlcNAcylation sites in the regulatory domains of synapsin I suggests that O-GlcNAcylation may modulate phosphorylation and indirectly affect synapsin I interactions. With use of synthetic peptides, however, the presence of an O-GlcNAc at sites Thr562 and Ser576 resulted in only a 66% increase in the Km of calcium/calmodulin-dependent protein kinase II phosphorylation of site Ser566 with no effect on its Vmax. We conclude that O-GlcNAcylation likely plays a more direct role in synapsin I interactions than simply modulating the protein's phosphorylation.     

Identification of the major site of O-linked beta-N-acetylglucosamine modification in the C terminus of insulin receptor substrate-1

Signal transduction from the insulin receptor to downstream effectors is attenuated by phosphorylation at a number of Ser/Thr residues of insulin receptor substrate-1 (IRS-1) resulting in resistance to insulin action, the hallmark of type II diabetes. Ser/Thr residues can also be reversibly glycosylated by O-linked beta-N-acetylglucosamine (O-GlcNAc) monosaccharide, a dynamic posttranslational modification that offers an alternative means of protein regulation to phosphorylation. To identify sites of O-GlcNAc modification in IRS-1, recombinant rat IRS-1 isolated from HEK293 cells was analyzed by two complementary mass spectrometric methods. Using data-dependent neutral loss MS3 mass spectrometry, MS/MS data were scanned for peptides that exhibited a neutral loss corresponding to the mass of N-acetylglucosamine upon dissociation in an ion trap. This methodology provided sequence coverage of 84% of the protein, permitted identification of a novel site of phosphorylation at Thr-1045, and facilitated the detection of an O-GlcNAc-modified peptide of IRS-1 at residues 1027-1073. The level of O-GlcNAc modification of this peptide increased when cells were grown under conditions of high glucose with or without chronic insulin stimulation or in the presence of an inhibitor of the O-GlcNAcase enzyme. To map the exact site of O-GlcNAc modification, IRS-1 peptides were chemically derivatized with dithiothreitol following beta-elimination and Michael addition prior to LC-MS/MS. This approach revealed Ser-1036 as the site of O-GlcNAc modification. Site-directed mutagenesis and Western blotting with an anti-O-GlcNAc antibody suggested that Ser-1036 is the major site of O-GlcNAc modification of IRS-1. Identification of this site will facilitate exploring the biological significance of the O-GlcNAc modification.     

A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer's disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.     

Glycosylation of the murine estrogen receptor-alpha

O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic and abundant modification found on nuclear and cytoplasmic proteins of nearly all eukaryotes. O-GlcNAc addition is required for life at the single cell level and is analogous to protein phosphorylation in most respects. In a previous study (M.S. Jiang, G.W. Hart, A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. J. Biol. Chem. 270 (1997) 2421-2428), we demonstrated that a subpopulation of the murine estrogen receptor-alpha (mER-alpha) is modified by O-GlcNAc at Thr(575). Here we mutated mER-alpha to convert Thr(575) and Ser(576) to Val and Ala, respectively. Surprisingly, this glycosylation-site mutant is still extensively modified by O-GlcNAc. Analyses of glycopeptides identified two additional sites of modification on mER-alpha, at Ser(10) and Thr(50) near the N-terminus. The major glycosylation sites are within or near PEST regions, suggesting that O-GlcNAc may regulate mER-alpha turnover.     

O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic beta cells

O-GlcNAc transferase (OGT)-mediated modification of protein Ser/Thr residues with O-GlcNAc influences protein activity, similar to the effects of phosphorylation. The anti-apoptotic Akt1 is both activated by phosphorylation and modified with O-GlcNAc. However, the nature and significance of the Akt1 O-GlcNAc modification is unknown. The relationship of O-GlcNAc modification and phosphorylation at Akt1 Ser473 was examined with respect to apoptosis of murine beta-pancreatic cells. Glucosamine treatment induced apoptosis, which correlated with enhanced O-GlcNAc modification of Akt1 and concomitant reduction in Ser473 phosphorylation. Pharmacological inhibition of OGT or O-GlcNAcase revealed an inverse correlation between O-GlcNAc modification and Ser473 phosphorylation of Akt1. MALDI-TOF/TOF mass spectrometry analysis of Akt1 immunoprecipitates from glucosamine-treated cells, but not untreated controls, showed a peptide containing S473/T479 that was presumably modified with O-GlcNAc. Furthermore, in vitro O-GlcNAc-modification analysis of wildtype and mutant Akt1 revealed that S473 was targeted by recombinant OGT. A S473A Akt1 mutant demonstrated reduced basal and glucosamine-induced Akt1 O-GlcNAc modification compared with wildtype Akt1. Furthermore, wildtype Akt1, but not the S473A mutant, appeared to be associated with OGT following glucosamine treatment. Together, these observations suggest that Akt1 Ser473 may undergo both phosphorylation and O-GlcNAc modification, and the balance between these may regulate murine beta-pancreatic cell fate.     

