Characterization of Interactions between the Anti-apoptotic Protein BAG-1 and Hsc70 Molecular Chaperones

Abstract

The anti-cell death protein BAG-1 binds to 70-kDa heat shock proteins (Hsp70/Hsc70) and modulates their chaperone activity. Among other facilitory roles, BAG-1 may serve as a nucleotide exchange factor for Hsp70/Hsc70 family proteins and thus represents the first example of a eukaryotic homologue of the bacterial co-chaperone GrpE. In this study, the interactions between BAG-1 and Hsc70 are characterized and compared with the analogous GrpE-DnaK bacterial system. In contrast to GrpE, which binds DnaK as a dimer, BAG-1 binds to Hsc70 as a monomer with a 1:1 stoichiometry. Dynamic light scattering, sedimentation equilibrium, and circular dichroism measurements provided evidence that BAG-1 exists as an elongated, highly helical monomer in solution. Isothermal titration microcalorimetry was used to determine the complex stoichiometry and an equilibrium dissociation constant, K D , of 100 nM. Kinetic analysis using surface plasmon resonance yielded a K D consistent with the calorimetrically determined value. Molecular modeling permitted a comparison of structural features between the functionally homologous BAG-1 and GrpE proteins. These data were used to propose a mechanism for BAG-1 in the regulation of Hsp70/Hsc70 chaperone activity.

CAIR-1/BAG-3 forms an EGF-regulated ternary complex with phospholipase C-γ and Hsp70/Hsc70

“…BAG-1 was identi®ed in a screen for bcl-2 binding proteins and has been shown to inhibit apoptosis and promote tumorigenesis (Stuart et al, 1998;Takayama et al, 1995Takayama et al, , 1997. Recent homology cloning identi®ed fragments of at least four other BAG family members, all of which contain a conserved Cterminal domain, the BAG domain (Takayama et al, 1999).…”

“…There is little other homology between BAG-1 and BAG-3, including lack of the BAG-1L nuclear localization signal or the BAG-1 ubiquitin region. The BAG domain was shown by pull down experiments and functional assays to bind to and promote substrate release from both HSP-70 and HSC-70 (Stuart et al, 1998;Takayama et al, 1997Takayama et al, , 1999. Direct binding of BAG-1 to bcl-2 could not be demonstrated upon direct confrontation, however addition of ATP markedly enhanced BAG-1/bcl-2 binding, suggesting the requirement for an HSP-70 intermediate (Takayama et al, 1997).…”

“…They bind ATP through an ATPase domain and have a separate substrate binding domain (Bukau and Horwich, 1998;Pilon and Schekman, 1999). The site of Hsp70/Hsc70 binding to BAG-1 has been de®ned as the ATPase domain (Bukau and Horwich, 1998;Takayama et al, 1997); a 1 : 1 molar binding results in reduction in the refolding function of HSP-70 (Stuart et al, 1998). While other functions for BAG-1 such as binding to and activating Raf-1 have been reported, no link between those functions and HSP-70 have been demonstrated.…”

BAG-1 accelerates cell motility of human gastric cancer cells

“…BAG-1 proteins contain a ubiquitin-like domain, the signi®cance of which is presently unknown, and several copies of a EE (A/V/L/ M)T(Q/R/K)(S/T) repeat. The carboxyl-domain of BAG-1 is required for Hsp70/Hsc70 binding and shows weak amino acid sequence homology with bacterial GrpE, an ADP/ATP exchange factor for the prokaryotic chaperone DNaJ (Stuart et al, 1998;Hartl, 1996). In this regard, BAG-1 has been shown to bind the ATPase domain of Hsc70, promote ADP/ ATP exchange and thereby modulating the chaperone activity in vitro HoÈ hfeld and Jentsch, 1997;Zeiner et al, 1997).…”

Positive and negative regulation of apoptotic pathways by cytotoxic agents in hematological malignancies

“…The constitutively expressed Hsc70 was also shown to interact with BAG-1, an anti-apoptotic protein that binds to Bcl-2, although the functional impact of this interaction remains elusive. 122 Recently, it was shown that Hsp70 could interfere with the signal transduction Leukemia pathway leading to activation of JNK in response to stress. [123][124][125] Hsp27 belongs to the small Hsp subfamily, a group of proteins that vary in size from 15 to 30 kDa and share sequence homologies and biochemical properties such as phosphorylation and oligomerization.…”