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Vol. 15, Issue 9, 4003-4010, September 2004
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Institut für Zellbiologie and Bonner Forum Biomedizin, Rheinische Friedrich-Wilhelms-Universität Bonn, D-53121 Bonn, Germany
Submitted April 8, 2004;
Revised May 14, 2004;
Accepted June 11, 2004
Monitoring Editor: Peter Walter
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Hartl and Hayer-Hartl, 2002). However, the cochaperone CHIP is able to switch chaperone activity from protein folding to protein degradation by virtue of its ubiquitin ligase activity (Connell et al., 2001; Demand et al., 2001; Höhfeld et al., 2001; Meacham et al., 2001; Murata et al., 2001). CHIP interacts with the carboxy termini of the cytoplasmic and nuclear chaperones Hsc70 and Hsp90, and mediates the covalent attachment of the degradation marker ubiquitin to chaperone-bound client proteins. In this way, CHIP triggers the turnover of chaperone clients such as the glucocorticoid hormone receptor and the oncogenic receptor tyrosine kinase ErbB2 by the 26S proteasome (Connell et al., 2001; Demand et al., 2001; Xu et al., 2002). CHIP apparently alters the balance between folding and degradation during the Hsc70- and Hsp90-mediated regulation of signaling pathways. In addition, CHIP plays a prominent role in protein quality control, as is illustrated by its involvement in the degradation of immature forms of cystic fibrosis transmembrane conductance regulator (CFTR) (Meacham et al., 2001). CFTR is an ion channel in the plasma membrane of epithelial cells and loss-of-function mutations in the gene encoding CFTR cause cystic fibrosis (Kopito, 1999; Riordan, 1999). The most common disease-causing allele, 
F508, drastically interferes with the protein's ability to fold, essentially barring it from functional expression in the plasma membrane. However, also wild-type CFTR folds very inefficiently, and <30% of the protein reaches the plasma membrane (Kopito, 1999). While trafficking through the endoplasmic reticulum (ER), immaturely glycosylated and aberrantly folded forms of CFTR are recognized by quality control systems and are eventually directed to the proteasome for degradation (Jensen et al., 1995; Ward et al., 1995; Kostova and Wolf, 2003). Hsc70 and Hsp90 participate in CFTR quality control, because the membrane protein exposes large domains into the cytoplasm. The chaperones assist the folding of these cytoplasmic domains (Yang et al., 1993; Loo et al., 1998; Meacham et al., 1999; Farinha et al., 2002). However, through binding to Hsc70, CHIP is able to gain access to immature CFTR and induces the ubiquitylation and proteasomal degradation of the membrane protein (Meacham et al., 2001).
During the sorting of chaperone clients to the proteasome, CHIP can cooperate with the Hsc70 cochaperone BAG-1. The two cochaperones interact simultaneously with Hsc70, as BAG-1 binds to the amino terminal ATPase domain of the chaperone and leaves the carboxy terminus of Hsc70 available for an association with CHIP (Demand et al., 2001; Alberti et al., 2002). BAG-1 is intimately involved in the regulation of the ATP-dependent peptide binding and release cycle of Hsc70 by stimulating nucleotide exchange (Höhfeld and Jentsch, 1997; Sondermann et al., 2001). Furthermore, BAG-1 associates with the proteasome through an integrated ubiquitin-like domain and thereby recruits Hsc70 chaperone complexes to the proteasome (Lüders et al., 2000). As a consequence BAG-1 stimulates the CHIP-induced degradation of the glucocorticoid hormone receptor (Demand et al., 2001). A cooperation of diverse cochaperones apparently provides additional levels of regulation to alter chaperone-assisted folding and degradation pathways. Conceivably, such a cooperation of cochaperones also may allow to confine the destructive activity of CHIP. We therefore investigated how cochaperones other than BAG-1 influence CHIP-mediated degradation. We were particularly interested in the cochaperone HspBP1, which is a nucleotide release factor of Hsc70 of unknown cellular function (Raynes and Guerriero, 1998; Kabani et al., 2002). HspBP1 is expressed at a similar level as CHIP in human HeLa cells (0.05% of cellular protein; Alberti, unpublished observation) and displays a similar tissue distribution (Raynes and Guerriero, 1998; Ballinger et al., 1999). Furthermore, HspBP1 interacts with the ATPase domain of Hsc70, like BAG-1, and may therefore modulate the function of the Hsc70/CHIP complex. In fact, here we identify HspBP1 as an inhibitor of the CHIP ubiquitin ligase and as an essential component of the cellular surveillance system that monitors the folded state of CFTR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), anti-FLAG (M2; Sigma-Aldrich, St. Louis, MO), anti-Hsc/Hsp70 (SPA-820; StressGen Biotechnologies, San Diego, CA), anti-HspBP1 (FL4; Delta Biolabs, Campbell, CA), anti-luciferase (Promega, Madison, WI), and anti-raf-1 (Santa Cruz).
Rat Hsc70, wheat E1, and BAG-1S, Hsp40, UbcH5b, and CHIP (all of human origin) were purified after recombinant expression as described previously (Höhfeld and Jentsch, 1997; Lüders et al., 2000; Demand et al., 2001; Alberti et al., 2002). Purified bovine ubiquitin was purchased from Sigma-Aldrich. For recombinant expression of His-tagged HspBP1 Escherichia coli BL21(DE3) cells were transformed with pET28a-hspBP1 (Raynes and Guerriero, 1998). Bacterial lysates were applied to Ni2+-NTA agarose chromatography. Affinity purified HspBP1 was further purified through anion exchange chromatography on a DEAE Sepharose column. For purification of untagged HspBP1, the hspBP1 coding region was cloned into pTYB12 and purified via a self-cutting intein/chitin binding domain as described by the manufacturer (New England BioLabs, Beverly, MA).