A novel post-translational modification in nerve terminals: O-linked N-acetylglucosamine phosphorylation

Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved in the assembly of clathrin coated vesicles in synaptic vesicle endocytosis. Unlike other types of O-glycosylation, O-GlcNAc is nucleocytoplasmic and reversible. It was thought to be a terminal modification, that is, the O-GlcNAc was not found to be additionally modified in any way. We now show that AP180 purified from rat brain contains a phosphorylated O-GlcNAc (O-GlcNAc-P) within a highly conserved sequence. O-GlcNAc or O-GlcNAc-P, but not phosphorylation alone, was found at Thr-310. Analysis of synthetic GlcNAc-6-P produced identical fragmentation products to GlcNAc-P from AP180. Direct O-linkage of GlcNAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification.     

Effect on the Ras/Raf signaling pathway of post‐translational modifications of neurofibromin: In silico study of protein modification responsible for regulatory pathways

Mapping and chemical characterization of post‐translational modifications (PTMs) in proteins are critical to understand the regulatory mechanisms involving modified proteins and their role in disease. Neurofibromatosis type 1 (NF‐1) is an autosomal dominantly inherited disorder, where NF1 mutations usually result in a reduced level of the tumor suppressor protein, neurofibromin (NF). NF is a multifunctional cytoplasmic protein that regulates microtubule dynamics and participates in several signaling pathways, particularly the RAS signaling pathway. NF is a Ras GTPase‐activating protein (GAP) that prevents oncogenesis by converting GTP‐Ras to GDP‐Ras. This function of NF is regulated by phosphorylation. Interplay of phosphorylation with O‐GlcNAc modification on the same or vicinal Ser/Thr residues, the Yin Yang sites, is well known in cytoplasmic and nuclear proteins. The dynamic aspects of PTMs and their interplay being difficult to follow in vivo, we undertook this in silico work to predict and define the possible role of Yin Yang sites in NF‐1. Interplay of phosphorylation and O‐GlcNAc modification is proposed as a mechanism controlling the Ras signaling pathway. J. Cell. Biochem. 108: 816–824, 2009. © 2009 Wiley‐Liss, Inc.     

O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress

Myriad nuclear and cytoplasmic proteins in metazoans are modified on Ser and Thr residues by the monosaccharide O-linked beta-N-acetylglucosamine (O-GlcNAc). The rapid and dynamic change in O-GlcNAc levels in response to extracellular stimuli, morphogens, the cell cycle and development suggests a key role for O-GlcNAc in signal transduction pathways. Modulation of O-GlcNAc levels has profound effects on the functioning of cells, in part mediated through a complex interplay between O-GlcNAc and O-phosphate. In many well-studied proteins, the O-GlcNAc modification and phosphorylation are reciprocal. That is, they occur on different subsets of the protein population, as the site of attachment occurs on the same or adjacent Ser/Thr residues. Recently, O-GlcNAc has been implicated in the etiology of type II diabetes, the regulation of stress response pathways, and in the regulation of the proteasome.     

Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis

Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344). We use protein semisynthesis to show that phosphorylation of Thr344 increases the cellular stability of CK2α by strengthening its interaction with Pin1, whereas glycosylation of Ser347 seems to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with site-specifically phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that the kinase substrate selectivity of CK2 is modulated by these specific post-translational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.     

Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation

Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is generally considered the major regulatory posttranslational modification in eukaryotic cells. Increasing evidence at the genome and proteome level shows that this modification is also present and functional in prokaryotes. We have recently reported the first in-depth phosphorylation site-resolved dataset from the model Gram-positive bacterium, Bacillus subtilis, showing that Ser/Thr/Tyr phosphorylation is also present on many essential bacterial proteins. To test whether this modification is common in Eubacteria, here we use a recently developed proteomics approach based on phosphopeptide enrichment and high accuracy MS to analyze the phosphoproteome of the model Gram-negative bacterium Escherichia coli. We report 81 phosphorylation sites on 79 E. coli proteins, with distribution of Ser/Thr/Tyr phosphorylation sites 68%/23%/9%. Despite their phylogenetic distance, phosphoproteomes of E. coli and B. subtilis show striking similarity in size, classes of phosphorylated proteins, and distribution of Ser/Thr/Tyr phosphorylation sites. By combining the two datasets, we created the largest phosphorylation site-resolved database of bacterial phosphoproteins to date (available at www.phosida.com) and used it to study evolutionary conservation of bacterial phosphoproteins and phosphorylation sites across the phylogenetic tree. We demonstrate that bacterial phosphoproteins and phosphorylated residues are significantly more conserved than their nonphosphorylated counterparts, with a number of potential phosphorylation sites conserved from Archaea to humans. Our results establish Ser/Thr/Tyr phosphorylation as a common posttranslational modification in Eubacteria, present since the onset of cellular life.     

