DOI QR코드

DOI QR Code

Identification of Potential Substrates of N-acteylglucosamine Kinase by a Proteomic Approach

프로테오믹스를 이용한 N-아세틸글루코사민 인산화효소 기질단백질의 동정

  • Lee, HyunSook (Neuroscience Section, Medical Institute of Dongguk University) ;
  • Moon, Il Soo (Neuroscience Section, Medical Institute of Dongguk University)
  • 이현숙 (동국대학교 의과대학 의학연구소) ;
  • 문일수 (동국대학교 의과대학 의학연구소)
  • Received : 2013.02.23
  • Accepted : 2013.02.28
  • Published : 2013.04.30

Abstract

Post-translational O-GlcNAc modification (O-GlcNAcylation) of serine or threonine is a new protein modulation mechanism. In contrast to the classical glycosylation, O-GlcNAcylation occurs in a one-step transfer of O-GlcNAc on both nuclear and cytoplasmic proteins. In contrast to the general consensus that O-GlcNAc is a final modification, a recent paper (J Proteome Res. 2011 10:2725-2733) showed the presence of O-GlcNAc-P on a synaptic assembly protein AP180. This finding raises a fundamental question about its prevalence. To address this question, we used proteomics to identify those proteins that were phospho-signal enriched by GlcNAc kinase (NAGK). Comparison of pDsRed2-$NAGK_{WT}$-transfected HEK293T cell extract with pDsRed2-$NAGK_{D107A}$-transfected control culture revealed 15 phospho-signal increased spots. Excluding those spots that had no detectable amount of protein expression yielded 7 spots, which were selected for ID determination. Among these, two duplicate spots (two $HSP90{\beta}$ and two ENO1 spots) were shown to be O-GlcNAcylated, two (dUTP nucleotidohydrolase mitochondrial isoform 2, glutathione S-transferase P) were not known to be involved in O-GlcNAcylation, and one (heat shock protein gp96 precursor or grp94) was a glycoprotein. The increase in the phospho-levels of O-GlcNAc by NAGK strongly indicates that these proteins are phosphorylated on O-GlcNAc. Our present data support the idea that O-GlcNAc is not a terminal modification.

단백질 번역 후 O-GlcNAc 수식은 단백질 조절의 새로운 기전으로 대두되고 있다. 전통적인 당수식과 달리 O-GlcNAc 수식은 단 한번의 O-GlcNAc 전달로 이루어지며, 핵 및 세포질단백질 모두에 수식될 수 있다. O-GlcNAc은 이 분자를 끝으로 하는 최종수식으로 생각되어 왔으나, 최근의 논문(J Proteome Res. 2011 10:2725-2733)은 AP180 단백질에 O-GlcNAc-P가 존재함을 보고하였다. 이 논문은 O-GlcNAc-P가 일반적인 단백질수식인지에 대한 중요한 질문을 던진다. 이에 답하고자 저자들은 HEK293T 세포에 O-GlcNAc 인산화효소 NAGK를 DsRed2에 연결한 DsRed2-$NAGK_{WT}$ 혹은 효소활성이 없는 돌연변이 NAGK를 표현하는 DsRed2-$NAGK_{D107A}$를 표현시키고, 단백질 추출물을 얻어 2D-PAGE로 분리한 후 인산화 정도를 측정하여, $NAGK_{WT}$에 의하여 인산화가 증가되는 15개의 단백질 스폿을 선별하였다. 이 가운데 7개 스팟을 동정한 결과 2개의 스폿은 O-GlcNAc 수식 단백질인 $HSP90{\beta}$, 다른 2개의 스폿도 O-GlcNAc 수식 단백질인 ENO1로 동정되었으며, 나머지(dUTP nucleotidohydrolase mitochondrial isoform 2, glutathione S-transferase P, grp94)는 O-GlcNAc 수식 여부를 아직 모르는 단백질이였다. NAGK에 의하여 O-GlcNAc 단백질의 인산화가 증가된다는 사실은 O-GlcNAc이 인산화되어 O-GlcNAc-P로 수식됨을 시사하며, 따라서 본 연구의 결과는 O-GlcNAc이 최종 수식이 아님을 지지한다.

