Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Aug 1;90(15):7074–7078. doi: 10.1073/pnas.90.15.7074

Heat-shock protein hsp90 governs the activity of pp60v-src kinase.

Y Xu 1, S Lindquist 1
PMCID: PMC47078  PMID: 7688470

Abstract

During or immediately after synthesis in vertebrate cells, the oncogenic protein-tyrosine kinase pp60v-src associates with the approximately 90-kDa heat-shock protein (hsp90). In this complex, pp60v-src is not functional as a kinase. When pp60v-src is subsequently found inserted into the plasma membrane, it is active as a kinase and is no longer associated with hsp90. We have taken advantage of genetic manipulations possible in Saccharomyces cerevisiae to investigate the function and specificity of the association between hsp90 and pp60v-src. Expression of pp60v-src is known to be toxic to S. cerevisiae cells. We find that this toxicity is due to a very specific effect on growth, arrest at a particular point in the cell cycle. In cells expressing v-src, a mutation that lowers the level of hsp90 expression (i) relieves cell cycle arrest and rescues growth, (ii) reduces the level of tyrosine phosphorylation mediated by pp60v-src, (iii) changes the pattern of tyrosine phosphorylation, and (iv) reduces the concentration of pp60v-src. We conclude that hsp90 does not simply suppress pp60v-src kinase activity during transit to the plasma membrane, as previously suggested, but also stabilizes the protein and affects both its activity and specificity. This function of hsp90 is highly selective for pp60v-src: the same hsp90 mutation has no effect on the activity or specificity of the exogenous pp160v-abl tyrosine kinase; similarly, it does not affect the specificity and has only a very small effect on the activity of the exogenous pp60c-src kinase.

