BAG-1 modulates the chaperone activity of Hsp70/Hsc70
1997; Springer Nature; Volume: 16; Issue: 16 Linguagem: Inglês
10.1093/emboj/16.16.4887
ISSN1460-2075
Autores Tópico(s)Heat shock proteins research
ResumoArticle15 August 1997free access BAG-1 modulates the chaperone activity of Hsp70/Hsc70 Shinichi Takayama Shinichi Takayama The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author David N. Bimston David N. Bimston Northwestern University, Department of Biochemistry, Molecular and Cell Biology, Rice Institute for Biomedical Research, Evanston, IL, 60208 USA Search for more papers by this author Shu-ichi Matsuzawa Shu-ichi Matsuzawa The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Brian C. Freeman Brian C. Freeman The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Northwestern University, Department of Biochemistry, Molecular and Cell Biology, Rice Institute for Biomedical Research, Evanston, IL, 60208 USA Search for more papers by this author Christine Aime-Sempe Christine Aime-Sempe The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Zhihua Xie Zhihua Xie The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Richard I. Morimoto Richard I. Morimoto Northwestern University, Department of Biochemistry, Molecular and Cell Biology, Rice Institute for Biomedical Research, Evanston, IL, 60208 USA Search for more papers by this author John C. Reed Corresponding Author John C. Reed The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Shinichi Takayama Shinichi Takayama The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author David N. Bimston David N. Bimston Northwestern University, Department of Biochemistry, Molecular and Cell Biology, Rice Institute for Biomedical Research, Evanston, IL, 60208 USA Search for more papers by this author Shu-ichi Matsuzawa Shu-ichi Matsuzawa The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Brian C. Freeman Brian C. Freeman The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Northwestern University, Department of Biochemistry, Molecular and Cell Biology, Rice Institute for Biomedical Research, Evanston, IL, 60208 USA Search for more papers by this author Christine Aime-Sempe Christine Aime-Sempe The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Zhihua Xie Zhihua Xie The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Richard I. Morimoto Richard I. Morimoto Northwestern University, Department of Biochemistry, Molecular and Cell Biology, Rice Institute for Biomedical Research, Evanston, IL, 60208 USA Search for more papers by this author John C. Reed Corresponding Author John C. Reed The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA Search for more papers by this author Author Information Shinichi Takayama1, David N. Bimston2, Shu-ichi Matsuzawa1, Brian C. Freeman1,2,3, Christine Aime-Sempe1, Zhihua Xie1, Richard I. Morimoto2 and John C. Reed 1 1The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, CA, 92037 USA 2Northwestern University, Department of Biochemistry, Molecular and Cell Biology, Rice Institute for Biomedical Research, Evanston, IL, 60208 USA 3University of California, San Francisco, Department of Biochemistry and Biophysics, Box 0448, San Francisco, CA, 94143-0448 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:4887-4896https://doi.org/10.1093/emboj/16.16.4887 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The 70 kDa heat shock family of molecular chaperones is essential to a variety of cellular processes, yet it is unclear how these proteins are regulated in vivo. We present evidence that the protein BAG-1 is a potential modulator of the molecular chaperones, Hsp70 and Hsc70. BAG-1 binds to the ATPase domain of Hsp70 and Hsc70, without requirement for their carboxy-terminal peptide-binding domain, and can be co-immunoprecipitated with Hsp/Hsc70 from cell lysates. Purified BAG-1 and Hsp/Hsc70 efficiently form heteromeric complexes in vitro. BAG-1 inhibits Hsp/Hsc70-mediated in vitro refolding of an unfolded protein substrate, whereas BAG-1 mutants that fail to bind Hsp/Hsc70 do not affect chaperone activity. The binding of BAG-1 to one of its