Posttranslational modifications on protein kinase c isozymes. Effects of epinephrine and phorbol esters

The posttranslational modifications induced on PKC isozymes as result of their activation were investigated. Reciprocal immunoprecipitations followed by Western blot analysis demonstrated that all PKC isozymes expressed in rat hepatocytes are modified by tyrosine nitration and tyrosine phosphorylation in different ways upon exposure of cells to a direct PKC activator (TPA), or to an extracellular ligand known to activate PKC-dependent pathways (epinephrine). Our data demonstrate for the first time that all PKC isozymes are also dynamically modified by O-linked beta-N-acetylglucosamine (O-GlcNAc); the presence of this modification was confirmed in part by FT-ICR mass spectrometry analysis. Interestingly, the O-GlcNAc modified Ser or Thr were mapped at similar positions in several PKC isozymes. The biochemical meaning of these posttranslational modifications was investigated for PKC alpha and delta. It was found that the PKC phosphorylation status of both isozymes in tyrosine and serine residues seems to regulate directly the enzyme activity since catalytic inactivation correlate with dephosphorylation of Ser at the C-terminus autophosphorylation sites of each PKC isozyme, and with an increase in the level of tyrosine phosphorylation. Whereas none of the other posttranslational modifications showed per se a direct effect in PKC delta activity, increased tyrosine nitration and O-GlcNAc modifications correlate negatively with PKCalpha activity.     

Identification of phosphorylation sites in native lamina-associated polypeptide 2 beta.

Lamina-associated polypeptide 2 beta (LAP 2 beta), an integral protein of the inner nuclear membrane, appears to be involved in the spatial organization of the interface between nucleoplasma, lamina, and nuclear envelope. Its ability to interact with other proteins and the structural integrity of the nuclear envelope is probably regulated by phosphorylation. Here, we report nonmitotic LAP 2 beta phosphorylation sites that are phosphorylated in the native protein when purified from nuclear envelopes of mouse neuroblastoma Neuro2a cells. Five phosphorylation sites were detected by nano-electrospray mass spectrometric analysis of tryptic LAP 2 beta peptides using parent ion scans specific for phosphopeptides. By mass spectrometric sequencing of these peptides, we identified as phosphorylated residues Thr 74, Thr 159, Ser 176, and Ser 179. Two of the phosphorylation sites, Thr 74 (within a region known to bind chromatin) and Thr 159, are part of consensus sequences of proline-directed kinases. Ser 179 is part of a consensus site for protein kinase C which is able to highly phosphorylate LAP 2 beta in vitro. Three phosphorylation sites, Thr 159, Ser 176, and Ser 179, are located within a stretch of 20 amino acids, thereby forming a highly phosphorylated protein domain which may integrate signaling by multiple protein kinases. Additionally, we identified for the first time at the protein level the LAP 2 splice variant LAP 2 epsilon in nuclear envelopes.     

Phosphorylation and glycosylation interplay: protein modifications at hydroxy amino acids and prediction of signaling functions of the human beta3 integrin family

Protein functions are determined by their three-dimensional structures and the folded 3-D structure is in turn governed by the primary structure and post-translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post-translational modifications, like phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer-assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi-directional (outside-in and inside-out) signaling events. The beta3 integrin family, including, alpha(IIb)beta3 and alpha(v)beta3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the beta3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside-out and outside-in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin-associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay.     

Differential effects of an O-GlcNAcase inhibitor on tau phosphorylation

Abnormal hyperphosphorylation of microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD). The aggregation of hyperphosphorylated tau into neurofibrillary tangles is also a hallmark brain lesion of AD. Tau phosphorylation is regulated by tau kinases, tau phosphatases, and O-GlcNAcylation, a posttranslational modification of proteins on the serine or threonine residues with β-N-acetylglucosamine (GlcNAc). O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase, the enzyme catalyzing the transfer of GlcNAc to proteins, and N-acetylglucosaminidase (OGA), the enzyme catalyzing the removal of GlcNAc from proteins. Thiamet-G is a recently synthesized potent OGA inhibitor, and initial studies suggest it can influence O-GlcNAc levels in the brain, allowing OGA inhibition to be a potential route to altering disease progression in AD. In this study, we injected thiamet-G into the lateral ventricle of mice to increase O-GlcNAcylation of proteins and investigated the resulting effects on site-specific tau phosphorylation. We found that acute thiamet-G treatment led to a decrease in tau phosphorylation at Thr181, Thr212, Ser214, Ser262/Ser356, Ser404 and Ser409, and an increase in tau phosphorylation at Ser199, Ser202, Ser396 and Ser422 in the mouse brain. Investigation of the major tau kinases showed that acute delivery of a high dose of thiamet-G into the brain also led to a marked activation of glycogen synthase kinase-3β (GSK-3β), possibly as a consequence of down-regulation of its upstream regulating kinase, AKT. However, the elevation of tau phosphorylation at the sites above was not observed and GSK-3β was not activated in cultured adult hippocampal progenitor cells or in PC12 cells after thiamet-G treatment. These results suggest that acute high-dose thiamet-G injection can not only directly antagonize tau phosphorylation, but also stimulate GSK-3β activity, with the downstream consequence being site-specific, bi-directional regulation of tau phosphorylation in the mammalian brain.