Abstract
The attachment of O-linked β-N-acetylglucosamine (O-GlcNAc) to proteins is an abundant and reversible modification that involves many cellular processes including transcription, translation, cell proliferation, apoptosis, and signal transduction. Here, we found that the O-GlcNAc modification pattern was altered during all-trans retinoic acid (tRA)-induced neurite outgrowth in the MN9D neuronal cell line. We identified several O-GlcNAcylated proteins using mass spectrometric analysis, including α- and β-tubulin. Further analysis of α- and β-tubulin revealed that O-GlcNAcylated peptides mapped between residues 173 and 185 of α-tubulin and between residues 216 and 238 of β-tubulin, respectively. We found that an increase in α-tubulin O-GlcNAcylation reduced heterodimerization and that O-GlcNAcylated tubulin did not polymerize into microtubules. Consequently, when O-GlcNAcase inhibitors were co-incubated with tRA, the extent of neurite outgrowth was decreased by 20% compared to control. Thus, our data indicate that the O-GlcNAcylation of tubulin negatively regulates microtubule formation.
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Abbreviations
- O-GlcNAc:
-
O-Linked β-N-acetylglucosamine
- tRA:
-
All-trans retinoic acid
- PTM:
-
Post-translational modification
- MAP:
-
Microtubule-associated protein
- OGT:
-
O-GlcNAc transferase
- OGA:
-
O-GlcNAcase
- NButGT:
-
1,2-Dideoxy-2′-propyl-α-d-glucopyranoso[2,1-d]-Δ2-thiazoline
- PUGNAc:
-
O-(2-Acetamido-2-deoxy-d-glycopyranosylidene)amino-N-phenylcarbamate
- ESI:
-
Electrospray ionization
- 2-DE:
-
Two-dimensional electrophoresis
- sWGA:
-
Succinylated wheat germ agglutinin
- CBB:
-
Coomassie Brilliant Blue
- IgG HC:
-
Immunoglobulin heavy chain
References
Al-Chalabi A, Miller CC (2003) Neurofilaments and neurological disease. Bioessays 25:346–355
Arnold CS, Johnson GV, Cole RN, Dong DL, Lee M, Hart GW (1996) The microtubule-associated protein tau is extensively modified with O-linked N-acetylglucosamine. J Biol Chem 271:28741–28744
Audebert S, Koulakoff A, Berwald-Netter Y, Gros F, Denoulet P, Edde B (1994) Developmental regulation of polyglutamylated alpha- and beta-tubulin in mouse brain neurons. J Cell Sci 107(Pt 8):2313–2322
Baas PW (1997) Microtubules and axonal growth. Curr Opin Cell Biol 9:29–36
Bhamidipati A, Lewis SA, Cowan NJ (2000) ADP ribosylation factor-like protein 2 (Arl2) regulates the interaction of tubulin-folding cofactor D with native tubulin. J Cell Biol 149:1087–1096
Castro DS, Hermanson E, Joseph B, Wallen A, Aarnisalo P, Heller A, Perlmann T (2001) Induction of cell cycle arrest and morphological differentiation by Nurr1 and retinoids in dopamine MN9D cells. J Biol Chem 276:43277–43284
Cheung WD, Hart GW (2008) AMP-activated protein kinase and p38 MAPK activate O-GlcNAcylation of neuronal proteins during glucose deprivation. J Biol Chem 283:13009–13020
Choi HK, Won LA, Kontur PJ, Hammond DN, Fox AP, Wainer BH, Hoffmann PC, Heller A (1991) Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion. Brain Res 552:67–76
Chou CF, Smith AJ, Omary MB (1992) Characterization and dynamics of O-linked glycosylation of human cytokeratin 8 and 18. J Biol Chem 267:3901–3906
Conde C, Caceres A (2009) Microtubule assembly, organization and dynamics in axons and dendrites. Nat Rev Neurosci 10:319–332
Cowan NJ, Lewis SA (2001) Type II chaperonins, prefoldin, and the tubulin-specific chaperones. Adv Protein Chem 59:73–104
Ding M, Vandre DD (1996) High molecular weight microtubule-associated proteins contain O-linked-N-acetylglucosamine. J Biol Chem 271:12555–12561
Dong DL, Xu ZS, Chevrier MR, Cotter RJ, Cleveland DW, Hart GW (1993) Glycosylation of mammalian neurofilaments. Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M. J Biol Chem 268:16679–16687
Dong DL, Xu ZS, Hart GW, Cleveland DW (1996) Cytoplasmic O-GlcNAc modification of the head domain and the KSP repeat motif of the neurofilament protein neurofilament-H. J Biol Chem 271:20845–20852
Edde B, Rossier J, Le Caer JP, Desbruyeres E, Gros F, Denoulet P (1990) Posttranslational glutamylation of alpha-tubulin. Science 247:83–85
Eom DS, Choi WS, Ji S, Cho JW, Oh YJ (2005) Activation of c-Jun N-terminal kinase is required for neurite outgrowth of dopaminergic neuronal cells. Neuroreport 16:823–828
Fourest-Lieuvin A, Peris L, Gache V, Garcia-Saez I, Juillan-Binard C, Lantez V, Job D (2006) Microtubule regulation in mitosis: tubulin phosphorylation by the cyclin-dependent kinase Cdk1. Mol Biol Cell 17:1041–1050
Gambetta MC, Oktaba K, Muller J (2009) Essential role of the glycosyltransferase sxc/Ogt in polycomb repression. Science (New York, NY) 325:93–96
Gordon-Weeks PR (2004) Microtubules and growth cone function. J Neurobiol 58:70–83
Hammond JW, Cai D, Verhey KJ (2008) Tubulin modifications and their cellular functions. Curr Opin Cell Biol 20:71–76