known cellular targets, Bcl-2, in cell lysates was found to be dependent on ATP, consistent with the possible involvement of Hsp/Hsc70 in complex formation. Overexpression of BAG-1 also protected certain cell lines from heat shock-induced cell death. The identification of Hsp/Hsc70 as a partner protein for BAG-1 may explain the diverse interactions observed between BAG-1 and several other proteins, including Raf-1, steroid hormone receptors and certain tyrosine kinase growth factor receptors. The inhibitory effects of BAG-1 on Hsp/Hsc70 chaperone activity suggest that BAG-1 represents a novel type of chaperone regulatory proteins and thus suggest a link between cell signaling, cell death and the stress response. Introduction BAG-1 is a novel multifunctional protein that was first identified based on its ability to bind the anti-apoptotic protein Bcl-2 and to promote cell survival (Takayama et al., 1995). Since its initial discovery as a Bcl-2-binding protein, however, BAG-1 has been reported to interact with and modulate the activities of other proteins. For example, BAG-1 can bind to the catalytic domain of the serine/threonine-specific protein kinase Raf-1, based on yeast two-hybrid experiments, in vitro protein-binding assays and co-immunoprecipitation experiments. Moreover, BAG-1 can stimulate Raf-1 kinase activity through a Ras-independent mechanism (Wang et al., 1996a). Raf-1 also binds to Bcl-2 and cooperates in the suppression of apoptosis (Wang et al., 1994, 1996b); therefore, the interaction of BAG-1 with Raf-1 has been hypothesized to provide a mechanism for local action of this kinase in the vicinity of Bcl-2. Recently, however, BAG-1 was also reported to bind the plasma membrane-associated tyrosine kinase growth factor receptors for hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF), enhancing the ability of these receptors to deliver signals for cell survival in the apparent absence of effects on signal transduction pathways linked to mitogenesis such as the Ras/Raf/MEK cascade (Bardelli et al., 1996). BAG-1 can also bind to several steroid hormone receptors (Zeiner and Gehring, 1995), though the functional significance of this interaction remains unknown. These observations suggest a general facilitatory role for BAG-1 in signal transduction pathways involved in the regulation of cell survival and possibly in the control of other cellular processes as well. How BAG-1 accomplishes these disparate biochemical functions, however, is unclear. Using interaction cloning techniques, we have identified the molecular chaperones, Hsp70 and Hsc70, as BAG-1-binding proteins. The identification of Hsp/Hsc70 as an accessory protein of BAG-1 suggests a possible role for chaperone interactions in regulating the activities of BAG-1. Furthermore, BAG-1 exhibits a novel activity, inasmuch as it functions as a potent inhibitor of chaperone-mediated folding reactions in vitro. Hitherto, no inhibitors of chaperone activity have been described, making BAG-1 the first such protein to be identified. Results BAG-1 interacts with the ATPase domain of Hsc70 During ligand blotting experiments using recombinant BAG-1 protein as a probe, we noted an ∼70 kDa BAG-1-binding protein in extracts prepared from many different types of cells derived from several species (unpublished data). We therefore attempted to identify cDNAs encoding this evolutionarily conserved protein by screening a λgt-11 expression cDNA library using GST–BAG-1 protein as a probe, as well as by yeast two-hybrid methods (Takayama et al., 1995; Takayama and Reed, 1996). Both approaches resulted in the cloning of cDNAs encoding Hsc70, with the overlapping region common to these cDNAs corresponding to amino acids 186–377 of the ATP-binding domain of human Hsc70 (Figure 1A). Indeed, two of the cDNA clones encoded only the ATPase domain of Hsc70 (clones #16 and #23), and were devoid of sequences corresponding to the peptide-binding domain which can interact with proteins in non-native conformations. Figure 1.Interaction cloning results for BAG-1. (A) The results of the interaction cloning experiments are