Cell Stress and Chaperones

, Volume 14, Issue 3, pp 253–263 | Cite as

Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis

Original Paper

Abstract

In addition to its role as a molecular chaperone, heat shock protein 72 (Hsp72) protects cells against a wide range of apoptosis inducing stresses. However, it is unclear if these two roles are functionally related or whether Hsp72 inhibits apoptosis by a mechanism independent of chaperone activity. The N-terminal adenosine triphosphatase domain, substrate-binding domain and the C-terminal EEVD regulatory motif of Hsp72 are all essential for chaperone activity. In this study, we show that Hsp72 mutants with a functional substrate-binding domain but lacking chaperone activity retain their ability to protect cells against apoptosis induced by heat and tumor necrosis factor alpha. In contrast, a deletion mutant lacking a functional substrate-binding domain has no protective capacity. The ability of the Hsp72 substrate-binding domain to inhibit apoptosis independent of the regulatory effects of the adenosine triphosphate-binding domain indicates that the inhibition of apoptosis may involve a stable binding interaction with a regulatory substrate rather than Hsp72 chaperone activity.

Keywords

Hsp72 Functional analysis Apoptosis Chaperone activity 

Notes

Acknowledgments

This study was supported by a grant from the NIH/NCI CA81421. The authors wish to acknowledge the kind gifts of reagents from Dr. R. Morimoto, Dr. Lois Greene, Dr. C. Hunt, Dr. S. Jane, Dr. W. Welch, Dr. P. Darcy, and Dr. N. Hoogenraad and technical assistance from Ms. C. Restall.

