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. 1994 Jan 4;91(1):340–344. doi: 10.1073/pnas.91.1.340

In vivo functional protein-protein interaction: nuclear targeted hsp90 shifts cytoplasmic steroid receptor mutants into the nucleus.

K I Kang 1, J Devin 1, F Cadepond 1, N Jibard 1, A Guiochon-Mantel 1, E E Baulieu 1, M G Catelli 1
PMCID: PMC42943  PMID: 8278390

Abstract

In target tissue extracts, heat shock protein hsp90 has been found associated to all unliganded steroid receptors. Modulation of important functions of these receptors, including prevention of DNA binding and optimization of transcriptional activity, has been attributed to hsp90. However no unequivocal in vivo demonstration of interaction between receptors and hsp90 has been presented. We targeted chicken hsp90, a mainly cytoplasmic protein, with the nucleoplasmin nuclear localization signal (90NLS). After transfection into COS-7 cells, 90NLS was found in the nucleus with specific immunofluorescence and confocal microscopy techniques. A human glucocorticosteroid receptor mutant devoid of NLS sequence was also expressed in COS-7 cells and found exclusively cytoplasmic. Coexpression of 90NLS and of the cytoplasmic human glucocorticosteroid receptor mutant led to complete nuclear localization of the receptor, indicating its piggyback transport by 90NLS and thus physical and functional interaction between the two proteins in the absence of hormone. The same nuclear localization was obtained after cotransfection of 90NLS and a cytoplasmic rabbit progesterone receptor mutant. Finally, coexpression of wild-type rabbit progesterone receptor (nuclear) and wildtype hsp90 (cytoplasmic) into COS-7 cells provoked partial relocalization of hsp90 into the nucleus. These experiments lay the groundwork on which to study hsp90 as a chaperone, regulating activities of steroid receptors and possibly participating in their nuclear-cytoplasmic shuttling.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Binart N., Chambraud B., Dumas B., Rowlands D. A., Bigogne C., Levin J. M., Garnier J., Baulieu E. E., Catelli M. G. The cDNA-derived amino acid sequence of chick heat shock protein Mr 90,000 (HSP 90) reveals a "DNA like" structure: potential site of interaction with steroid receptors. Biochem Biophys Res Commun. 1989 Feb 28;159(1):140–147. doi: 10.1016/0006-291x(89)92415-7. [DOI] [PubMed] [Google Scholar]
  2. Biou V., Gibrat J. F., Levin J. M., Robson B., Garnier J. Secondary structure prediction: combination of three different methods. Protein Eng. 1988 Sep;2(3):185–191. doi: 10.1093/protein/2.3.185. [DOI] [PubMed] [Google Scholar]
  3. Cadepond F., Binart N., Chambraud B., Jibard N., Schweizer-Groyer G., Segard-Maurel I., Baulieu E. E. Interaction of glucocorticosteroid receptor and wild-type or mutated 90-kDa heat shock protein coexpressed in baculovirus-infected Sf9 cells. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10434–10438. doi: 10.1073/pnas.90.22.10434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cadepond F., Gasc J. M., Delahaye F., Jibard N., Schweizer-Groyer G., Segard-Maurel I., Evans R., Baulieu E. E. Hormonal regulation of the nuclear localization signals of the human glucocorticosteroid receptor. Exp Cell Res. 1992 Jul;201(1):99–108. doi: 10.1016/0014-4827(92)90352-9. [DOI] [PubMed] [Google Scholar]
  5. Cadepond F., Schweizer-Groyer G., Segard-Maurel I., Jibard N., Hollenberg S. M., Giguère V., Evans R. M., Baulieu E. E. Heat shock protein 90 as a critical factor in maintaining glucocorticosteroid receptor in a nonfunctional state. J Biol Chem. 1991 Mar 25;266(9):5834–5841. [PubMed] [Google Scholar]
  6. Callebaut I., Renoir J. M., Lebeau M. C., Massol N., Burny A., Baulieu E. E., Mornon J. P. An immunophilin that binds M(r) 90,000 heat shock protein: main structural features of a mammalian p59 protein. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6270–6274. doi: 10.1073/pnas.89.14.6270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Catelli M. G., Binart N., Feramisco J. R., Helfman D. M. Cloning of the chick hsp 90 cDNA in expression vector. Nucleic Acids Res. 1985 Sep 11;13(17):6035–6047. doi: 10.1093/nar/13.17.6035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Catelli M. G., Binart N., Jung-Testas I., Renoir J. M., Baulieu E. E., Feramisco J. R., Welch W. J. The common 90-kd protein component of non-transformed '8S' steroid receptors is a heat-shock protein. EMBO J. 1985 Dec 1;4(12):3131–3135. doi: 10.1002/j.1460-2075.1985.tb04055.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dingwall C., Dilworth S. M., Black S. J., Kearsey S. E., Cox L. S., Laskey R. A. Nucleoplasmin cDNA sequence reveals polyglutamic acid tracts and a cluster of sequences homologous to putative nuclear localization signals. EMBO J. 1987 Jan;6(1):69–74. doi: 10.1002/j.1460-2075.1987.tb04720.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garcia-Bustos J., Heitman J., Hall M. N. Nuclear protein localization. Biochim Biophys Acta. 1991 Mar 7;1071(1):83–101. doi: 10.1016/0304-4157(91)90013-m. [DOI] [PubMed] [Google Scholar]