presented schematically, showing the portions of human Hsc70 encoded by the cDNAs which were obtained either by screening a λgt-11 human fetal brain cDNA library using GST–BAG-1 protein as a probe (clones #16 and #23) or by yeast two-hybrid screening of a human T cell cDNA library using LexA–BAG-1 as a bait (clones #13 and #25). The deduced minimal region of Hsc70 that should be necessary for BAG-1 binding is indicated and corresponds to the ATPase domain. (B) EGY48 strain yeast were transformed with 1 μg each of the indicated LexA DNA-binding domain (pGilda) and transactivation domain (pJG4.5) plasmids and plated initially on medium containing glucose to repress the Gal1 promoters in the two-hybrid plasmids and leucine to permit growth of auxotrophs in the absence of two-hybrid interactions. Three independent colonies were chosen randomly from each transformation, patched to medium containing glucose and leucine, and then replica plated onto medium containing galactose (for induction of the two-hybrid plasmids) and leucine. Two-hybrid-based interactions were detected by β-galactosidase filter assays. Data shown represent color development after 1 h incubation with X-gal. The BAG-1 (ΔC) mutant is missing the last 47 amino acids of the murine BAG-1 protein. v-Ras is presented as a typical example of a negative control protein. The Hsc70 partial cDNA clones #13 and #16 are depicted in (A). Download figure Download PowerPoint The interaction of these fragments of Hsc70 with BAG-1 was readily demonstrable by two-hybrid assays, regardless of whether Hsc70 and BAG-1 were expressed as fusion proteins with an appended DNA-binding domain or with an attached transactivation domain (Figure 1B). In contrast, neither Hsc70 nor BAG-1 interacted in two-hybrid assays with a number of control proteins, including Ras, lamin-B or the cytosolic domains of Fas (CD95) and CD40 (Figure 1B and data not presented). Moreover, certain deletion mutants of BAG-1 failed to display interactions with Hsc70 in two-hybrid assays, such as a carboxy-terminal truncation mutant missing the last 47 amino acids of BAG-1 (Figure 1B), further implying a specific interaction between these two proteins. To confirm the interaction of BAG-1 with Hsc70 by an independent method, a GST–BAG-1 fusion protein was produced in bacteria and examined for its ability to bind the in vitro translated 35S-labeled ATPase domain of Hsc70 (Figure 2, left panel). Hsc70(ATPase) bound to GST–BAG-1 but not to several control GST proteins and not to the glutathione–Sepharose beads used to recover GST fusion proteins. Conversely, when the ATPase domain of Hsc70 was produced as a GST fusion protein and assayed for binding to various 35S-labeled in vitro translated proteins, interaction with only BAG-1 was detected. (Figure 2, right panel). Figure 2.BAG-1 binds to the ATPase domain of Hsp/Hsc70. BAG-1 interactions with the ATPase domain of Hsc70 were tested by in vitro binding assays where BAG-1 or various BAG-1 mutants were expressed as GST fusion proteins (left and middle panels) or in vitro translated (IVT) in the presence of [35S]L-methionine (right panel) and incubated with either 35S-labeled, IVT Hsc70 [ATPase domain (residues 1–386)] or with GST–Hsc70(ATPase), respectively. GST fusions representing the cytosolic domain of CD40 or the baculovirus p35 protein were included as controls. Download figure Download PowerPoint The BAG-1 protein contains a domain with similarity to ubiquitin (residues 48–89 in the 219 amino acid long murine BAG-1 protein). Previous studies have shown that the first 89 amino acids of BAG-1 are expendable for its binding to Bcl-2 and to the kinase Raf-1 (Wang et al., 1996a and unpublished data). Similarly, a GST–BAG-1 mutant lacking residues 1–89 also bound to [35S]Hsc70(ATP-binding domain) with efficiency comparable with the full-length BAG-1 (Figure 2). In contrast, a mutant of BAG-1 containing only residues 90–172 failed to interact with Hsc70(ATP-binding domain), implying that the region of BAG-1 from 172 to 219 is essential for Hsc70 binding. A C-terminal truncation mutant of BAG-1 lacking only the