Animal cells and systems

The Korean Society for Integrative Biology (한국통합생물학회)
권호 표지

Identification of Ku70/Ku80 as ADD1/SREBP1c Interacting Proteins

  • Lee, Yun Sok (School of Biological Sciences, Seoul National University) ;
  • Koh, Hae-Young (Department of Physiology and Biophysics, Mount Sinai School of Medicine) ;
  • Park, Sang Dai (International Vaccine Institute) ;
  • Kim, Jae Bum (School of Biological Sciences, Seoul National University)
  • Published : 2004.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

In vertebrates, multisubunit cofactors regulate gene expression through interacting with cell-type- and gene-specific DNA-binding proteins in a chromatin-selective manner. ADD1/SREBP1c regulates fatty acid metabolism and insulin-dependent gene expression through binding to SRE and E-box motif with dual DNA binding specificity. Although its transcriptional and post-translational regulation has been extensively studied, its regulation by interacting proteins is not well understood. To identify cellular proteins that associate with nuclear form of ADD1/SEBP1c, we employed the GST pull-down system with Hela cell nuclei extract. In this study, we demonstrated that Ku proteins interact specifically with ADD1/SREP1c protein. GST pull-down combined with peptide sequencing analysis revealed that Ku80 binds to ADD1/SREBP1c in vitro. Additionally, western blot analysis showed that Ku70, a heterodimerizing partner of Ku80, also associates with ADD1/SREBP1c. Furthermore, co-transfection of Ku70/Ku80 with ADD1/SREBP1c enhanced the transcriptional activity of ADD1/SREBP1c. Taken together, these results suggest that the Ku proteins might be involved in the lipogenic and/or adipogenic gene expression through interacting with ADD1/SREBP1c.

