Hsp70–RAP46 interaction in downregulation of DNA binding by glucocorticoid receptor

Receptor‐associating protein 46 (RAP46) is a cochaperone that regulates the transactivation function of several steroid receptors. It is transported into the nucleus by a liganded glucocorticoid receptor where it downregulates DNA binding and transactivation by this receptor. The N‐ and C‐termini of RAP46 are both implicated in its negative regulatory function. In metabolic labelling experiments, we have shown that the N‐terminus of RAP46 is modified by phosphorylation, but this does not contribute to the downregulation of glucocorticoid receptor activity. However, deletion of a sequence that binds 70 kDa heat shock protein (Hsp70) and the constitutive isoform of Hsp70 (Hsc70) at the C‐terminus of RAP46 abrogated its negative regulatory action. Surface plasmon resonance studies showed that RAP46 binds the glucocorticoid receptor only when it has interacted with Hsp70/Hsc70, and confocal immunofluorescence analyses revealed a nuclear transport of Hsp70/Hsc70 by the liganded receptor. Together these findings demonstrate an important contribution of Hsp70/Hsc70 in the binding of RAP46 to the glucocorticoid receptor and suggest a role for this molecular chaperone in the RAP46‐mediated downregulation of glucocorticoid receptor activity.

[1]  E. Eisenberg,et al.  The Molecular Chaperones Hsp90 and Hsc70 Are Both Necessary and Sufficient to Activate Hormone Binding by Glucocorticoid Receptor* , 2000, The Journal of Biological Chemistry.

[2]  A. Silverstein,et al.  The Hsp Organizer Protein Hop Enhances the Rate of but Is Not Essential for Glucocorticoid Receptor Folding by the Multiprotein Hsp90-based Chaperone System* , 2000, The Journal of Biological Chemistry.

[3]  K. Yamamoto,et al.  The p23 molecular chaperones act at a late step in intracellular receptor action to differentially affect ligand efficacies. , 2000, Genes & development.

[4]  H. Kampinga,et al.  Bag1 Functions In Vivo as a Negative Regulator of Hsp70 Chaperone Activity , 2000, Molecular and Cellular Biology.

[5]  G. Giannoukos,et al.  The Seven Amino Acids (547–553) of Rat Glucocorticoid Receptor Required for Steroid and Hsp90 Binding Contain a Functionally Independent LXXLL Motif That Is Critical for Steroid Binding* , 1999, The Journal of Biological Chemistry.

[6]  John Calvin Reed,et al.  Differential Effects of the hsp70-binding Protein BAG-1 on Glucocorticoid Receptor Folding by the hsp90-based Chaperone Machinery* , 1999, The Journal of Biological Chemistry.

[7]  J. Schneikert,et al.  A Nuclear Action of the Eukaryotic Cochaperone Rap46 in Downregulation of Glucocorticoid Receptor Activity , 1999, The Journal of cell biology.

[8]  Y. Niyaz,et al.  The hsp70-associating protein Hap46 binds to DNA and stimulates transcription. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  M. Green,et al.  A human nuclear-localized chaperone that regulates dimerization, DNA binding, and transcriptional activity of bZIP proteins. , 1999, Molecular cell.

[10]  J. Buchner,et al.  Hsp90 & Co. - a holding for folding. , 1999, Trends in biochemical sciences.

[11]  S. Mohr,et al.  Vitamin D receptor interacts with DnaK/heat shock protein 70: identification of DnaK interaction site on vitamin D receptor. , 1999, Archives of biochemistry and biophysics.

[12]  E. Baulieu,et al.  The molecular chaperone Hsp90 can negatively regulate the activity of a glucocorticosteroid-dependent promoter. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[13]  J. Song,et al.  BAG‐1, a negative regulator of Hsp70 chaperone activity, uncouples nucleotide hydrolysis from substrate release , 1998, The EMBO journal.

[14]  Z. Ding,et al.  Human BAG-1/RAP46 protein is generated as four isoforms by alternative translation initiation and overexpressed in cancer cells , 1998, Oncogene.

[15]  K. R. Ely,et al.  Characterization of Interactions between the Anti-apoptotic Protein BAG-1 and Hsc70 Molecular Chaperones* , 1998, The Journal of Biological Chemistry.

[16]  J C Reed,et al.  Expression and location of Hsp70/Hsc-binding anti-apoptotic protein BAG-1 and its variants in normal tissues and tumor cell lines. , 1998, Cancer research.

[17]  John Calvin Reed,et al.  Interaction of BAG-1 with Retinoic Acid Receptor and Its Inhibition of Retinoic Acid-induced Apoptosis in Cancer Cells* , 1998, The Journal of Biological Chemistry.

[18]  S. Heck,et al.  RAP46 Is a Negative Regulator of Glucocorticoid Receptor Action and Hormone-induced Apoptosis* , 1998, The Journal of Biological Chemistry.

[19]  J C Reed,et al.  BAG-1L Protein Enhances Androgen Receptor Function* , 1998, The Journal of Biological Chemistry.

[20]  J L Cleveland,et al.  Mammalian cells express two differently localized Bag-1 isoforms generated by alternative translation initiation. , 1997, The Biochemical journal.

[21]  S. Jentsch,et al.  GrpE‐like regulation of the Hsc70 chaperone by the anti‐apoptotic protein BAG‐1 , 1997, The EMBO journal.

[22]  U. Gehring,et al.  Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins , 1997, The EMBO journal.

[23]  R. Ellis Molecular chaperones: Avoiding the crowd , 1997, Current Biology.

[24]  John Calvin Reed,et al.  BAG‐1 modulates the chaperone activity of Hsp70/Hsc70 , 1997, The EMBO journal.

[25]  W. Pratt,et al.  Steroid receptor interactions with heat shock protein and immunophilin chaperones. , 1997, Endocrine reviews.

[26]  W. Pratt,et al.  Folding of the Glucocorticoid Receptor by the Reconstituted hsp90-based Chaperone Machinery , 1997, The Journal of Biological Chemistry.

[27]  S. Gottesman,et al.  Protein quality control: triage by chaperones and proteases. , 1997, Genes & development.

[28]  Bernd Bukau,et al.  Substrate specificity of the DnaK chaperone determined by screening cellulose‐bound peptide libraries , 1997, The EMBO journal.

[29]  J. Frydman,et al.  Chaperones get in touch: the Hip-Hop connection. , 1997, Trends in biochemical sciences.

[30]  S. Lindquist,et al.  A Cyclophilin Function in Hsp90-Dependent Signal Transduction , 1996, Science.

[31]  I. Macara,et al.  Evidence using a green fluorescent protein-glucocorticoid receptor chimera that the Ran/TC4 GTPase mediates an essential function independent of nuclear protein import , 1996, The Journal of cell biology.

[32]  U. Gehring,et al.  A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[33]  K. Yamamoto,et al.  Hold 'em and fold 'em: chaperones and signal transduction. , 1995, Science.

[34]  H. Klocker,et al.  A single amino acid exchange abolishes dimerization of the androgen receptor and causes Reifenstein syndrome , 1995, Molecular and Cellular Endocrinology.

[35]  S. Long,et al.  The Rhizobium meliloti groELc locus is required for regulation of early nod genes by the transcription activator NodD. , 1995, Genes & development.

[36]  John Calvin Reed,et al.  Cloning and functional analysis of BAG-1: A novel Bcl-2-binding protein with anti-cell death activity , 1995, Cell.

[37]  J. Blow,et al.  Inhibition of cyclin-dependent kinases by purine analogues. , 1994, European journal of biochemistry.

[38]  N. Sreerama,et al.  Poly(pro)II helices in globular proteins: identification and circular dichroic analysis. , 1994, Biochemistry.

[39]  E. Thompson,et al.  Heat shock protein is tightly associated with the recombinant human glucocorticoid receptor:glucocorticoid response element complex. , 1994, Molecular endocrinology.

[40]  T. Schmidt,et al.  Heat shock protein 70 is associated in substoichiometric amounts with the rat hepatic glucocorticoid receptor. , 1993, Biochemistry.

[41]  N. Sreerama,et al.  A self-consistent method for the analysis of protein secondary structure from circular dichroism. , 1993, Analytical biochemistry.

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

[43]  H. Bujard,et al.  Physical interaction between heat shock proteins DnaK, DnaJ, and GrpE and the bacterial heat shock transcription factor σ 32 , 1992, Cell.

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

[45]  E. Bresnick,et al.  Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor. , 1989, The Journal of biological chemistry.

[46]  R. Evans,et al.  Functional domains of the human glucocorticoid receptor , 1986, Cell.

[47]  C. Anfinsen Principles that govern the folding of protein chains. , 1973, Science.

[48]  H. Gronemeyer,et al.  Reappraisal of the role of heat shock proteins as regulators of steroid receptor activity. , 1998, Critical reviews in biochemistry and molecular biology.

[49]  M. Jäättelä,et al.  HSP27 and HSP70 increase the survival of WEHI-S cells exposed to hyperthermia. , 1996, International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group.

[50]  R. Ashley Ion channels : a practical approach , 1995 .

[51]  D. Toft,et al.  Steroid receptors and their associated proteins. , 1993, Molecular endocrinology.