Heat Shock Protein 90 Regulates the Stability of c-Jun in HEK293 Cells

  • Lu, Chen (Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University) ;
  • Chen, Dan (Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University) ;
  • Zhang, Zhengping (Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University) ;
  • Fang, Fang (Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University) ;
  • Wu, Yifan (Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University) ;
  • Luo, Lan (State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University) ;
  • Yin, Zhimin (Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University)
  • Received : 2007.02.04
  • Accepted : 2007.05.09
  • Published : 2007.10.31

Abstract

The 90-kDa heat shock protein (HSP90) normally functions as a molecular chaperone participating in folding and stabilizing newly synthesized proteins, and refolding denatured proteins. The HSP90 inhibitor geldanamycin (GA) occupies the ATP/ADP binding pocket of HSP90 so inhibits its chaperone activity and causes subsequent degradation of HSP90 client proteins by proteasomes. Here we show that GA reduces the level of endogenous c-Jun in human embryonic kidney 293 (HEK293) cells in a time and dose dependent manner, and that this decrease can be reversed by transfection of HSP90 plasmids. Transfection of HSP90 plasmids in the absence of GA increases the level of endogenous c-Jun protein, but has no obvious affect on c-Jun mRNA levels. We also showed that HSP90 prolongs the half-life of c-Jun by stabilizing the protein; the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocks the degradation of c-Jun promoted by GA. Transfection of HSP90 plasmids did not obviously alter phosphorylation of c-Jun, and a Jun-2 luciferase activity assay indicated that over-expression of HSP90 elevated the total protein activity of c-Jun in HEK293 cells. All our evidence indicates that HSP90 stabilizes c-Jun protein, and so increases the total activity of c-Jun in HEK293 cells.

Keywords

c-Jun;Geldanamycin;Heat Shock Protein 90;HEK293 Cells;Jun-2 Luciferase Reporter;MG132

Acknowledgement

Supported by : National Nature Science Foundation of China, Jiangsu Major Nature Science Foundation of High Education