Hart GW, Housley MP, Slawson C (2007) Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 446:1017–1022
Howard J, Hyman AA (2003) Dynamics and mechanics of the microtubule plus end. Nature 422:753–758
Kang JG, Park SY, Ji S, Jang I, Park S, Kim HS, Kim SM, Yook JI, Park YI, Roth J, Cho JW (2009) O-GlcNAc protein modification in cancer cells increases in response to glucose deprivation through glycogen degradation. J Biol Chem 284:34777–34784
Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC (2004) Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proc Natl Acad Sci USA 101:13132–13137
Ku NO, Omary MB (1994) Expression, glycosylation, and phosphorylation of human keratins 8 and 18 in insect cells. Exp Cell Res 211:24–35
Liu F, Iqbal K, Grundke-Iqbal I, Hart GW, Gong CX (2004) O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer’s disease. Proc Natl Acad Sci USA 101:10804–10809
Love DC, Hanover JA (2005) The hexosamine signaling pathway: deciphering the “O-GlcNAc code”. Sci STKE 2005:re13
Lubas WA, Smith M, Starr CM, Hanover JA (1995) Analysis of nuclear pore protein p62 glycosylation. Biochemistry 34:1686–1694
Mandell JW, Banker GA (1995) The microtubule cytoskeleton and the development of neuronal polarity. Neurobiol Aging 16:229–237 (discussion 238)
Marshall S, Nadeau O, Yamasaki K (2004) Dynamic actions of glucose and glucosamine on hexosamine biosynthesis in isolated adipocytes: differential effects on glucosamine 6-phosphate, UDP-N-acetylglucosamine, and ATP levels. J Biol Chem 279:35313–35319
Minotti AM, Barlow SB, Cabral F (1991) Resistance to antimitotic drugs in Chinese hamster ovary cells correlates with changes in the level of polymerized tubulin. J Biol Chem 266:3987–3994
Redeker V, Levilliers N, Schmitter JM, Le Caer JP, Rossier J, Adoutte A, Bre MH (1994) Polyglycylation of tubulin: a posttranslational modification in axonemal microtubules. Science 266:1688–1691
Slawson C, Zachara NE, Vosseller K, Cheung WD, Lane MD, Hart GW (2005) Perturbations in O-linked beta-N-acetylglucosamine protein modification cause severe defects in mitotic progression and cytokinesis. J Biol Chem 280:32944–32956
Slawson C, Lakshmanan T, Knapp S, Hart GW (2008) A mitotic GlcNAcylation/phosphorylation signaling complex alters the posttranslational state of the cytoskeletal protein vimentin. Mol Biol Cell 19:4130–4140
Tian G, Lewis SA, Feierbach B, Stearns T, Rommelaere H, Ampe C, Cowan NJ (1997) Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors. J Cell Biol 138:821–832
Verhey KJ, Gaertig J (2007) The tubulin code. Cell Cycle 6:2152–2160
Walgren JL, Vincent TS, Schey KL, Buse MG (2003) High glucose and insulin promote O-GlcNAc modification of proteins, including alpha-tubulin. Am J Physiol 284:E424–E434
Wang Y, Tian G, Cowan NJ, Cabral F (2006) Mutations affecting beta-tubulin folding and degradation. J Biol Chem 281:13628–13635
Wang Z, Pandey A, Hart GW (2007) Dynamic interplay between O-linked N-acetylglucosaminylation and glycogen synthase kinase-3-dependent phosphorylation. Mol Cell Proteomics 6:1365–1379
Wells L, Vosseller K, Cole RN, Cronshaw JM, Matunis MJ, Hart GW (2002) Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1:791–804
Westermann S, Weber K (2003) Post-translational modifications regulate microtubule function. Nat Rev Mol Cell Biol 4:938–947
Yang WH, Kim JE, Nam HW, Ju JW, Kim HS, Kim YS, Cho JW (2006) Modification of p53 with O-linked N-acetylglucosamine regulates p53 activity and stability. Nat Cell Biol 8:1074–1083
Yang WH, Park SY, Nam HW, Kim do H, Kang JG, Kang ES, Kim YS, Lee HC, Kim KS, Cho JW (2008) NFkappaB activation is associated with its O-GlcNAcylation state under hyperglycemic conditions. Proc Natl Acad Sci USA 105:17345–17350
Yang WH, Park SY, Ji S, Kang JG, Kim JE, Song H, Mook-Jung I, Choe KM, Cho JW (2010) O-GlcNAcylation regulates hyperglycemia-induced GPX1 activation. Biochem Biophys Res Commun 391:756–761
Zachara NE, O’Donnell N, Cheung WD, Mercer JJ, Marth JD, Hart GW (2004) Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress A survival response of mammalian cells. J Biol Chem 279:30133–30142
Acknowledgments
This work was supported by grants from National Research Foundation funded by the Ministry of Education, Science and Technology Grant (R0A-2007-000-20011-0), and WCU project (R31-2008-000-10086-0). In addition, S.J., S.Y.P. and J.G.K. are fellowship awardees of the Brain Korea 21 program. This work was made possible through the use of research facilities in the Yonsei Center for Biotechnology.
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Ji, S., Kang, J.G., Park, S.Y. et al. O-GlcNAcylation of tubulin inhibits its polymerization. Amino Acids 40, 809–818 (2011). https://doi.org/10.1007/s00726-010-0698-9
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DOI: https://doi.org/10.1007/s00726-010-0698-9