References

  1. Agashe VR, Hartl FU (2000) Roles of molecular chaperones in cytoplasmic protein folding. Semin Cell Dev Biol 11:15–25 doi:10.1006/scdb.1999.0347 PubMedCrossRefGoogle Scholar
  2. Ahn JH, Ko YG, Park WY, Kang YS, Chung HY, Seo JS (1999) Suppression of ceramide-mediated apoptosis by HSP70. Mol Cells 9:200–206PubMedGoogle Scholar
  3. Beckmann RP, Mizzen LE, Welch WJ (1990) Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly. Science 248:850–854 doi:10.1126/science.2188360 PubMedCrossRefGoogle Scholar
  4. Beere HM (2005) Death versus survival: functional interaction between the apoptotic and stress-inducible heat shock protein pathways. J Clin Invest 115:2633–2639 doi:10.1172/JCI26471 PubMedCrossRefGoogle Scholar
  5. Boice JA, Hightower LE (1997) A mutational study of the peptide-binding domain of Hsc70 guided by secondary structure prediction. J Biol Chem 272:24825–24831 doi:10.1074/jbc.272.40.24825 PubMedCrossRefGoogle Scholar
  6. Brinker A, Scheufler C, Von Der Mulbe F, Fleckenstein B, Herrmann C et al (2002) Ligand discrimination by TPR domains. Relevance and selectivity of EEVD-recognition in Hsp70 x Hop x Hsp90 complexes. J Biol Chem 277:19265–19275 doi:10.1074/jbc.M109002200 PubMedCrossRefGoogle Scholar
  7. Buzzard KA, Giaccia AJ, Killender M, Anderson RL (1998) Heat shock protein 72 modulates pathways of stress-induced apoptosis. J Biol Chem 273:17147–17153 doi:10.1074/jbc.273.27.17147 PubMedCrossRefGoogle Scholar
  8. Chappell TG, Welch WJ, Schlossman DM, Palter KB, Schlesinger MJ, Rothman JE (1986) Uncoating ATPase is a member of the 70 kilodalton family of stress proteins. Cell 45:3–13 doi:10.1016/0092-8674(86)90532-5 PubMedCrossRefGoogle Scholar
  9. Ciocca DR, Calderwood SK (2005) Heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications. Cell Stress Chaperones 10:86–103 doi:10.1379/CSC-99r.1 PubMedCrossRefGoogle Scholar
  10. Craig EA (1985) The heat shock response. CRC Crit Rev Biochem 18:239–80 doi:10.3109/10409238509085135 PubMedCrossRefGoogle Scholar
  11. De Los Rios P, Ben-Zvi A, Slutsky O, Azem A, Goloubinoff P (2006) Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling. Proc Natl Acad Sci U S A 103:6166–71 doi:10.1073/pnas.0510496103 PubMedCrossRefGoogle Scholar
  12. Demidenko ZN, Vivo C, Halicka HD, Li CJ, Bhalla K et al (2006) Pharmacological induction of Hsp70 protects apoptosis-prone cells from doxorubicin: comparison with caspase-inhibitor- and cycle-arrest-mediated cytoprotection. Cell Death Differ 13:1434–1441 doi:10.1038/sj.cdd.4401812 PubMedCrossRefGoogle Scholar
  13. Dice JF (2007) Chaperone-mediated autophagy. Autophagy 3:295–299PubMedGoogle Scholar
  14. Diehl JA, Yang W, Rimerman RA, Xiao H, Emili A (2003) Hsc70 regulates accumulation of cyclin D1 and cyclin D1-dependent protein kinase. Mol Cell Biol 23:1764–1774 doi:10.1128/MCB.23.5.1764-1774.2003 PubMedCrossRefGoogle Scholar
  15. Dittmar KD, Pratt WB (1997) Folding of the glucocorticoid receptor by the reconstituted Hsp90-based chaperone machinery. The initial hsp90.p60.hsp70-dependent step is sufficient for creating the steroid binding conformation. J Biol Chem 272:13047–13054 doi:10.1074/jbc.272.20.13047 PubMedCrossRefGoogle Scholar
  16. Dressel R, Grzeszik C, Kreiss M, Lindemann D, Herrmann T et al (2003) Differential effect of acute and permanent heat shock protein 70 overexpression in tumor cells on lysability by cytotoxic T lymphocytes. Cancer Res 63:8212–8220PubMedGoogle Scholar
  17. Esser C, Alberti S, Hohfeld J (2004) Cooperation of molecular chaperones with the ubiquitin/proteasome system. Biochim Biophys Acta 1695:171–188 doi:10.1016/j.bbamcr.2004.09.020 PubMedCrossRefGoogle Scholar
  18. Fourie AM, Hupp TR, Lane DP, Sang BC, Barbosa MS et al (1997) HSP70 binding sites in the tumor suppressor protein p53. J Biol Chem 272:19471–19479 doi:10.1074/jbc.272.31.19471 PubMedCrossRefGoogle Scholar
  19. Freeman BC, Myers MP, Schumacher R, Morimoto RI (1995) Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1. EMBO J 14:2281–2292PubMedGoogle Scholar
  20. Frydman J, Nimmesgern E, Ohtsuka K, Hartl FU (1994) Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature 370:111–117 doi:10.1038/370111a0 PubMedCrossRefGoogle Scholar