  11. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  12. Guiochon-Mantel A., Lescop P., Christin-Maitre S., Loosfelt H., Perrot-Applanat M., Milgrom E. Nucleocytoplasmic shuttling of the progesterone receptor. EMBO J. 1991 Dec;10(12):3851–3859. doi: 10.1002/j.1460-2075.1991.tb04954.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guiochon-Mantel A., Loosfelt H., Lescop P., Sar S., Atger M., Perrot-Applanat M., Milgrom E. Mechanisms of nuclear localization of the progesterone receptor: evidence for interaction between monomers. Cell. 1989 Jun 30;57(7):1147–1154. doi: 10.1016/0092-8674(89)90052-4. [DOI] [PubMed] [Google Scholar]
  14. Howard K. J., Distelhorst C. W. Evidence for intracellular association of the glucocorticoid receptor with the 90-kDa heat shock protein. J Biol Chem. 1988 Mar 5;263(7):3474–3481. [PubMed] [Google Scholar]
  15. Joab I., Radanyi C., Renoir M., Buchou T., Catelli M. G., Binart N., Mester J., Baulieu E. E. Common non-hormone binding component in non-transformed chick oviduct receptors of four steroid hormones. 1984 Apr 26-May 2Nature. 308(5962):850–853. doi: 10.1038/308850a0. [DOI] [PubMed] [Google Scholar]
  16. Madan A. P., DeFranco D. B. Bidirectional transport of glucocorticoid receptors across the nuclear envelope. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3588–3592. doi: 10.1073/pnas.90.8.3588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nadeau K., Das A., Walsh C. T. Hsp90 chaperonins possess ATPase activity and bind heat shock transcription factors and peptidyl prolyl isomerases. J Biol Chem. 1993 Jan 15;268(2):1479–1487. [PubMed] [Google Scholar]
  18. Picard D., Khursheed B., Garabedian M. J., Fortin M. G., Lindquist S., Yamamoto K. R. Reduced levels of hsp90 compromise steroid receptor action in vivo. Nature. 1990 Nov 8;348(6297):166–168. doi: 10.1038/348166a0. [DOI] [PubMed] [Google Scholar]
  19. Picard D., Yamamoto K. R. Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J. 1987 Nov;6(11):3333–3340. doi: 10.1002/j.1460-2075.1987.tb02654.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pratt W. B. Control of steroid receptor function and cytoplasmic-nuclear transport by heat shock proteins. Bioessays. 1992 Dec;14(12):841–848. doi: 10.1002/bies.950141209. [DOI] [PubMed] [Google Scholar]
  21. Pratt W. B., Jolly D. J., Pratt D. V., Hollenberg S. M., Giguere V., Cadepond F. M., Schweizer-Groyer G., Catelli M. G., Evans R. M., Baulieu E. E. A region in the steroid binding domain determines formation of the non-DNA-binding, 9 S glucocorticoid receptor complex. J Biol Chem. 1988 Jan 5;263(1):267–273. [PubMed] [Google Scholar]
  22. Radanyi C., Renoir J. M., Sabbah M., Baulieu E. E. Chick heat-shock protein of Mr = 90,000, free or released from progesterone receptor, is in a dimeric form. J Biol Chem. 1989 Feb 15;264(5):2568–2573. [PubMed] [Google Scholar]
  23. Renoir J. M., Radanyi C., Jung-Testas I., Faber L. E., Baulieu E. E. The nonactivated progesterone receptor is a nuclear heterooligomer. J Biol Chem. 1990 Aug 25;265(24):14402–14406. [PubMed] [Google Scholar]
  24. Robbins J., Dilworth S. M., Laskey R. A., Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. doi: 10.1016/0092-8674(91)90245-t. [DOI] [PubMed] [Google Scholar]
  25. Sanchez E. R., Toft D. O., Schlesinger M. J., Pratt W. B. Evidence that the 90-kDa phosphoprotein associated with the untransformed L-cell glucocorticoid receptor is a murine heat shock protein. J Biol Chem. 1985 Oct 15;260(23):12398–12401. [PubMed] [Google Scholar]
  26. Schuh S., Yonemoto W., Brugge J., Bauer V. J., Riehl R. M., Sullivan W. P., Toft D. O. A 90,000-dalton binding protein common to both steroid receptors and the Rous sarcoma virus transforming protein, pp60v-src. J Biol Chem. 1985 Nov 15;260(26):14292–14296. [PubMed] [Google Scholar]
  27. Smith D. F., Toft D. O. Steroid receptors and their associated proteins. Mol Endocrinol. 1993 Jan;7(1):4–11. doi: 10.1210/mend.7.1.8446107. [DOI] [PubMed] [Google Scholar]
  28. Tuohimaa P., Pekki A., Bläuer M., Joensuu T., Vilja P., Ylikomi T. Nuclear progesterone receptor is mainly heat shock protein 90-free in vivo. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5848–5852. doi: 10.1073/pnas.90.12.5848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ylikomi T., Bocquel M. T., Berry M., Gronemeyer H., Chambon P. Cooperation of proto-signals for nuclear accumulation of estrogen and progesterone receptors. EMBO J. 1992 Oct;11(10):3681–3694. doi: 10.1002/j.1460-2075.1992.tb05453.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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