last 47 amino acids of BAG-1, GST–BAG-1(1–172), also was unable to bind Hsc70(ATP-binding domain), further demonstrating the importance of the region from 172 to the C-terminus. Though important for BAG-1–Hsc70 interactions, the last 48 amino acids of BAG-1 were insufficient for Hsc70(ATPase) binding (Figure 2), implying that additional residues upstream of amino acid 172 are necessary for binding or for proper folding of the domain in BAG-1 which interacts with Hsc70(ATPase). Taken together, these in vitro binding studies confirm the ability of BAG-1 to interact with the N-terminal ATPase domain of Hsc70, independently of its C-terminal peptide-binding domain. Evidence for BAG-1 interactions with Hsc70 in cells Co-immunoprecipitation assays were performed to explore whether BAG-1 associated with the full-length Hsc70 protein in lysates of mammalian cells. For these experiments, a cDNA encoding a FLAG epitope-tagged BAG-1 protein or the parental plasmid was stably transfected into 293 human kidney epithelial cells. Immunoprecipitations were performed using the anti-FLAG monoclonal antibody M2, and the resulting immune complexes were subjected to SDS–PAGE/immunoblotting using the anti-Hsp/Hsc70 antibody 3a3 or the anti-FLAG monoclonal antibody M2. As shown in Figure 3, Hsc70 was recovered in association with the anti-FLAG immune complexes prepared from the FLAG-BAG-1-expressing 293 cells but not from the control 293-Neo cells. Figure 3.Hsp/Hsc70 co-immunoprecipitates with BAG-1 in mammalian cells. Cell lysates were prepared with or without addition of 10 mM ATP from FLAG-BAG-1-expressing or Neo control 293 cells transfectants and subjected to immunoprecipitation using anti-FLAG tag antibody followed by SDS–PAGE/immunoblotting using either anti-Hsp70/Hsc70 or anti-FLAG antibodies (top and bottom portions of the blot, respectively). Lysates (one-tenth input) were also loaded directly in the gel, without immunoprecipitation (far right; last two lanes) as a control. Download figure Download PowerPoint Previous investigations of Hsc/Hsp70 family proteins have shown that ATP binding induces a conformational change in the ATPase domain (Ha and McKay, 1995; Fung et al., 1996). Addition of 10 mM ATP to cell lysates prior to immunoprecipitation reduced the association of Hsc70 with BAG-1 (Figure 3), suggesting a conformational dependence of this interaction. Similar co-immunoprecipitation results were obtained using monoclonal antibodies directed against the BAG-1 protein (Takayama et al., 1996) rather than relying upon epitope tags and when employing cell lines (such as the Jurkat T-cell leukemia line) that contain relatively high levels of endogenous BAG-1 protein (data not presented). Thus, gene transfer-mediated overexpression of epitope-tagged BAG-1 is not required for demonstrating interactions with Hsc70 in vivo. BAG-1 binds directly to Hsp70 and Hsc70 The association of BAG-1 with Hsc70 could be direct or mediated by accessory proteins. To distinguish between these possibilities, purified GST–BAG-1 was tested for binding in vitro to recombinant, purified Hsc70 and Hsp70. For these experiments, 5 μg of purified Hsc70 or Hsp70 was incubated with 10 μg of GST–BAG-1 immobilized on glutathione–Sepharose, and the BAG-1-associated Hsc/Hsp70 proteins were detected by immunoblotting using an anti-Hsp/Hsc70 antibody (Figure 4). Both Hsc70 and Hsp70 bound to GST–BAG-1, with ∼20% of the input chaperone recovered in association with BAG-1, but not to a control GST fusion protein (CD40). The addition of ATP to these binding assays significantly reduced binding of Hsc70 to GST–BAG-1, whereas ADP and non-hydrolyzable ATPγS had less effect. Figure 4.BAG-1 interaction with Hsc70 is regulated by ATP hydrolysis. Equivalent amounts of purified GST–BAG-1 or GST–CD40 cytosolic domain (negative control) immobilized on glutathione–Sepharose were incubated with either purified Hsp70 or Hsc70 (5 μg) in the presence of 10 mM ATP, ADP or ATPγS as indicated. After washing the beads extensively, associated Hsp/Hsc70 was detected by immunoblotting. One μg of purified Hsc70 or Hsp70 was loaded