  21. Gabai VL, Meriin AB, Mosser DD, Caron AW, Rits S et al (1997) Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance. J Biol Chem 272:18033–18037 doi:10.1074/jbc.272.29.18033 PubMedCrossRefGoogle Scholar
  22. Gabai VL, Budagova KR, Sherman MY (2005) Increased expression of the major heat shock protein Hsp72 in human prostate carcinoma cells is dispensable for their viability but confers resistance to a variety of anticancer agents. Oncogene 24:3328–3338 doi:10.1038/sj.onc.1208495 PubMedCrossRefGoogle Scholar
  23. Gotoh T, Terada K, Oyadomari S, Mori M (2004) hsp70–DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria. Cell Death Differ 11:390–402 doi:10.1038/sj.cdd.4401369 PubMedCrossRefGoogle Scholar
  24. Herbst R, Schafer U, Seckler R (1997) Equilibrium intermediates in the reversible unfolding of firefly (Photinus pyralis) luciferase. J Biol Chem 272:7099–7105 doi:10.1074/jbc.272.11.7099 PubMedCrossRefGoogle Scholar
  25. Jaattela M, Wissing D, Bauer PA, Li GC (1992) Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity. EMBO J 11:3507–3512PubMedGoogle Scholar
  26. Kang PJ, Ostermann J, Shilling J, Neupert W, Craig EA, Pfanner N (1990) Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature 348:137–143 doi:10.1038/348137a0 PubMedCrossRefGoogle Scholar
  27. Kobayashi Y, Kume A, Li M, Doyu M, Hata M et al (2000) Chaperones Hsp70 and Hsp40 suppress aggregate formation and apoptosis in cultured neuronal cells expressing truncated androgen receptor protein with expanded polyglutamine tract. J Biol Chem 275:8772–8778 doi:10.1074/jbc.275.12.8772 PubMedCrossRefGoogle Scholar
  28. Kosano H, Stensgard B, Charlesworth MC, McMahon N, Toft D (1998) The assembly of progesterone receptor-hsp90 complexes using purified proteins. J Biol Chem 273:32973–32979 doi:10.1074/jbc.273.49.32973 PubMedCrossRefGoogle Scholar
  29. Lee YJ, Dewey WC (1987) Effect of cycloheximide or puromycin on induction of thermotolerance by heat in Chinese hamster ovary cells: dose fractionation at 45.5 degrees C1. Cancer Res 47:5960–5966PubMedGoogle Scholar
  30. Lelekakis M, Moseley JM, Martin TJ, Hards D, Williams E, Ho P, Lowen D, Javni J, Miller FR, Slavin J, Anderson RL (1999) A novel orthotopic model of breast cancer metastasis to bone. Clin Exp Metastasis 17:163–170 doi:10.1023/A:1006689719505 PubMedCrossRefGoogle Scholar
  31. Lepock JR, Frey HE, Ritchie KP (1993) Protein denaturation in intact hepatocytes and isolated cellular organelles during heat shock. J Cell Biol 122:1267–1276 doi:10.1083/jcb.122.6.1267 PubMedCrossRefGoogle Scholar
  32. Li GC, Li L, Liu RY, Rehman M, Lee WM (1992) Heat shock protein hsp70 protects cells from thermal stress even after deletion of its ATP-binding domain. Proc Natl Acad Sci U S A 89:2036–2040 doi:10.1073/pnas.89.6.2036 PubMedCrossRefGoogle Scholar
  33. Li L, Shen G, Li GC (1995) Effects of expressing human Hsp70 and its deletion derivatives on heat killing and on RNA and protein synthesis. Exp Cell Res 217:460–468 doi:10.1006/excr.1995.1110 PubMedCrossRefGoogle Scholar
  34. Mayer MP, Bukau B (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 62:670–684 doi:10.1007/s00018-004-4464-6 PubMedCrossRefGoogle Scholar
  35. Minton KW, Karmin P, Hahn GM, Minton AP (1982) Nonspecific stabilization of stress-susceptible proteins by stress-resistant proteins: a model for the biological role of heat shock proteins. Proc Natl Acad Sci U S A 79:7107–7111 doi:10.1073/pnas.79.23.7107 PubMedCrossRefGoogle Scholar
  36. Morshauser RC, Hu W, Wang H, Pang Y, Flynn GC, Zuiderweg ER (1999) High-resolution solution structure of the 18 kDa substrate-binding domain of the mammalian chaperone protein Hsc70. J Mol Biol 289:1387–1403 doi:10.1006/jmbi.1999.2776 PubMedCrossRefGoogle Scholar
  37. Mosser DD, Caron AW, Bourget L, Denis-Larose C, Massie B (1997) Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol Cell Biol 17:5317–5327PubMedGoogle Scholar
  38. Mosser DD, Caron AW, Bourget L, Meriin AB, Sherman MY et al (2000) The chaperone function of hsp70 is required for protection against stress-induced apoptosis. Mol Cell Biol 20:7146–7159 doi:10.1128/MCB.20.19.7146-7159.2000 PubMedCrossRefGoogle Scholar
  39. Nollen EA, Brunsting JF, Roelofsen H, Weber LA, Kampinga HH (1999) In vivo chaperone activity of heat shock protein 70 and thermotolerance. Mol Cell Biol 19:2069–2079PubMedGoogle Scholar
