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,William B Pratt

doi:10.1074/jbc.274.48.34134

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 approximately 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.

Highlights

A number of signaling proteins, including several members of the nuclear receptor family, the dioxin receptor, nitric-oxide synthase, and several protein kinases, exist in cytosolic complexes with the ubiquitous and abundant heat shock protein1 hsp90
We have shown previously that immunoadsorption of hsp organizer protein (Hop) from reticulocyte lysate is accompanied by co-immunoadsorption of hsp90, hsp70, and hsp40 as an hsp901⁄7Hop1⁄7hsp701⁄7hsp40 heterocomplex [8]
Hip and BAG-1 compete for binding to the ATPase domain of hsp70 [28], and they must exist in separate hsp901⁄7Hop1⁄7hsp701⁄7hsp40 heterocomplexes

Summary

Introduction

A number of signaling proteins, including several members of the nuclear receptor family, the dioxin receptor, nitric-oxide synthase, and several protein kinases, exist in cytosolic complexes with the ubiquitous and abundant heat shock protein (hsp) hsp (for review, see Refs. 1 and 2). A number of signaling proteins, including several members of the nuclear receptor family, the dioxin receptor, nitric-oxide synthase, and several protein kinases, exist in cytosolic complexes with the ubiquitous and abundant heat shock protein (hsp) hsp BAG-1 was originally cloned from a mouse library as a Bcl-2-binding protein and shown to have anti-apoptotic activity [13]. BAG-1 was shown to form complexes with some signaling proteins, including receptors for hepatocyte growth factor and platelet-derived growth factor [14], the serine/threonine protein kinase Raf-1 [15], and the retinoic acid receptor [16]. An isoform of the human BAG-1 protein called RAP46 (46-kDa receptor-associated protein; BAG-1M) was cloned and shown to bind to members of the nuclear receptor family, including the glucocorticoid receptor (GR) [17]. BAG-1M (RAP46) was found to be a negative regulator of GR activity [23], whereas BAG-1L, but not BAG-1 or BAG-1M, forms complexes with the androgen receptor and enhances androgen receptor-mediated transactivation of a reporter gene [22]

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Results

Conclusion