directly in the gels as a control. Download figure Download PowerPoint Molecular sieve chromatography was employed as an additional means of evaluating the BAG-1–Hsp70 interaction. When analyzed by itself, Hsp70 eluted from a Sepharose-6 column with a peak corresponding to Hsp70 homodimers, and BAG-1 eluted from the column with a peak corresponding to BAG-1 homodimers (Figure 5). When BAG-1 and Hsp70 were co-incubated in equimolar quantities and chromatographed, all of the BAG-1 and Hsc70 proteins eluted in complexes >120 kDa, thus confirming the efficient formation of BAG-1–Hsp70 complexes. The elution characteristics of the BAG-1–Hsp70 complexes were consistent with a minimal stoichiometry of 2:2 (heterotetramer), though larger complexes were also present. Dimer formation by BAG-1 alone was confirmed by dynamic light-scattering experiments (not shown). Figure 5.Gel-sieve chromatographic analysis of BAG-1–Hsp70 complexes. Purified BAG-1, Hsp70 or the combination of BAG-1 and Hsc70 were subjected to gel-sieve chromatography and the eluted fractions were analyzed for the presence of BAG-1 and Hsp70 by SDS–PAGE with Coomassie staining. The elution positions of molecular weight standard proteins are indicated. Dark arrows indicate Hsp70, while open arrows denote BAG-1. Download figure Download PowerPoint BAG-1 modulates the in vitro chaperone activity of Hsp70 Unlike other members of the chaperone family (Hsp90, cyclophilin 40, p23) which maintain unfolded proteins in a transitional state amenable to subsequent refolding, Hsp/Hsc70 can also complete the process of refolding non-native proteins to their native structures through a ATP and co-chaperone (Hdj-1)-dependent mechanism (Freeman and Morimoto, 1996; Freeman et al., 1996). We therefore evaluated the effects of purified GST–BAG-1 on the ability of Hsc70 and Hsp70 to assist in the refolding of denatured β-galactosidase. In the absence of molecular chaperones, denatured β-galactosidase aggregates and does not regain enzymatic activity. In contrast, in the presence of ATP and Hdj-1, denatured, enzymatically inactive β-galactosidase can be refolded by Hsp/Hsc70 with recovery of ∼50% of the enzymatically active protein. When BAG-1 was added to the assay, Hsc70-mediated refolding of denatured β-galactosidase was inhibited in a concentration-dependent manner, with complete inhibition achieved at a 1:1 molar ratio (Figure 6A). Essentially identical results were noted for BAG-1 inhibition of Hsp70-mediated refolding of β-galactosidase (Figure 6B) Figure 6.BAG-1 inhibits Hsc70- and Hsp70-mediated refolding of denatured β-galactosidase. (A) BAG-1-mediated inhibition of Hsc70-dependent refolding was examined by diluting denatured β-galactosidase (3.4 μM final) into refolding buffer containing 1.6 μM Hsc70 and 3.2 μM Hdj-1 without (I) or with increasing concentrations of full-length BAG-1: 0.05 (III), 0.1 (IV), 0.2 (V), 0.4 (VI), 0.8 (VII), 1.6 (VIII), 3.2 μM BAG-1 (IX). As a control for inhibition of refolding, denatured β-galactosidase was diluted into refolding buffer containing Hsc70, Hdj-1 and 3.2 μM BSA (II). (B) The effect of BAG-1 on Hsp70-mediated β-galactosidase refolding was examined in a similar fashion by adding increasing concentrations of BAG-1 to a refolding reaction containing 1.6 μM Hsp70 and 3.2 μM hdj-1 (I): 0.2 (III), 0.4 (IV), 0.8 (V), 1.6 (VI), 3.2 μM BAG-1 (VII). As a control for spontaneous refolding, denatured β-galactosidase was diluted into refolding buffer containing 1.6 μM BSA (II). (C) The effects of BAG-1 ΔC (contains amino acids 1–172) and BAG-1 ΔN (contains amino acids 90–219) deletion mutants on chaperone-mediated refolding were examined: 0.4 μM BAG-1 ΔC mutant (III), 0.8 μM BAG-1 ΔC mutant (IV), 1.6 μM BAG-1 ΔC mutant (V), 3.2 μM BAG-1 ΔC mutant (VI), 0.4 μM BAG-1 ΔN mutant (VII), 0.8 μM BAG-1 ΔN mutant (VIII), 1.6 μM BAG-1 ΔN mutant (X), 3.2 μM BAG-1 ΔN mutant (XI). Denatured β-galactosidase was diluted into refolding buffer containing either Hsc70 and Hdj-1 (I) or Hsc70, Hdj-1 and 3.2 μM (IX) as positive controls and 1.6 μM BSA (II) as a negative control and β-galactosidase assays