Role of HSP90 in Salt Stress Tolerance via Stabilization and Regulation of Calcineurin

ABSTRACT The role of HSP90 in stress tolerance was investigated inSaccharomyces cerevisiae. Cells showing 20-fold overexpression of Hsc82, an HSP90 homologue in yeast, were hypersensitive to high-NaCl or H-LiCl stresses. Hsc82-overexpressing cells appeared similar to calcineurin-defective cells in salt sensitivity and showed reduced levels of calcineurin-dependent gene expression. Co-overexpression of Cna2, the catalytic subunit of calcineurin, suppressed the hypersensitivity. Cna2 and Hsc82 coimmunoprecipitated from control cells grown under normal conditions but not from stressed cells. In contrast, coimmunoprecipitation was detected with Hsc82-overexpressing cells even after exposure to stresses. Cna2 immune complexes from stressed control cells showed a significant level of calcineurin activity, whereas those from stressed Hsc82-overexpressing cells did not. Treatment of extracts from Hsc82-overexpressing cells with Ca2+-calmodulin increased the calcineurin activity associated with Cna2 immune complexes. Geldanamycin, an inhibitor of HSP90 abolished the coimmunoprecipitation but did not activate calcineurin. When the expression level of Hsc82 decreased to below 30% of the normal level, cells also became hypersensitive to salt stress. In these cells, the amount of Cna2 was reduced, likely as a result of degradation. The present results showed that Hsc82 binds to and stabilizes Cna2 and that dissociation of Cna2 from Hsc82 is necessary for its activation.

[1]  J. Buchner,et al.  Hsp90 chaperones protein folding in vitro , 1992, Nature.

[2]  R. Morimoto,et al.  The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj‐1 have distinct roles in recognition of a non‐native protein and protein refolding. , 1996, The EMBO journal.

[3]  S. Lindquist,et al.  In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[4]  M. Welsh,et al.  Interaction of the glucocorticoid receptor with the Mr 90,000 heat shock protein: an evolving model of ligand-mediated receptor transformation and translocation. , 1989, Cancer research.

[5]  M. Cyert,et al.  Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation. , 1999, Genes & development.

[6]  J. Brugge,et al.  Interaction of the Rous sarcoma virus protein pp60src with the cellular proteins pp50 and pp90. , 1986, Current topics in microbiology and immunology.

[7]  P. Cohen,et al.  Discovery of A Ca2+‐and calmodulin‐dependent protein phosphatase , 1982, FEBS letters.

[8]  F. Hartl,et al.  Molecular chaperone functions of heat-shock proteins. , 1993, Annual review of biochemistry.

[9]  S. Meshinchi,et al.  Structural and functional reconstitution of the glucocorticoid receptor-hsp90 complex. , 1990, The Journal of biological chemistry.

[10]  L. Guarente.  Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. , 1983, Methods in enzymology.

[11]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[12]  I. Yahara,et al.  Heat-induced Chaperone Activity of HSP90 (*) , 1996, The Journal of Biological Chemistry.

[13]  S. Koyasu,et al.  A heat shock-resistant variant of Chinese hamster cell line constitutively expressing heat shock protein of Mr 90,000 at high level. , 1986, Cell structure and function.

[14]  J. Tumlin,et al.  Heat shock proteins 70 and 90 increase calcineurin activity in vitro through calmodulin-dependent and independent mechanisms. , 1999, Biochemical and biophysical research communications.

[15]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations. , 1984, Journal of bacteriology.

[16]  P. Piper,et al.  Authentic temperature-regulation of a heat shock gene inserted into yeast on a high copy number vector. Influences of overexpression of HSP90 protein on high temperature growth and thermotolerance. , 1992, Biochimica et biophysica acta.

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

[18]  Y. Liu,et al.  Protein phosphatase type 2B (calcineurin)‐mediated, FK506‐sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions. , 1993, The EMBO journal.

[19]  C. Zuker,et al.  Protein folding and the regulation of signaling pathways , 1994, Cell.

[20]  H. Kawasaki,et al.  A Critical Role for the Proteasome Activator PA28 in the Hsp90-dependent Protein Refolding* , 2000, The Journal of Biological Chemistry.

[21]  R. Müller,et al.  Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. , 1995, Gene.

[22]  M. Cyert,et al.  Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast. , 1997, Genes & development.

[23]  L. Langeberg,et al.  Coordination of Three Signaling Enzymes by AKAP79, a Mammalian Scaffold Protein , 1996, Science.

[24]  M. Goebl,et al.  Yeast CAL1 is a structural and functional homologue to the DPR1 (RAM) gene involved in ras processing. , 1991, The Journal of biological chemistry.

[25]  K. Yamamoto,et al.  13 Modulation of Steroid Receptor Signal Transduction by Heat Shock Proteins , 1994 .

[26]  S. Snyder,et al.  Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca2+ flux , 1995, Cell.

[27]  H. Erdjument-Bromage,et al.  FK506 binding protein associated with the calcium release channel (ryanodine receptor). , 1992, The Journal of biological chemistry.

[28]  T. Kuno,et al.  cDNA cloning of a calcineurin B homolog in Saccharomyces cerevisiae. , 1991, Biochemical and biophysical research communications.

[29]  S. Lindquist,et al.  Heat-shock protein hsp90 governs the activity of pp60v-src kinase. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[30]  A. Marks,et al.  The calcium release channel of sarcoplasmic reticulum is modulated by FK-506-binding protein. Dissociation and reconstitution of FKBP-12 to the calcium release channel of skeletal muscle sarcoplasmic reticulum. , 1993, The Journal of biological chemistry.

[31]  S. Snyder,et al.  FKBP12 Binds the Inositol 1,4,5-Trisphosphate Receptor at Leucine-Proline (1400–1401) and Anchors Calcineurin to this FK506-like Domain* , 1997, The Journal of Biological Chemistry.

[32]  Y. Miyata,et al.  The 90-kDa heat shock protein, HSP90, binds and protects casein kinase II from self-aggregation and enhances its kinase activity. , 1992, The Journal of biological chemistry.

[33]  W. Pratt.  The role of the hsp90-based chaperone system in signal transduction by nuclear receptors and receptors signaling via MAP kinase. , 1997, Annual review of pharmacology and toxicology.

[34]  M. Cyert,et al.  Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone , 1992, Molecular and cellular biology.

[35]  G. Fink,et al.  Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases , 1994, The Journal of cell biology.

[36]  T. Soderling,et al.  Identification of an autoinhibitory domain in calcineurin. , 1990, The Journal of biological chemistry.

[37]  C. Klee,et al.  Activation of calcineurin by limited proteolysis. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[38]  M. Cyert,et al.  Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[39]  S. Lindquist,et al.  Reduced levels of hsp90 compromise steroid receptor action in vivo , 1990, Nature.

[40]  S. Lindquist,et al.  hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures , 1989, Molecular and cellular biology.

[41]  F. Rubio,et al.  The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae. , 1994, The Journal of biological chemistry.

[42]  D. Matheos,et al.  Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae. , 1997, Genes & development.

[43]  Anna Tempczyk,et al.  Crystal structures of human calcineurin and the human FKBP12–FK506–calcineurin complex , 1995, Nature.

[44]  J. Buchner,et al.  Transient Interaction of Hsp90 with Early Unfolding Intermediates of Citrate Synthase , 1995, The Journal of Biological Chemistry.

[45]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.