Full text

PDF
7074

Images in this article

7076Fig. 1
on p.7076
7076Fig. 2
on p.7076
7077Fig. 3
on p.7077

Selected References

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

  1. Adkins B., Hunter T., Sefton B. M. The transforming proteins of PRCII virus and Rous sarcoma virus form a complex with the same two cellular phosphoproteins. J Virol. 1982 Aug;43(2):448–455. doi: 10.1128/jvi.43.2.448-455.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Borkovich K. A., Farrelly F. W., Finkelstein D. B., Taulien J., Lindquist S. hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol Cell Biol. 1989 Sep;9(9):3919–3930. doi: 10.1128/mcb.9.9.3919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brugge J. S., Erikson E., Erikson R. L. The specific interaction of the Rous sarcoma virus transforming protein, pp60src, with two cellular proteins. Cell. 1981 Aug;25(2):363–372. doi: 10.1016/0092-8674(81)90055-6. [DOI] [PubMed] [Google Scholar]
  4. Brugge J. S. Interaction of the Rous sarcoma virus protein pp60src with the cellular proteins pp50 and pp90. Curr Top Microbiol Immunol. 1986;123:1–22. doi: 10.1007/978-3-642-70810-7_1. [DOI] [PubMed] [Google Scholar]
  5. Brugge J. S., Jarosik G., Andersen J., Queral-Lustig A., Fedor-Chaiken M., Broach J. R. Expression of Rous sarcoma virus transforming protein pp60v-src in Saccharomyces cerevisiae cells. Mol Cell Biol. 1987 Jun;7(6):2180–2187. doi: 10.1128/mcb.7.6.2180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hoffman-Falk H., Einat P., Shilo B. Z., Hoffmann F. M. Drosophila melanogaster DNA clones homologous to vertebrate oncogenes: evidence for a common ancestor to the src and abl cellular genes. Cell. 1983 Feb;32(2):589–598. doi: 10.1016/0092-8674(83)90478-6. [DOI] [PubMed] [Google Scholar]
  7. Hoffmann F. M. Drosophila abl and genetic redundancy in signal transduction. Trends Genet. 1991 Nov-Dec;7(11-12):351–355. doi: 10.1016/0168-9525(91)90254-f. [DOI] [PubMed] [Google Scholar]
  8. Hutchison K. A., Brott B. K., De Leon J. H., Perdew G. H., Jove R., Pratt W. B. Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system. J Biol Chem. 1992 Feb 15;267(5):2902–2908. [PubMed] [Google Scholar]
  9. Iba H., Takeya T., Cross F. R., Hanafusa T., Hanafusa H. Rous sarcoma virus variants that carry the cellular src gene instead of the viral src gene cannot transform chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4424–4428. doi: 10.1073/pnas.81.14.4424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jove R., Kornbluth S., Hanafusa H. Enzymatically inactive p60c-src mutant with altered ATP-binding site is fully phosphorylated in its carboxy-terminal regulatory region. Cell. 1987 Sep 11;50(6):937–943. doi: 10.1016/0092-8674(87)90520-4. [DOI] [PubMed] [Google Scholar]
  12. Kawai S., Hanafusa H. The effects of reciprocal changes in temperature on the transformed state of cells infected with a rous sarcoma virus mutant. Virology. 1971 Nov;46(2):470–479. doi: 10.1016/0042-6822(71)90047-x. [DOI] [PubMed] [Google Scholar]
  13. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  14. Kornbluth S., Jove R., Hanafusa H. Characterization of avian and viral p60src proteins expressed in yeast. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4455–4459. doi: 10.1073/pnas.84.13.4455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lindquist S., Craig E. A. The heat-shock proteins. Annu Rev Genet. 1988;22:631–677. doi: 10.1146/annurev.ge.22.120188.003215. [DOI] [PubMed] [Google Scholar]
  16. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Oppermann H., Levinson W., Bishop J. M. A cellular protein that associates with the transforming protein of Rous sarcoma virus is also a heat-shock protein. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1067–1071. doi: 10.1073/pnas.78.2.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Parsons J. T., Weber M. J. Genetics of src: structure and functional organization of a protein tyrosine kinase. Curr Top Microbiol Immunol. 1989;147:79–127. doi: 10.1007/978-3-642-74697-0_3. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Pratt W. B., Sanchez E. R., Bresnick E. H., Meshinchi S., Scherrer L. C., Dalman F. C., Welsh M. J. Interaction of the glucocorticoid receptor with the Mr 90,000 heat shock protein: an evolving model of ligand-mediated receptor transformation and translocation. Cancer Res. 1989 Apr 15;49(8 Suppl):2222s–2229s. [PubMed] [Google Scholar]
  21. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  22. Scherrer L. C., Dalman F. C., Massa E., Meshinchi S., Pratt W. B. Structural and functional reconstitution of the glucocorticoid receptor-hsp90 complex. J Biol Chem. 1990 Dec 15;265(35):21397–21400. [PubMed] [Google Scholar]
  23. Schieven G., Thorner J., Martin G. S. Protein-tyrosine kinase activity in Saccharomyces cerevisiae. Science. 1986 Jan 24;231(4736):390–393. doi: 10.1126/science.2417318. [DOI] [PubMed] [Google Scholar]
  24. Shilo B. Z., Weinberg R. A. DNA sequences homologous to vertebrate oncogenes are conserved in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6789–6792. doi: 10.1073/pnas.78.11.6789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Simon M. A., Drees B., Kornberg T., Bishop J. M. The nucleotide sequence and the tissue-specific expression of Drosophila c-src. Cell. 1985 Oct;42(3):831–840. doi: 10.1016/0092-8674(85)90279-x. [DOI] [PubMed] [Google Scholar]
  26. Smith D. F., Sullivan W. P., Marion T. N., Zaitsu K., Madden B., McCormick D. J., Toft D. O. Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70. Mol Cell Biol. 1993 Feb;13(2):869–876. doi: 10.1128/mcb.13.2.869. [DOI] [PMC free article] [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. Snyder M. A., Bishop J. M., McGrath J. P., Levinson A. D. A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity. Mol Cell Biol. 1985 Jul;5(7):1772–1779. doi: 10.1128/mcb.5.7.1772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wiech H., Buchner J., Zimmermann R., Jakob U. Hsp90 chaperones protein folding in vitro. Nature. 1992 Jul 9;358(6382):169–170. doi: 10.1038/358169a0. [DOI] [PubMed] [Google Scholar]
  30. Williamson D. H., Fennell D. J. The use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA. Methods Cell Biol. 1975;12:335–351. doi: 10.1016/s0091-679x(08)60963-2. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES