The Chaperone Function of hsp70 Is Required for Protection against Stress-Induced Apoptosis

ABSTRACT Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochromec-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.

[1]  Dick D. Mosser,et al.  Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome , 2000, Nature Cell Biology.

[2]  Young-Gyu Ko,et al.  Heat Shock Protein 70 Inhibits Apoptosis Downstream of Cytochrome c Release and Upstream of Caspase-3 Activation* , 2000, The Journal of Biological Chemistry.

[3]  Y Li,et al.  [Mitochondria and apoptosis]. , 2000, Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine].

[4]  Gerard I. Evan,et al.  The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant , 2000, Nature Cell Biology.

[5]  Matthew G. Vander Heiden,et al.  Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis? , 1999, Nature Cell Biology.

[6]  M. Sherman,et al.  ATPase activity of the heat shock protein Hsp72 is dispensable for its effects on dephosphorylation of stress kinase JNK and on heat‐induced apoptosis , 1999, FEBS letters.

[7]  S. Korsmeyer,et al.  BCL-2 family members and the mitochondria in apoptosis. , 1999, Genes & development.

[8]  M. Sherman,et al.  The Function of HSP72 in Suppression of c-Jun N-terminal Kinase Activation Can Be Dissociated from Its Role in Prevention of Protein Damage* , 1999, The Journal of Biological Chemistry.

[9]  S. Srinivasula,et al.  Cytochrome c and dATP-mediated Oligomerization of Apaf-1 Is a Prerequisite for Procaspase-9 Activation* , 1999, The Journal of Biological Chemistry.

[10]  M. Sherman,et al.  Oncogenic potential of Hsp72 , 1999, Oncogene.

[11]  B. Zhivotovsky,et al.  Presence of a pre‐apoptotic complex of pro‐caspase‐3, Hsp60 and Hsp10 in the mitochondrial fraction of Jurkat cells , 1999, The EMBO journal.

[12]  P. Tawa,et al.  Hsp60 accelerates the maturation of pro‐caspase‐3 by upstream activator proteases during apoptosis , 1999, The EMBO journal.

[13]  L. Zon,et al.  Protein-Damaging Stresses Activate c-Jun N-Terminal Kinase via Inhibition of Its Dephosphorylation: a Novel Pathway Controlled by HSP72 , 1999, Molecular and Cellular Biology.

[14]  A. Eastman,et al.  The temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis. , 1999, Experimental cell research.

[15]  R. Morimoto,et al.  Regulation of the Heat Shock Transcriptional Response: Cross Talk between a Family of Heat Shock Factors, Molecular Chaperones, and Negative Regulators the Heat Shock Factor Family: Redundancy and Specialization , 2022 .

[16]  M. Sherman,et al.  Reduced thermotolerance in aged cells results from a loss of an hsp72-mediated control of JNK signaling pathway. , 1998, Cell stress & chaperones.

[17]  Lee,et al.  Expression of heat shock protein 70 blocks thymic differentiation of T cells in transgenic mice , 1998, Immunology.

[18]  M. Jäättelä,et al.  Hsp70 exerts its anti‐apoptotic function downstream of caspase‐3‐like proteases , 1998, The EMBO journal.

[19]  M. Sherman,et al.  Role of Hsp70 in regulation of stress‐kinase JNK: implications in apoptosis and aging , 1998, FEBS letters.

[20]  W. Park,et al.  The role of inducible 70-kDa heat shock protein in cell cycle control, differentiation, and apoptotic cell death of the human myeloid leukemic HL-60 cells. , 1998, Cellular immunology.

[21]  A. Giaccia,et al.  Heat Shock Protein 72 Modulates Pathways of Stress-induced Apoptosis* , 1998, The Journal of Biological Chemistry.

[22]  B. Polla,,et al.  In vivo heat shock protects rat myocardial mitochondria. , 1998, Biochemical and biophysical research communications.

[23]  R. Ezzell,et al.  Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70. , 1998, Genes & development.

[24]  J. Höhfeld,et al.  The Carboxy-Terminal Domain of Hsc70 Provides Binding Sites for a Distinct Set of Chaperone Cofactors , 1998, Molecular and Cellular Biology.

[25]  Y. Ip,et al.  Signal transduction by the c-Jun N-terminal kinase (JNK)--from inflammation to development. , 1998, Current opinion in cell biology.

[26]  R. Morimoto,et al.  Molecular chaperones as HSF1-specific transcriptional repressors. , 1998, Genes & development.

[27]  Bernd Bukau,et al.  The Hsp70 and Hsp60 Chaperone Machines , 1998, Cell.

[28]  S. Srinivasula,et al.  Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade , 1997, Cell.

[29]  L. Bourget,et al.  Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis , 1997, Molecular and cellular biology.

[30]  Dick D. Mosser,et al.  Hsp70 Prevents Activation of Stress Kinases , 1997, The Journal of Biological Chemistry.

[31]  X. Ding,et al.  Overexpression of the heat shock protein 70 enhances the TCR/CD3- and Fas/Apo-1/CD95-mediated apoptotic cell death in Jurkat T cells. , 1997, Journal of immunology.

[32]  D. Green,et al.  The Release of Cytochrome c from Mitochondria: A Primary Site for Bcl-2 Regulation of Apoptosis , 1997, Science.

