Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system.
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.
Hutchison
K A
KA
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
Brott
B K
BK
De Leon
J H
JH
Perdew
G H
GH
Jove
R
R
Pratt
W B
WB
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D003600:Cytosol / Q000378:metabolism; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015151:Immunoblotting; D046911:Macromolecular Substances; D008970:Molecular Weight; D008982:Molybdenum / Q000494:pharmacology; D046912:Multiprotein Complexes; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D008982:Molybdenum; C024687:sodium molybdate(VI); D015688:Oncogene Protein pp60(v-src)