Keywords

References

  1. Bene H, Lasky D, and Ntambi JM (2001) Cloning and characterization of the human stearyl-CoA desaturase gene promoter: transcriptional activation by sterol regulatory element binding protein and repression by polyunsaturated fatty acids and cholesterol. Biochem Biophys Res Commun 284: 1194-1198 https://doi.org/10.1006/bbrc.2001.5102
  2. Briggs MR, Yokoyama C, Wang X, Brown MS, and Goldstein JL (1993) Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. I. Identification of the protein and delineation of its target nucleotide sequence. J Biol Chem 268: 14490-14496
  3. Brown MS and Goldstein JL (1999) A proteolytic pathway that controls the cholesterol content of membranes, cells and blood. Proc Natl Acad Sci USA 96: 11041-11048 https://doi.org/10.1073/pnas.96.20.11041
  4. deVries E, Driel WV, Bergsma WG, Arnberg AC, and van der Vliet PC (1989) Hela nuclear protein recognizing DNA termini and translocating on DNA forming a regular DNA forming a regular DNA multimeric protein complex. J Mol Biol 208: 65-78 https://doi.org/10.1016/0022-2836(89)90088-0
  5. Dignam JD, Martin PL, Shastry BS, and Roeder RG (1983a) Eukaryotic gene transcription with purified components. Methods Enzymol 101: 582-598 https://doi.org/10.1016/0076-6879(83)01039-3
  6. Dignam JD, Lebovitz RM, and Roeder RG (1983b) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acid Res 11: 1475-1489 https://doi.org/10.1093/nar/11.5.1475
  7. Dooley KA, Millinder S, and Osborne TF (1998) Sterol regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene through a direct interaction between sterol regulatory element binding protein and the trimeric CCAAT-binding factor/nuclear factor Y. J Biol Chem 273: 1349-1356 https://doi.org/10.1074/jbc.273.3.1349
  8. Dvir A, Peterson SR, Kunth MW, Lu H, and Dyan WS (1992) Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II. Proc Natl Acad Sci USA 89: 11920-11924 https://doi.org/10.1073/pnas.89.24.11920
  9. Falzon M, Fewell JW, and Kuff EL (1993) EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single to double-strand transitions in DNA. J Biol Chem 268: 10546-10552
  10. Falzon M and Kuff EL (1989) Isolation and characterization of a protein fracion that binds to enhancer core sequences in intracisternal A particle long terminal repeats. J Biol Chem 264: 21915-21922
  11. Giffin W, Torrance H, Rodda DJ, Prefontaine GG, Pope L, and Hache RJG (1996) Sequence-specific DNA binding by Ku autoantigen and its effects on transcription. Nature 380: 265-268 https://doi.org/10.1038/380265a0
  12. Gottlieb TM and Jackson SP (1993) The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 72: 131-142 https://doi.org/10.1016/0092-8674(93)90057-W
  13. Horton JD, Goldstein JL, and Brown MS (2002) SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109: 1125-1131 https://doi.org/10.1172/JCI200215593
  14. Hua X, Nohturfft A, Goldstein JL, and Brown MS (1996) Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein. Cell 87: 415-426 https://doi.org/10.1016/S0092-8674(00)81362-8
  15. Inoue J, Kumagai H, Terada T, Maeda M, Shimizu M, and Sato R (2001) Proteolytic activation of SREBPs during adipocyte differentiation. Biochem Biophys Res Commun 283: 1157-1161 https://doi.org/10.1006/bbrc.2001.4915
  16. Ito M, Yuan C, Okano HJ, Darnell RB, and Roeder RG (2000) Involvement of the TRAP220 component of the TRAP/SMCC coactivator complex in embryonic development and thyroid hormone action. Mol Cell 5: 683-693 https://doi.org/10.1016/S1097-2765(00)80247-6
  17. Kim D, Ouyang H, Yang SH, Nussenzweig A, Burgman P, and Li GC (1995) A constitutive heat shock element-binding factor is immunologically identical to the Ku autoantigen. J Biol Chem 270: 15277-15284 https://doi.org/10.1074/jbc.270.25.15277
  18. Kim JB and Spiegelman BM (1996) ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. Genes Dev 10: 1096-1107 https://doi.org/10.1101/gad.10.9.1096
  19. Kim JB, Wright HM, Wright M, and Spiegelman BM (1998a) ADD1/SREBP1 activates PPARg through the production of endogenous ligand. Proc Natl Acad Sci USA 95: 4333-4337 https://doi.org/10.1073/pnas.95.8.4333
  20. Kim JB, Sarraf P, Wright M, Yao KM, Mueller E, Solanes G, Lowell BB, and Spiegelman BM (1998b) Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1. J Clin Invest 101: 1-9 https://doi.org/10.1172/JCI1411
  21. Knuth MW, Gunderson SI, Thompson NE, Strasheim LA, and Burgess RR (1990) Purification and characterization of proximal sequence element-binding protein I, a transcription activating protein related to Ku and TREF that binds the proximal sequence element of the human U1 promoter. J Biol Chem 265: 17911-17920
  22. Lee YS, Lee HH, Park J, Yoo EJ, Glackin CA, Choi YI, Jeon SH, Seong RH, Park SD, and Kim JB (2003) Twist2, a novel ADD1/SREBP1c interacting protein, represses the transcriptional activity of ADD1/SREBP1c. Nucleic Acids Res 31: 7165-7174 https://doi.org/10.1093/nar/gkg934
  23. Lees-Miller SP, Chen YR, and Anderson CW (1990) Human cells contain a DNA-activated protein kinase that phosphorylates Simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Mol Cell Biol 10: 6472-6481 https://doi.org/10.1128/MCB.10.12.6472
