Reconstitution of the multiprotein complex of pp60(src), hsp90, and p50 in a cell-free system

K. A. Hutchison, B. K. Brott, J. H. De Leon, G. H. Perdew, R. Jove, W. B. Pratt

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Abstract

A rabbit reticulocyte lysate system that has been used to reconstitute functional complexes between steroid receptors and the 90-kDa heat shock protein (hsp90) has been used here to form complexes between the pp60(src) tyrosine kinase and hsp90. Reticulocyte lysate forms complexes between hsp90 and a temperature-sensitive mutant of Rous sarcoma virus pp60(v-src), which is normally present in cytosol virtually entirely in the multiprotein complex form. In addition, hsp90 in the lysate complexes with wild-type pp60(v-src), of which only a small portion is normally recovered in cytosol in the native multiprotein complex, and with the cellular homolog, pp60(c-src), which has never been recovered in cytosol in the form of a native multiprotein complex with hsp90. Moreover, the reticulocyte lysate-reconstituted complex also contains the 50-kDa phosphoprotein component of the native pp60(v-src) multiprotein complex. The native and reconstituted pp60(src)-hsp90 complexes have similar thermal stability and, like steroid receptor heterocomplexes, they are stabilized by molybdate. As previously shown with reticulocyte lysate-reconstituted steroid receptor heteroprotein complexes, the reconstituted pp60(src) multiprotein complex contains hsp70, which is a major candidate for providing the protein unfoldase activity required for hsp90 association.

Original languageEnglish (US)
Pages (from-to)2902-2908
Number of pages7
JournalJournal of Biological Chemistry
Volume267
Issue number5
StatePublished - 1992

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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