A 50-kDa cytosolic protein complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus.
We have previously shown that a 50-kDa protein is one component of a heteromeric complex immunoprecipitated by the 90-kDa heat shock protein (hsp90) monoclonal antibodies 8D3 and 3G3 (Perdew, G. H., and Whitelaw, M. L. (1991) J. Biol. Chem. 266, 6708-6713). In this report, we compare the 50-kDa protein with that found in pp60v-src-hsp90-p50 complexes immunoprecipitated from Rous sarcoma virus-transformed cells with antibodies to pp60v-src. 35S- and 32P-labeled p50 proteins from each system were identical in their mobilities by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The profile of N-chlorosuccinimide cleavage products derived from each 32P-labeled p50 protein were also identical when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have developed a mouse monoclonal antibody, 3M/1B5p50, capable of detecting p50 on Western blots. This antibody detected the 50-kDa protein which co-purified with the pa104 pp60v-src mutant of the avian sarcoma virus oncoprotein in 44A rat fibroblasts. We did not detect p50 in association with native glucocorticoid receptor in L cells or with the overexpressed glucocorticoid receptor in Chinese hamster ovary cells. Two experiments utilizing immunochemical staining implied that essentially all cytosolic p50 is associated with hsp90. Firstly, immunoprecipitating hsp90 from Hepa 1 cytosol with monoclonal antibody 3G3 left the cytosol depleted of p50. Secondly, cytosol fractionated by sucrose gradient revealed that p50 cosedimented with hsp90, confirming the existence of p50 only in association with hsp90.
Whitelaw
M L
ML
Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana 47907.
Hutchison
K
K
Perdew
G H
GH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001358:Avian Sarcoma Viruses; D015153:Blotting, Western; D002460:Cell Line; D002472:Cell Transformation, Viral; D003600:Cytosol; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D015688:Oncogene Protein pp60(v-src) / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D011233:Precipitin Tests; D011506:Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D051381:Rats
D000911:Antibodies, Monoclonal; D006360:Heat-Shock Proteins; D011506:Proteins; D015688:Oncogene Protein pp60(v-src)