Keywords

References

  1. Asensio, C. and Ruiz-Amil, M. 1966. N-acetyl-D- glucosamine kinase. II. Escherichia coli, Methods Enzymol 9, 421-425. https://doi.org/10.1016/0076-6879(66)09086-4
  2. Blume, A., Berger, M., Benie, A. J., Peters, T. and Hinderlich, S. 2008. Characterization of ligand binding to N-acetylglucosamine kinase studied by STD NMR. Biochem 47, 13138-13146. https://doi.org/10.1021/bi8016894
  3. Copeland, R. J., Bullen, J. W. and Hart, G. W. 2008. Cross-talk between GlcNAcylation and phosphorylation:roles in insulin resistance and glucose toxicity. Am J Physiol Endocrinol Metab 295, E17-E28. https://doi.org/10.1152/ajpendo.90281.2008
  4. Dephoure, N., Zhou, C., Villén, J., Beausoleil, S. A., Bakalarski, C. E., Elledge, S. J. and Gygi, S. P. 2008. A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci USA 105, 10762-10767. https://doi.org/10.1073/pnas.0805139105
  5. Diaz-Ramos, A., Roig-Borrellas, A., Garcia-Melero, A. and Lopez-Alemany, R. 2012. ${\alpha}$-Enolase, a multifunctional protein: its role on pathophysiological situations. J Biomed Biotechnol 2012:156795. doi: 10.1155/2012/156795.
  6. Dong, D. L. and Hart, G. W. 1994. Purification and characterization of an O-GlcNAc selective N-acetyl-beta-D-glucosaminidase from rat spleen cytosol. J Biol Chem 269, 19321-19330.
  7. Fernandez, J., Gharahdaghi, F. and Mische, S. M. 1998. Routine identification of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels or polyvinyl difluoride membranes using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Electrophoresis 19, 1036-1045. https://doi.org/10.1002/elps.1150190619
  8. Gao, Y., Wells, L., Comer, F. I., Parker, G. J. and Hart, G. W. 2001. Dynamic Oglycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic beta-N-acetylglucosaminidase from human brain. J Biol Chem 276, 9838-9845. https://doi.org/10.1074/jbc.M010420200
  9. Graham, M. E, Thaysen-Andersen, M., Bache, N., Craft, G. E., Larsen, M. R, Packer, N, H. and Robinson P. J. 2011. A novel post-translational modification in nerve terminals: O-linked N-acetylglucosamine phosphorylation. J Proteome Res 10, 2725-2733. https://doi.org/10.1021/pr1011153
  10. Hahne, H., Sobotzki, N., Nyberg, T., Helm, D., Borodkin, V. S., van Aalten, D. M., Agnew, B. and Kuster, B. 2013. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927-936. https://doi.org/10.1021/pr300967y
  11. Haltiwanger, R, S., Grove, K. and Philipsberg, G. A. 1998. Modulation of O-linked Nacetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-N-acetylglucosaminidase inhibitor O-(2- acetamido-2-deoxy-D-glucopyranosylidene)amino-Nphenylcarbamate. J Biol Chem 273, 3611-3617. https://doi.org/10.1074/jbc.273.6.3611
  12. Haltiwanger, R. S., Blomberg, M. A. and Hart, G. W. 1992. Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho- N-acetylglucosamine: polypeptide beta-N-acetylglucosaminyltransferase. J Biol Chem 267, 9005-9013.
  13. Hanover, J. A. 2001. Glycan-dependent signaling: O-linked N-acetylglucosamine. FASEB J 15, 1865-1876. https://doi.org/10.1096/fj.01-0094rev
  14. Hart, G. W, Slawson, C., Ramirez-Correa, G. and Lagerlof, O. 2011. Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease. Annu Rev Biochem 80, 825-858. https://doi.org/10.1146/annurev-biochem-060608-102511
  15. Hart, G. W., Housley, M. P. and Slawson, C. 2007. Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 446, 1017-1022. https://doi.org/10.1038/nature05815
