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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.

Acknowledgement

Supported by : National Research Foundation of Korea (NRF)

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