  24. Liang G, Yang J, Horton JD, Hammer RE, Goldstein JL, and Brown MS (2002) Diminished hepatic response to fasting/refeeding and liver X receptor agonists in mice with selective deficiency of sterol regulatory element-binding protein-1c. J Biol Chem 277: 9520-9528 https://doi.org/10.1074/jbc.M111421200
  25. Lim JW, Kim H, and Kim KH (2004) The Ku antigen-recombination signal-binding protein J{${\kappa}$} complex binds to the nuclear factor-{${\kappa}$}B p50 promoter and acts as a positive regulator of p50 expression in human gastric cancer cells. J Biol Chem 279: 231-237 https://doi.org/10.1074/jbc.M308609200
  26. Lopez JM, Bennette MK, Sanchez HB, Rosenfeld JM, and Osborne TE (1996) Sterol regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate control of cellular lipid. Proc Natl Acad Sci USA 93: 1049-1053 https://doi.org/10.1073/pnas.93.3.1049
  27. Magana MM and Osborne TF (1996) Two tandem binding sites for sterol regulatory element binding proteins are required for sterol regulation of fatty-acid synthase promoter. J Biol Chem 271: 32689-32694 https://doi.org/10.1074/jbc.271.51.32689
  28. Magana MM, Koo SH, Towle HC, and Osborne TF (2000) Different sterol regulatory element-binding protein-1 isoforms utilize distinct co-regulatory factors to activate the promoter for fatty acid synthase. J Biol Chem 275: 4726-4733 https://doi.org/10.1074/jbc.275.7.4726
  29. Naar AM, Beaurang PA, Robinson KM, Oliner JD, Avizonis D, Scheek S, Zwicker J, Kadonaga JT, and Tjian R (1998) Chromatin, TAFs, and a novel multiprotein coactivator are required for synergistic activation by Sp1 and SREBP-1a in vitro. Genes Dev 12: 3020-3031 https://doi.org/10.1101/gad.12.19.3020
  30. Naar AM, Beaurang PA, Zhou S, Abraham S, Solomon W, and Tjian R (1999) Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398: 828-832 https://doi.org/10.1038/19789
  31. Quinn JB and Farira AR (1991) Autoimmune antigen Ku is enriched on oligonucleotide columns distinct from those containing the octamer binding protein DNA consensus sequence. FEBS Lett 286: 225-228 https://doi.org/10.1016/0014-5793(91)80979-D
  32. Rachez C, Lemon BD, Suldan Z, Bromleigh V, Gamble M, Naar AM, Erdjument-Bromage H, Tempst P, and Freedman LP (1999) Ligand-dependent activation by nuclear receptors requires the DRIP complex. Nature 398: 824-828 https://doi.org/10.1038/19783
  33. Sanchez HB, Yieh L, and Osborne TF (1995) Cooperation by sterol regulatory element-binding protein and Sp1 in sterol regulation of low density lipoprotein receptor gene. J Biol Chem 270: 1161-1169 https://doi.org/10.1074/jbc.270.3.1161
  34. Sato R, Yang J, Wang X, Evans MJ, Ho YK, Goldstein JL, and Brown MS (1994) Assignment of the membrane attachment, DNA binding, and transcriptional activation domains of sterol regulatory element-binding protein-1 (SREBP-1). J Biol Chem 269: 17267-17273
  35. Shimano H, Horton JD, Shimomura I, Hammer RE, Brown MS, and Goldstein JL (1997a) Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells. J Clin Invest 99: 846-854 https://doi.org/10.1172/JCI119248
  36. Shimano H, Shimomura I, Hammer RE, Herz J, Goldstein JL, Brown MS, and Horton JD (1997b) Elevated levels of SREBP-2 and cholesterol synthesis in livers of mice homozygous for a targeted disruption of the SREBP-1 gene. J Clin Invest 100: 2115-2124 https://doi.org/10.1172/JCI119746
  37. Shimomura I, Shimano H, Korn BS, Bashmakov Y, and Horton JD (1998a) Nuclear sterol regulatory element-binding proteins activate genes responsible for the entire program of unsaturated fatty acid biogenesis in transgenic mouse liver. J Biol Chem 273: 35299-35306 https://doi.org/10.1074/jbc.273.52.35299
  38. Shimomura I, Hammer RE, Richardson JA, Ikemoto S, Bashmakov Y, Goldstein JL, and Brown MS (1998b) Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy. Genes Dev 12: 3182-3194 https://doi.org/10.1101/gad.12.20.3182
  39. Tontonoz P, Kim JB, Graves RA, and Spiegelman BM (1993) ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation. Mol Cell Biol 13: 4753-4759 https://doi.org/10.1128/MCB.13.8.4753
  40. Tuteja R and Tuteja N (2000) Ku autoantigen: a multifunctional DNA-binding protein. Crit Rev Biochem Mol Biol 35: 1-33 https://doi.org/10.1080/10409230091169177
  41. Wang G, Caltin GT, Stevens JL, and Berk AJ (2001) Characterization of mediator complexes from Hela cell nuclear extract. Mol Cell Biol 21: 4604-4613 https://doi.org/10.1128/MCB.21.14.4604-4613.2001
  42. Wang X, Briggs MR, Hua X, Yokoyama C, Goldstein JL, and Brown MS (1993) Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization. J Biol Chem 268: 14497-14504
  43. Wang X, Sato R, Brown MS, Hua X, and Goldstein JL (1994) SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell 77: 53-62 https://doi.org/10.1016/0092-8674(94)90234-8
  44. Yabe D, Brown MS, and Goldstein JL (2002) Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins. Proc Natl Acad Sci USA 99: 12753-12758 https://doi.org/10.1073/pnas.162488899
  45. Yabe D, Komuro R, Liang G, Goldstein JL, and Brown MS (2003) Liver-specific mRNA for Insig-2 down-regulated by insulin: implications for fatty acid synthesis. Proc Natl Acad Sci USA 100: 3155-3160 https://doi.org/10.1073/pnas.0130116100
  46. Yang T, Espenshade PJ, Wright ME, Yabe D, Gong Y, Aebersold R, Goldstein JL, and Brown MS (2002) Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell 110: 489-500 https://doi.org/10.1016/S0092-8674(02)00872-3