  16. Hedou, J., Bastide, B., Page, A., Michalski, J. C. and Morelle, W. 2009. Mapping of O-linked beta-N-acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle. Proteomics 9, 2139-2148. https://doi.org/10.1002/pmic.200800617
  17. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. and Pease, L. R. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51-59. https://doi.org/10.1016/0378-1119(89)90358-2
  18. Kearse, K. P. and Hart, G. W. 1991. Lymphocyte activation induces rapid changes in nuclear and cytoplasmic glycoproteins. Proc Natl Acad Sci USA 88, 1701-1705. https://doi.org/10.1073/pnas.88.5.1701
  19. Khidekel, N., Ficarro, S. B., Clark, P. M., Bryan, M. C., Swaney, D. L., Rexach, J. E., Sun, Y. E., Coon, J. J., Peters, E. C. and Hsieh-Wilson, L. C. 2007. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat Chem Biol 3, 339-348. https://doi.org/10.1038/nchembio881
  20. Kreppel, L. K. and Hart, G. W. 1999. Regulation of a cytosolic and nuclear O-GlcNAc transferase. Role of the tetratricopeptide repeats. J Biol Chem 274, 32015-32022. https://doi.org/10.1074/jbc.274.45.32015
  21. Love, D. C. and Hanover, J. A. 2005. The hexosamine signaling pathway: deciphering the "O-GlcNAc code". Sci. STKE 312, re13.
  22. Marshall, S., Nadeau, O. and 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. https://doi.org/10.1074/jbc.M404133200
  23. Nettelblad, F. A. and Engstrom, L. 1987. The kinetic effects of in vitro phosphorylation of rabbit muscle enolase by protein kinase C. A possible new kind of enzyme regulation. FEBS Lett 214, 249-252. https://doi.org/10.1016/0014-5793(87)80064-9
  24. Overath, T., Kuckelkorn, U., Henklein, P., Strehl, B., Bonar, D., Kloss, A., Siele, D., Kloetzel, P. M. and Janek, K. 2012. Mapping of O-GlcNAc sites of 20S proteasome subunits and Hsp90 by a novel biotin-cystamine tag. Mol Cell Proteomics 11, 467-477. https://doi.org/10.1074/mcp.M111.015966
  25. Rambaruth, N. D., Greenwell, P. and Dwek, M. V. 2012. The lectin Helix pomatia agglutinin recognizes O-GlcNAc containing glycoproteins in human breast cancer. Glycobiol 22, 839-848. https://doi.org/10.1093/glycob/cws051
  26. Torres, C. R. and Hart, G. W. 1984. Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc. J Biol Chem 259, 3308-3317.
  27. Uehara, T. and Park, J. T. 2004. The N-acetyl-D-glucosamine kinase of Escherichia coli and its role in murein recycling. J Bacteriol 186, 7273-7279. https://doi.org/10.1128/JB.186.21.7273-7279.2004
  28. Wang, Z., Park, K., Comer, F., Hsieh-Wilson, L. C., Saudek, C. D. and Hart, G. W. 2009. Site-specific GlcNAcylation of human erythrocyte proteins: potential biomarker(s) for diabetes. Diabetes 58, 309-317. https://doi.org/10.2337/db08-0994
  29. Wells, L., Vosseller, K., Cole, R. N., Cronshaw, J. M., Matunis, M. J. and Hart, G. W. 2002. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791-804. https://doi.org/10.1074/mcp.M200048-MCP200
  30. Zachara, N. E., O'Donnell, N., Cheung, W. D., Mercer, J. J., Marth, J. D. and Hart, G. W. 2004. Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress. A survival response of mammalian cells. J Biol Chem 279, 30133-30142. https://doi.org/10.1074/jbc.M403773200
  31. Zeidan, Q. and Hart, G. W. 2010. The intersections between O-GlcNAcylation and phosphorylation: implications for multiple signaling pathways. J Cell Sci 123, 13-22. https://doi.org/10.1242/jcs.053678