were performed as described (Freeman and Morimoto, 1996). Download figure Download PowerPoint The specificity of the inhibitory effect of BAG-1 on the chaperoning activities of Hsc70 and Hsp70 was confirmed by use of a GST–BAG-1 (ΔC) mutant deleted for the C-terminal 47 residues of BAG-1. This mutant form of BAG-1 failed to bind to Hsc/Hsp70 and did not affect the refolding assay. In contrast, a GST–BAG-1 (ΔN) mutant retaining Hsp/Hsc70 binding ability was nearly as active as the full-length BAG-1 in inhibiting Hsc/Hsp70-mediated refolding of denatured β-galactosidase (Figure 6C and data not shown). Thus, BAG-1 binding to Hsp/Hsc70 correlates with BAG-1-mediated suppression of Hsp/Hsc70 chaperone activity. BAG-1 stably associates with the chaperone–substrate complex Hsp70 family members bind to non-native proteins via the C-terminal peptide-binding domain. To explore the effect of BAG-1 on interactions of Hsp70 with a denatured protein substrate, Hsp70 was incubated with the permanently unfolded substrate 125I-labeled reduced carboxymethylated α-lactalbumin (RCMLA). The formation of Hsp70–RCMLA heteromeric complexes in the presence or absence of purified GST–BAG-1 was monitored by native polyacrylamide gel electrophoresis. Hsp70, but not BAG-1 or bovine serum albumin (BSA), forms a complex with RCMLA, resulting in the appearance of a slower migrating band (Figure 7A). In contrast, when GST–BAG-1 was added at a 1:1 molar ratio with Hsp70, a ‘super-shifted’ complex was detected, consistent with the idea that BAG-1 stably associates with the Hsp70–RCMLA complex. Figure 7.Differential effects of BAG-1 on Hsp70 and DNaK interactions with a denatured protein substrate. 125I-labeled RCMLA was incubated with 14 μM Hsp70 alone or in combination with various molar ratios of (A) wild-type GST–BAG-1 or (B) GST–BAG-1 mutants lacking either the first 89 amino acids, GST–BAG-1 (ΔN), or the last 47 amino acids, GST–BAG-1 (ΔC), of the murine BAG-1 protein. In (C), 14 μM bacterial DNaK was employed instead of Hsp70. The samples were analyzed by non-denaturing polyacrylamide gel electrophoresis, followed by autoradiography. As controls, BSA or GST proteins were incubated with RCLMA, demonstrating no binding. Alternatively, RCLMA was added without other proteins. The positions of free and bound RCLMA are indicated. Download figure Download PowerPoint At higher BAG-1:Hsp70 ratios, typically little or no complexes with the [125I]RCMLA substrate were detected (see Figure 7A, lane 6), implying that BAG-1 either prevented the formation of Hsp70–RCMLA complexes or promoted their rapid disassembly under these conditions. We cannot exclude the rather unlikely possibility, however, that, at higher BAG-1 concentrations, the size of the trimeric BAG-1–Hsp70–RCMLA complexes impairs their ability to enter gels. The specificity of the interaction of BAG-1 with Hsp70–RCLMA complexes was confirmed by experiments employing deletion mutants of BAG-1. As shown in Figure 7B, the BAG-1 (ΔN) protein, which binds Hsc/Hsp70 and inhibits refolding, produced a super-shifted complex, whereas BAG-1 (ΔC) did not. These data strongly suggest that BAG-1 binds to chaperone–RCMLA heteromers and is a component of these super-shifted complexes. To exclude the possibility that BAG-1 merely competes with RCMLA for binding to the peptide-binding site of the 70 kDa chaperone, the effects of BAG-1 were tested on the binding of RCMLA by either a truncation mutant of Hsp70 consisting only of the peptide-binding domain [residues 386–640] and therefore lacking the ATPase domain needed for BAG-1 binding (not shown), or by DnaK, a bacterial homolog of the eukaryotic Hsp/Hsc70 family. The Hsp70 [386–640]–RCMLA (not shown) and DnaK–RCMLA complexes (Figure 7C) were unaffected by addition of GST–BAG-1, thus confirming the specificity of the functional interaction of BAG-1 with Hsc70 and Hsp70 and providing additional evidence that BAG-1 is not recognized as a denatured substrate by the 70 kDa chaperones. When taken together with the data demonstrating inhibition of Hsp/Hsc70 chaperone activity by BAG-1 (Figure 6), the finding