[33]  Dean P. Jones,et al.  Prevention of Apoptosis by Bcl-2: Release of Cytochrome c from Mitochondria Blocked , 1997, Science.

[34]  T. Mak,et al.  Stress-signalling kinase Sek1 protects thymocytes from apoptosis mediated by CD95 and CD3 , 1997, Nature.

[35]  R. Davis,et al.  The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and gamma radiation. Duration of JNK activation may determine cell death and proliferation. , 1996, The Journal of biological chemistry.

[36]  G. Kroemer,et al.  Bcl-2 inhibits the mitochondrial release of an apoptogenic protease , 1996, The Journal of experimental medicine.

[37]  J. Avruch,et al.  Protein kinase cascades activated by stress and inflammatory cytokines , 1996, BioEssays : news and reviews in molecular, cellular and developmental biology.

[38]  C. Franceschi,et al.  Mitochondria are selective targets for the protective effects of heat shock against oxidative injury. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[39]  F. Hartl.  Molecular chaperones in cellular protein folding , 1996, Nature.

[40]  L. Zon,et al.  The stress-activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation or heat , 1996, Current Biology.

[41]  L. Zon,et al.  Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis , 1996, Nature.

[42]  T G Cotter,et al.  Heat shock proteins increase resistance to apoptosis. , 1996, Experimental cell research.

[43]  Jong-il Kim,et al.  T Cell Lymphoma in Transgenic Mice Expressing the HumanHsp70Gene , 1996 .

[44]  Michael E. Greenberg,et al.  Opposing Effects of ERK and JNK-p38 MAP Kinases on Apoptosis , 1995, Science.

[45]  Bowler,et al.  The development of thermotolerance protects blowfly flight muscle mitochondrial function from heat damage , 1995, The Journal of experimental biology.

[46]  D. Green,et al.  Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl , 1995, The Journal of experimental medicine.

[47]  M. Gossen,et al.  Transcriptional activation by tetracyclines in mammalian cells. , 1995, Science.

[48]  R. Morimoto,et al.  Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ‐1. , 1995, The EMBO journal.

[49]  G. Li,et al.  Effects of expressing human Hsp70 and its deletion derivatives on heat killing and on RNA and protein synthesis. , 1995, Experimental cell research.

[50]  H. Ouyang,et al.  Heat shock protein hsp70 accelerates the recovery of heat-shocked mammalian cells through its modulation of heat shock transcription factor HSF1. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[51]  M. Jäättelä.  Over‐expression of hsp70 confers tumorigenicity to mouse fibrosarcoma cells , 1995, International journal of cancer.

[52]  F. Hartl,et al.  Molecular chaperones in cellular protein folding. , 1994, Nature.

[53]  H. Kampinga,et al.  Importance of the ATP-binding domain and nucleolar localization domain of HSP72 in the protection of nuclear proteins against heat-induced aggregation. , 1994, Experimental cell research.

[54]  D. D. Mosser,et al.  The DNA-binding activity of the human heat shock transcription factor is regulated in vivo by hsp70 , 1993, Molecular and cellular biology.

[55]  D. Latchman,et al.  Heat shock protects neuronal cells from programmed cell death by apoptosis , 1993, Neuroscience.

[56]  M. Jäättelä,et al.  Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity. , 1992, The EMBO journal.

[57]  S. Lindquist,et al.  The consequences of expressing hsp70 in Drosophila cells at normal temperatures. , 1992, Genes & development.

[58]  D. D. Mosser,et al.  Induced thermotolerance to apoptosis in a human T lymphocyte cell line , 1992, Journal of cellular physiology.

[59]  G. Li,et al.  Heat shock protein hsp70 protects cells from thermal stress even after deletion of its ATP-binding domain. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[60]  B. Palsson,et al.  Growth, Metabolic, And Antibody Production Kinetics Of Hybridoma Cell Culture: 1. Analysis Of Data From Controlled Batch Reactors , 1991, Biotechnology progress.

[61]  G. Li,et al.  Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[62]  C. Amici,et al.  Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[63]  J. Landry,et al.  Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells , 1989, The Journal of cell biology.

[64]  R. Morimoto,et al.  Cell cycle-dependent association of HSP70 with specific cellular proteins , 1989, The Journal of cell biology.

[65]  R. Morimoto,et al.  Expression of human HSP70 during the synthetic phase of the cell cycle. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[66]  D. D. Mosser,et al.  Use of a micromanipulator for high-efficiency cloning of cells co-expressing fluorescent proteins. , 2000, Methods in cell science : an official journal of the Society for In Vitro Biology.

[67]  D. Latchman,et al.  Heat shock proteins delivered with a virus vector can protect cardiac cells against apoptosis as well as against thermal or hypoxic stress. , 1999, Journal of molecular and cellular cardiology.

[68]  L. Bourget,et al.  Use of a dicistronic expression cassette encoding the green fluorescent protein for the screening and selection of cells expressing inducible gene products. , 1997, BioTechniques.

[69]  F. Gago,et al.  Heat shock proteins and cell proliferation in human breast cancer biopsy samples. , 1997, Cancer detection and prevention.

[70]  S. H. Kim,et al.  T cell lymphoma in transgenic mice expressing the human Hsp70 gene. , 1996, Biochemical and biophysical research communications.

[71]  Richard I. Morimoto,et al.  1 Progress and Perspectives on the Biology of Heat Shock Proteins and Molecular Chaperones , 1994 .