Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system

• Published in: The Journal of biological chemistry 1992-02, Vol.267 (5), p.2902-2908
• Main Authors: HUTCHISON, K. A, BROTT, B. K, DE LEON, J. H, PERDEW, G. H, JOVE, R, PRATT, W. B
• Format: Article
• Language: English
• Subjects: Animals; Avian Sarcoma Viruses - genetics; Biological and medical sciences; Cytosol - metabolism; Drug Stability; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Heat-Shock Proteins - isolation & purification; Heat-Shock Proteins - metabolism; Immunoblotting; Macromolecular Substances; Microbiology; Molecular Weight; Molybdenum - pharmacology; Morphology, structure, chemical composition, physicochemical properties; Multiprotein Complexes; Oncogene Protein pp60(v-src) - genetics; Oncogene Protein pp60(v-src) - isolation & purification; Oncogene Protein pp60(v-src) - metabolism; Phosphoproteins - isolation & purification; Phosphoproteins - metabolism; Rabbits; Reticulocytes - metabolism; Virology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)50671-8

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 pp60src tyrosine kinase and hsp90. Reticulocyte lysate forms complexes between hsp90 and a temperature-sensitive mutant of Rous sarcoma virus pp60v-src, which is normally present in cytosol virtually entirely in the multiprotein complex form. In addition, hsp90 in the lysate complexes with wild-type pp60v-src, of which only a small portion is normally recovered in cytosol in the native multiprotein complex, and with the cellular homolog, pp60c-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 pp60v-src multiprotein complex. The native and reconstituted pp60src-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 pp60src multiprotein complex contains hsp70, which is a major candidate for providing the protein unfoldase activity required for hsp90 association.