References

  1. Adler, V., Yin, Z., Fuchs, S. Y., Benezra, M., Rosario, L., et al. (1999) Regulation of JNK signaling by GSTp. EMBO J. 18, 1321−1334
  2. Basso, A. D., Solit, D. B., Chiosis, G., Giri, B., Tsichlis, P., et al. (2002) Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function. J. Biol. Chem. 277, 39858−39866
  3. Feder, M. E. and Hofmann, G. E. (1999) Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61, 243−282
  4. Hoyt, M. A. and Coffino, P. (2004) Ubiquitin-free routes into the proteasome. Cell Mol. Life Sci. 61, 1596−1600
  5. Jariel-Encontre, I., Pariat, M., Martin, F., Carillo, S., Salvat, C., et al. (1995) Ubiquitinylation is not an absolute requirement for degradation of c-Jun protein by the 26 S proteasome. J. Biol. Chem. 270, 11623−11627
  6. Lamph, W.W., Wamsley, P., Sassone-Corsi, P., and Verma, I. M. (1988) Induction of proto-oncogene Jun/AP-1 by serum and TPA. Nature 334, 629−631
  7. Pearl, L. H. and Prodromou, C. (2001) Structure, function and mechanism of the Hsp90 molecular chaperone. Adv. Prot. Chem. 59, 157−186
  8. Picard, D. (2002) Heat-shock protein 90, a chaperone for folding and regulation. Cell Mol. Life Sci. 59, 1640−1648
  9. Schulte, T. W., Akinaga, S., Soga, S., Sullivan, W., Stensgard, B., et al. (1998) Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin. Cell Stress Chaperones 3, 100−108
  10. Smith, D. F. (2000) Chaperones in progesterone receptor complexes. Semin. Cell Dev. Biol. 11, 45−52
  11. Stebbins, C. E., Russo, A. A., Schneider, C., Rosen, N., Hartl, F. U., et al. (1997) Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell 89, 239−250
  12. Treier, M., Staszewski, L. M., and Bohmann, D. (1994) Ubiquitin- dependent c-Jun degradation in vivo is mediated by the delta domain. Cell 78, 787−798
  13. Welch, W. J. and Feramisco, J. R. (1982) Purification of the major mammalian heat shock proteins. J. Biol. Chem. 257, 14949− 14959
  14. Wiech, H., Buchner, J., Zimmermann, R., and Jakob, U. (1992) Hsp90 chaperones protein folding in vitro. Nature 358, 169− 170
  15. Zou, J., Guo, Y., Guettouche, T., Smith, D. F., and Voellmy, R. (1998) Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stresssensitive complex with HSF1. Cell 94, 471−480
  16. Giguere, V., Hollenberg, S. M., Rosenfeld, M. G., and Evans, R. M. (1986) Functional domains of the human glucocorticoid receptor. Cell 46, 645−652
  17. Mayer, M. P. and Bukau, B. (1999) Molecular chaperones: the busy life of Hsp90. Curr. Biol. 9, R322−R325 https://doi.org/10.1016/S0960-9822(99)80142-0
  18. Welch, W. J. (1991) The role of heat-shock proteins as molecular chaperones. Curr. Opin. Cell Biol. 3, 1033−1038
  19. Schneider, C., Sepp-Lorenzino, L., Nimmesgern, E., Ouerfelli, O., Danishefsky, S., et al. (1996) Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90. Proc. Natl. Acad. Sci. USA 93, 14536−14541
  20. Cadepond, F., Schweizer-Groyer, G., Segard-Maurel, I., Jibard, N., Hollen-berg, S. M., et al. (1991) Heat shock protein 90 as a critical factor in maintaining glucocorticosteroid receptor in a nonfunctional state. J. Biol. Chem. 266, 5834−5841
  21. Srivastava, P. (2002) Roles of heat-shock proteins in innate and adaptive immunity. Nat. Rev. Immunol. 2, 185−194
  22. Neckers, L., Mimnaugh, E., and Schulte, T. W. (1999) The Hsp90 chaperone family. Handb. Exp. Pharmacol. 136, 9−42
  23. Pearl, L. H. and Prodromou, C. (2000) Structure and in vivo function of Hsp90. Curr. Opin. Struct. Biol. 10, 46−51
  24. Salvat, C., Aquaviva, C., Jariel-Encontre, I., Ferrara, P., Pariat, M., et al. (1999) Are there multiple proteolytic pathways contributing to c-Fos, c-Jun and p53 protein degradation in vivo? Mol. Biol. Rep. 26, 45−51
  25. Wu, Y. F., Fan, Y. M., Xue, B., Luo, L., Shen, J. Y., et al. (2006) Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2–ASK1 signals. Oncogene 25, 5787− 5800 https://doi.org/10.1038/sj.onc.1209021
  26. Wang, T. Y., Wu, N. H., and Shen, Y. F. (2001) Gene expression changes after heat shock by cDNA array analysis. Acta Academiae Medicinae Sinicae 23, 361−364
  27. Jariel-Encontre, I., Salvat, C., Steff, A. M., Pariat, M., Acquaviva, C., et al. (1997 ) Complex mechanisms for c-fos and c-jun degradation. Mol. Biol. Rep. 24, 51−56
  28. Chen, H. Q., Wu, Y. F., Zhang, Y. Q., Jin, L. N., Luo, L., et al. (2006) Hsp70 inhibits lipopolysaccharide-induced NF-${\kappa}B$ activation by interacting with TRAF6 and inhibiting its ubiquitination. Febs. Lett. 580, 3145−3152
  29. Lindquist, S. and Craig, E. A. (1988) The heat-shock proteins. Annu. Rev. Genet. 22, 631−677
  30. Craig, E. A., Gambill, B. D., and Nelson, R. J. (1993) Heat shock proteins: molecular chaperones of protein biogenesis. Microbiol. Rev. 57, 402−414
  31. Caplan, A. J. (1999) Hsp90's secrets unfold: new insights from structural and functional studies. Trends Cell Biol. 9, 262− 268 https://doi.org/10.1016/S0962-8924(99)01578-0
  32. Musti, A. M., Treier, M., Peverali, F. A., and Bohmann, D. (1996) Differential regulation of c-Jun and JunD by ubiquitindependent protein degradation. J. Biol. Chem. 377, 619−624
  33. Sepp-Lorenzino, L., Ma, Z., Lebwohl, D. E., Vinitsky, A., and Rosen, N. (1995) Herbimycin A induces the 20 S proteasomeand ubiquitin-dependent degradation of receptor tyrosine kinases. J. Biol. Chem. 270, 16580−16587
  34. Marije, M., Vleugel, A. E., Greijer, R. B., Elsken, V. W., and Paul, J. D. (2006) c-Jun activation is associated with proliferation and angiogenesis in invasive breast cancer. Hum. Pathol. 37, 668−674
  35. Pratt, W. B. (1998) The hsp90-based chaperone system: involvement in signal transduction from a variety of hormone and growth factor receptors. Proc. Soc. Exp. Biol Med. 217, 420− 434
  36. Buchner, J. (1999) Hsp90 & Co.-a holding for folding. Trends Biochem. Sci. 24, 136−141
  37. Xu, W., Yu, F., Yan, M., Lu, L., Zou, W., et al. (2004) Geldanamycin, a heat shock protein 90-binding agent, disrupts Stat5 activation in IL-2-stimulated cells. J. Cell. Physiol. 198, 188− 196
  38. Zhao, X., Fan, Y. M., Shen, J. Y., Wu, Y. F., and Yin, Z. M. (2006) Human glutathione S-transferase P1 suppresses MEKK1- mediated apoptosis by regulating MEKK1 kinase activity in HEK293 cells. Mol. Cells 21, 395−400
  39. Prodromou, C., Roe, S. M., O'Brien, R., Ladbury, J. E., Piper, P. W., et al. (1997) Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 90, 65−75
  40. Pratt, W. B. and Toft, D. O. (2003) Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp. Biol. Med. 228, 111−133