Suchergebnisse

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a typical glycolytic enzyme comprised of four identical 37 kDa subunits. In addition to its glycolytic function, GAPDH has a number of biological functions which are related to its subcellular localization. Generally, protein O-linked N-acetylglucosamine modification (O-GlcNAcylation) is considered, among other effects, to mediate the nuclear transportation of cytosolic proteins. To elucidate the effect of O-GlcNAcylation on GAPDH. we determined the location of the O-GlcNAcylation site by tandem mass spectrometry, and subsequently examined the biological significance of this derivatization. The site involved was identified to be Thr227 by beta-elimination and Michael addition. Transient transfection assays demonstrated that the T227A mutation induced the cytoplasmic accumulation of GAPDH, whereas the wild type was present in the cytoplasm and nuclei. Structural modeling, mutagenesis of Thr227 to Lys and Arg, and gel filtration chromatography of mutated and wild type GAPDH, together suggested that O-GlcNAcylation at Thr227 interrupts the hydrophobic interfaces formed between GAPDH monomers in its tetrameric state. The present study identified Thr227 as the major GAPDH O-GlcNAcylation site, which suggests that this modification mediates the nuclear translocation of GAPDH, presumably by disrupting the conformation of tetrameric GAPDH. (C) 2008 Elsevier B.V. All rights reserved. ; This work was supported by a grant from the Innovative Research Institute for Cell Therapy, Republic of Korea (#A062260) and by a grant from the Korean Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MOST; R01-2006-000-10248-0). ; Barbini L, 2007, MOL CELL BIOCHEM, V300, P19, DOI 10.1007/s11010-006-9341-1 ; Andrali SS, 2007, J BIOL CHEM, V282, P15589, DOI 10.1074/jbc.M701762200 ; Raman P, 2007, J BIOL CHEM, V282, P5704, DOI 10.1074/jbc.M610965200 ; Carujo S, 2006, ONCOGENE, V25, P4033, DOI 10.1038/sj.onc.1209433 ; Kim HS, 2006, FEBS LETT, V580, P2311, DOI 10.1016/j.febslet.2006.03.043 ; Jenkins JL, 2006, ACTA CRYSTALLOGR D, V62, P290, DOI 10.1107/S0907444905042289 ; Majumdar G, 2006, J BIOL CHEM, V281, P3642, DOI 10.1074/jbc.M51123200 ; Bae Byoung-Il, 2006, Proc Natl Acad Sci U S A, V103, P3405, DOI 10.1073/pnas.0511316103 ; Whelan SA, 2006, METHOD ENZYMOL, V415, P113, DOI 10.1016/S0076-6879(06)15008-9 ; Perani M, 2005, J BIOL CHEM, V280, P42863, DOI 10.1074/jbc.M502963200 ; Park J, 2005, J BIOCHEM MOL BIOL, V38, P725 ; Hara MR, 2005, NAT CELL BIOL, V7, P665, DOI 10.1038/ncb1268 ; Sirover MA, 2005, J CELL BIOCHEM, V95, P45, DOI 10.1002/jcb.20399 ; Mazzola JL, 2005, BBA-GEN SUBJECTS, V1722, P168, DOI 10.1016/j.bbagen.2004.12.010 ; Vosseller K, 2005, PROTEOMICS, V5, P388, DOI 10.1002/pmic.200401066 ; Chuang DM, 2005, ANNU REV PHARMACOL, V45, P269, DOI 10.1146/annurev.pharmtox.45.120403.095902 ; Love D. C., 2005, SCI STKE, pre13, DOI DOI 10.1126/STKE.3122005RE13 ; Liu F, 2004, P NATL ACAD SCI USA, V101, P10804, DOI 10.1073/pnas.0400348101 ; Zachara NE, 2004, BBA-GEN SUBJECTS, V1673, P13, DOI 10.1016/j.bbagen.2004.03.016 ; Monsigny M, 2004, BBA-GEN SUBJECTS, V1673, P94, DOI 10.1016/j.bbagen.2004.03.015 ; Gewinner C, 2004, J BIOL CHEM, V279, P3563, DOI 10.1074/jbc.M306449200 ; Zheng L, 2003, CELL, V114, P255, DOI 10.1016/S0092-8674(03)00552-X ; Mazzola JL, 2003, J NEUROSCI RES, V71, P279, DOI 10.1002/jnr.10484 ; Wells L, 2002, MOL CELL PROTEOMICS, V1, P791, DOI 10.1074/mcp.M200048-MCP200 ; Mazzola JL, 2002, NEUROTOXICOLOGY, V23, P603, DOI 10.1016/S0161-813X(02)00062-1 ; Liu K, 2000, P NATL ACAD SCI USA, V97, P2820, DOI 10.1073/pnas.97.6.2820 ; Carlile GW, 2000, MOL PHARMACOL, V57, P2 ; Sirover MA, 1999, BBA-PROTEIN STRUCT M, V1432, P159, DOI 10.1016/S0167-4838(99)00119-3 ; Hanover JA, 1999, ARCH BIOCHEM BIOPHYS, V362, P38, DOI 10.1006/abbi.1998.1016 ; Petersen HV, 1998, FEBS LETT, V431, P362, DOI 10.1016/S0014-5793(98)00776-5 ; Sirover MA, 1997, J CELL BIOCHEM, V66, P133, DOI 10.1002/(SICI)1097-4644(19970801)66:23.3.CO ; 2-V ; Arnold CS, 1996, J BIOL CHEM, V271, P28741 ; Hart GW, 1996, GLYCOBIOLOGY, V6, P711, DOI 10.1093/glycob/6.7.711 ; DUVERGER E, 1995, J CELL SCI, V108, P1325 ; MANSUR NR, 1993, NUCLEIC ACIDS RES, V21, P993, DOI 10.1093/nar/21.4.993 ; ANDREWS NC, 1991, NUCLEIC ACIDS RES, V19, P2499, DOI 10.1093/nar/19.9.2499 ; TORRES CR, 1984, J BIOL CHEM, V259, P3308 ; 6