that BAG-1 binds to Hsp/Hsc70–substrate complexes suggests that the interaction between chaperone and substrate is affected by BAG-1 such that chaperone folding activity is inhibited. ATP-dependent interaction of BAG-1 with Bcl-2 suggests a role for chaperone proteins Attempts to demonstrate binding of BAG-1 to Bcl-2 using purified recombinant proteins have been unsuccessful, suggesting the need for an additional protein or proteins (unpublished observations). These findings prompted us to explore the possibility that the 70 kDa chaperones might regulate interactions between Bcl-2 and BAG-1. The ability of Hsp70 and Hsc70 to alter the conformations of proteins and to promote assembly of protein complexes is dependent on ATP hydrolysis (reviewed in Pratt and Welsh, 1994). We therefore examined the ATP dependence of Bcl-2 interactions with BAG-1, using cell lysates that contain chaperone components including Hsc70. When purified GST–BAG-1 was incubated with cell lysates derived from insect Sf9 cells infected with a recombinant baculovirus encoding Bcl-2, >10-fold more Bcl-2 bound to GST–BAG-1 when 10 mM ATP was added compared with extracts without ATP supplementation (Figure 8A). In contrast, Bcl-2 did not bind to GST–CD40 or other control GST fusion proteins, regardless of ATP levels; nor did other control proteins such as β-galactosidase bind to GST–BAG-1 (Figure 8A and data not shown). Treating cell lysates with apyrase to consume endogenous ATP completely abolished the small amount of Bcl-2 binding to GST–BAG-1 that occurred in the absence of added ATP (data not presented). Figure 8.Interaction of BAG-1 with Bcl-2 displays ATP dependence. (A) Lysates from Sf9 insect cells that had been infected with either Bcl-2- or β-galactosidase-producing recombinant baculoviruses (Wang et al., 1996a) were incubated directly or after adding 10 mM ATP with either GST–BAG-1 or GST–CD40 immobilized on glutathione–Sepharose. BAG-1-associated Bcl-2 protein was detected by immunoblotting. (B) Lysates were prepared from the human B cell lymphoma cell line RS11846 (contains high levels of Bcl-2 due to t[14;18] translocation) and incubated with GST fusion proteins immobilized on glutathione–Sepharose with or without 10 mM ATP or ATPγS. BAG-1-associated Bcl-2 was detected by immunoblotting. Lysate from cells was also run directly in the gel (first lane) as a control. (C) Lysates were prepared with or without 10 mM ATP from Jurkat T-cells which had been stably transfected with either Bcl-2 or Neo control expression plasmids (Takayama et al., 1995) and immunoprecipitations were performed using either the IgG1 anti-BAG-1 monoclonal KS6C8 (Takayama et al., 1996) or an IgG1 control antibody. Immune complexes were analyzed by SDS–PAGE/immunoblot assay using anti-Bcl-2 polyclonal antiserum. Lysates from cells (one-tenth input) were also run directly in the gel as a control (far left/first two lanes). Download figure Download PowerPoint Similarly, binding of GST–BAG-1 to Bcl-2 protein in mammalian cell lysates also was increased markedly by addition of ATP. Furthermore, a requirement for ATP hydrolysis or nucleotide-specific conformational changes was suggested by use of non-hydrolyzable ATPγS, which failed to promote BAG-1 interactions with Bcl-2 in either mammalian or insect cell lysates (Figure 8B and data not shown). In addition to experiments with GST–BAG-1 fusion protein, the ability of Bcl-2 to co-immunoprecipitate with endogenous BAG-1 was greatly enhanced if 10 mM ATP was included in the cell lysis buffer. Figure 8C, for example, shows the results derived from Jurkat T cells that had been stably transfected with either a Bcl-2-encoding plasmid (Jurkat-Bcl-2) or the same parental plasmid without an insert (Jurkat-Neo). When the endogenous BAG-1 protein was immunoprecipitated from these cells using a monoclonal antibody specific for the human BAG-1 protein (KS6C8), far more Bcl-2 protein was recovered in associated with BAG-1 when ATP was added to the lysates. Similar results were also obtained using lysates prepared from other mam
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