Differential Effects of the hsp70-binding Protein BAG-1 on Glucocorticoid Receptor Folding by the hsp90-based Chaperone Machinery*
- Kimon C. Kanelakis‡,
- Yoshihiro Morishima‡,
- Kurt D. Dittmar‡,
- Mario D. Galigniana‡,
- Shinichi Takayama§,
- John C. Reed§ and
- William B. Pratt‡¶
- From the ‡Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109 and the§Burnham Institute, Cancer Research Center, La Jolla, California 92037
Abstract
The heat shock protein hsp70/hsc70 is a required component of a five-protein (hsp90, hsp70, Hop, hsp40, and p23) minimal chaperone system reconstituted from reticulocyte lysate that forms glucocorticoid receptor (GR)·hsp90 heterocomplexes. BAG-1 is a cofactor that binds to the ATPase domain of hsp70/hsc70 and that modulates its chaperone activity. Inasmuch as BAG-1 has been found in association with several members of the steroid receptor family, we have examined the effect of BAG-1 on GR folding and GR·hsp90 heterocomplex assembly. BAG-1 was present in reticulocyte lysate at a BAG-1:hsp70/hsc70 molar ratio of ∼0.03, and its elimination by immunoadsorption did not affect GR folding and GR·hsp90 heterocomplex assembly. At low BAG-1:hsp70/hsc70 ratios, BAG-1 promoted the release of Hop from the hsp90-based chaperone system without inhibiting GR·hsp90 heterocomplex assembly. However, at molar ratios approaching stoichiometry with hsp70, BAG-1 produced a concentration-dependent inhibition of GR folding to the steroid-binding form with corresponding inhibition of GR·hsp90 heterocomplex assembly by the minimal five-protein chaperone system. Also, there was decreased steroid-binding activity in cells that were transiently or stably transfected with BAG-1. These observations suggest that, at physiological concentrations, BAG-1 modulates assembly by promoting Hop release from the assembly complex; but, at concentrations closer to those in transfected cells and some transformed cell lines, hsp70 is continuously bound by BAG-1, and heterocomplex assembly is blocked.
Footnotes
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↵* This work was supported by National Institutes of Health Grants DK31573 (to W. B. P.) and CA67329 (to J. C. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom to address correspondence: Dept. of Pharmacology, University of Michigan Medical School, 1301 Medical Science Research Bldg. III, Ann Arbor, MI 48109-0632. Tel.: 734-764-5414; Fax: 734-763-4450.
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↵2 In this paper, we use the term hsp70 collectively to refer to both the heat shock-induced hsp70 and the constitutively expressed heat shock cognate hsc70.
- Abbreviations:
- hsp
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heat shock protein
- GR
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glucocorticoid receptor
- GFP
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green fluorescent protein
- DMEM
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Dulbeccos' modified Eagle's medium
- TES
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2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid
- PAGE
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polyacrylamide gel electrophoresis
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- Received June 21, 1999.
- Revision received September 3, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