  40. Nylandsted J, Brand K, Jaattela M (2000) Heat shock protein 70 is required for the survival of cancer cells. Ann N Y Acad Sci 926:122–125PubMedCrossRefGoogle Scholar
  41. Nylandsted J, Gyrd-Hansen M, Danielewicz A, Fehrenbacher N, Lademann U et al (2004) Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization. J Exp Med 200:425–435 doi:10.1084/jem.20040531 PubMedCrossRefGoogle Scholar
  42. Palleros DR, Welch WJ, Fink AL (1991) Interaction of hsp70 with unfolded proteins: effects of temperature and nucleotides on the kinetics of binding. Proc Natl Acad Sci U S A 88:5719–5723 doi:10.1073/pnas.88.13.5719 PubMedCrossRefGoogle Scholar
  43. Prasad K, Heuser J, Eisenberg E, Greene L (1994) Complex formation between clathrin and uncoating ATPase. J Biol Chem 269:6931–6939PubMedGoogle Scholar
  44. Rajapandi T, Wu C, Eisenberg E, Greene L (1998) Characterization of D10S and K71E mutants of human cytosolic hsp70. Biochemistry 37:7244–7250 doi:10.1021/bi972252r PubMedCrossRefGoogle Scholar
  45. Ruchalski K, Mao H, Li Z, Wang Z, Gillers S et al (2006) Distinct hsp70 domains mediate apoptosis-inducing factor release and nuclear accumulation. J Biol Chem 281:7873–7880 doi:10.1074/jbc.M513728200 PubMedCrossRefGoogle Scholar
  46. Schumacher RJ, Hurst R, Sullivan WP, McMahon NJ, Toft DO, Matts RL (1994) ATP-dependent chaperoning activity of reticulocyte lysate. J Biol Chem 269:9493–9499PubMedGoogle Scholar
  47. Simon MM, Reikerstorfer A, Schwarz A, Krone C, Luger TA et al (1995) Heat shock protein 70 overexpression affects the response to ultraviolet light in murine fibroblasts. Evidence for increased cell viability and suppression of cytokine release. J Clin Invest 95:926–933 doi:10.1172/JCI117800 PubMedCrossRefGoogle Scholar
  48. Sliutz G, Karlseder J, Tempfer C, Orel L, Holzer G, Simon MM (1996) Drug resistance against gemcitabine and topotecan mediated by constitutive hsp70 overexpression in vitro: implication of quercetin as sensitiser in chemotherapy. Br J Cancer 74:172–177PubMedGoogle Scholar
  49. Sondermann H, Scheufler C, Schneider C, Hohfeld J, Hartl FU, Moarefi I (2001) Structure of a Bag/Hsc70 complex: convergent functional evolution of Hsp70 nucleotide exchange factors. Science 291:1553–1557 doi:10.1126/science.1057268 PubMedCrossRefGoogle Scholar
  50. Stankiewicz AR, Lachapelle G, Foo CP, Radicioni SM, Mosser DD (2005) Hsp70 inhibits heat-induced apoptosis upstream of mitochondria by preventing Bax translocation. J Biol Chem 280:38729–38739 doi:10.1074/jbc.M509497200 PubMedCrossRefGoogle Scholar
  51. Steel R, Doherty JP, Buzzard K, Clemons N, Hawkins CJ, Anderson RL (2004) Hsp72 inhibits apoptosis upstream of the mitochondria and not through interactions with Apaf-1. J Biol Chem 279:51490–51499 doi:10.1074/jbc.M401314200 PubMedCrossRefGoogle Scholar
  52. Sun Y, Ouyang YB, Xu L, Chow AM, Anderson R et al (2006) The carboxyl-terminal domain of inducible Hsp70 protects from ischemic injury in vivo and in vitro. J Cereb Blood Flow Metab 26:937–950 doi:10.1038/sj.jcbfm.9600246 PubMedCrossRefGoogle Scholar
  53. Volloch V, Gabai VL, Rits S, Sherman MY (1999) ATPase activity of the heat shock protein hsp72 is dispensable for its effects on dephosphorylation of stress kinase JNK and on heat-induced apoptosis. FEBS Lett 461:73–76 doi:10.1016/S0014-5793(99)01428-3 PubMedCrossRefGoogle Scholar
  54. Wang Q, Mosser DD, Bag J (2005) Induction of HSP70 expression and recruitment of HSC70 and HSP70 in the nucleus reduce aggregation of a polyalanine expansion mutant of PABPN1 in HeLa cells. Hum Mol Genet 14:3673–3684 doi:10.1093/hmg/ddi395 PubMedCrossRefGoogle Scholar
  55. Young JC, Barral JM, Ulrich Hartl F (2003) More than folding: localized functions of cytosolic chaperones. Trends Biochem Sci 28:541–547 doi:10.1016/j.tibs.2003.08.009 PubMedCrossRefGoogle Scholar
  56. Zhu X, Zhao X, Burkholder WF, Gragerov A, Ogata CM et al (1996) Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272:1606–1614 doi:10.1126/science.272.5268.1606 PubMedCrossRefGoogle Scholar

Copyright information

© Cell Stress Society International 2008

Authors and Affiliations

  • Ari M. Chow
    • 1
  • Rohan Steel
    • 1
  • Robin L. Anderson
    • 1
    • 2
  1. 1.Peter MacCallum Cancer CentreEast MelbourneAustralia
  2. 2.Peter MacCallum Cancer CentreMelbourneAustralia

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