Immunoprecipitation and Binding Experiments
To isolate HspBP1 and CHIP immunocomplexes, HeLa cells were transfected with the plasmids pCMVTag2-hspBP1 and pCMVTag2-CHIP, respectively, by using the CalPhos transfection system (BD Biosciences Clontech, Palo Alto, CA). When effects of HspBP1 on the association of CHIP with Hsc70 were investigated, cells were cotransfected with pCMVTag2-CHIP and pcDNA3.1-hspBP1 or an equal amount of empty pcDNA3.1. Two days after transfection, HeLa cells were lysed in 25 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.2, 100 mM KCl, and 0.5% Tween 20 (buffer A) containing 5 mM EDTA and Complete protease inhibitors (Roche Diagnostics, Indianapolis, IN). The lysate was centrifuged at 20,000 x g for 20 min at 4°C, and the soluble supernatant fraction was used for immunoprecipitation. Anti-FLAG antibody (M2; Sigma-Aldrich) was added at a concentration of 10 µg/ml, and samples were incubated for 1 h at 4°C. After addition of protein G-Sepharose, samples were incubated for another hour. Alternatively, M2-agarose (Sigma-Aldrich) was used at a concentration of 30 µl of agarose per milliliter of lysate, and samples were incubated for 3 h at 4°C. The Sepharose was collected by centrifugation and washed five times with buffer A and once with buffer A lacking detergent. Isolated immunocomplexes were incubated for 20 min at 30°C in detergent-free buffer A containing 2 mM MgCl2, 1 mM ATP, and 1 mM phenylmethylsulfonyl fluoride, followed by the precipitation of ATP-eluted proteins with trichloroacetic acid. The Sepharose beads were washed once more with buffer A, followed by elution of associated proteins by boiling in SDS-PAGE loading buffer. When M2-agarose was used, agarose-bound proteins were eluted with 0.1 M glycine, pH 3.5.
To test for interactions of HspBP1 with Hsc70, in vitro binding assays with immobilized His-tagged HspBP1 were performed. For immobilization of His-tagged HspBP1 10 µg of purified protein was incubated with 50 µl of Ni2+-NTA agarose in buffer B (20 mM Tris-HCl, pH 7.6, 100 mM KCl, 20 mM imidazole) for 2 h with gentle agitation, resulting in 
20% binding. Samples were washed once with buffer B, followed by addition of Hsc70, BAG-1S, and CHIP at one-fifth of the initial HspBP1 concentration. Samples were incubated for additional 2 h at 4°C, followed by four washing steps with buffer B. Proteins were eluted in 20 mM Tris, pH 7.6, 100 mM KCl, 200 mM imidazole for 15 min at room temperature and precipitated by the addition of trichloroacetic acid.
The competition of BAG-1 and HspBP1 in Hsc70 binding was analyzed with purified BAG-1 immobilized to protein G-Sepharose beads with a specific anti-BAG-1 antibody. The binding reaction was performed with 1.33 µg of BAG-1S and equimolar amounts of Hsc70 for 1 h at 4°C in 200 µl of buffer A containing 5 mM EDTA. When indicated HspBP1 was added in equimolar amounts or in a two- or fivefold molar excess over BAG-1. Samples were mixed with 4 µg of anti-BAG-1 antibody or control IgG and incubated for another hour at 4°C. After addition of protein G-Sepharose, samples were further incubated for 1 h at 4°C. The Sepharose was collected by centrifugation and was washed five times with buffer A. Sepharose-associated proteins were eluted by addition of SDS-PAGE loading buffer.
In Vitro Ubiquitylation
Ubiquitylation of Raf-1 in vitro was performed as described previously in the presence of 0.3 µM Hsp40, 3 µM Hsc70, 4 µM UbcH5, and 3 µM CHIP (Demand et al., 2001). When indicated, His-tagged HspBP1 was added. Samples were analyzed by SDS-PAGE and immunoblotting with anti-raf-1, anti-Hsc/Hsp70, and anti-HspBP1 antibodies.
To investigate the ubiquitylation of heat-denatured firefly luciferase, the protein (0.26 µM) was incubated at 43°C for 10 min in buffer C (20 mM MOPS, pH 7.2, 50 mM KCl, 4 mM MgCl2, 4 mM ATP, and 2 mM DTT) in the presence of Hsp40 (3.2 µM) and Hsc70 (4.7 µM); or in the presence of Hsp90 (11 µM) under conditions described previously (Murata et al., 2001). After heating, samples were chilled on ice, and subsequent ubiquitylation was performed under the same conditions as described above for Raf-1 without further addition of Hsc70 and Hsp40.
For reconstitution of CFTR ubiquitylation, in vitro transcription of CFTR was performed with linearized pcDNA3.1-CFTR as template by using the T7 RiboMax system according to the protocol of the manufacturer (Promega). Using nuclease-treated rabbit reticulocyte lysate (Promega), radiolabeled CFTR was synthesized in the presence of porcine pancreas microsomes (0.1 eq/µl), which were isolated according to Walter and Blobel (1983). After incubation at 30°C for 90 min, translation reactions were stopped by addition of 2 mM puromycin, 0.5 mM cycloheximide, 2 mM unlabeled methionine, and 10 µM MG-132 (Calbiochem, San Diego, CA). For ubiquitylation of CFTR, 20 µl of the translation reactions received 1 µM UbcH5, 1 µM CHIP, and increasing amounts of HspBP1 (1, 2, and 5 µM) as indicated. The total volume of the samples was adjusted to 25 µl with 20 mM MOPS, pH 7.2, 100 mM KCl containing 10 µM MG-132 and EDTA-free Complete protease inhibitor. Samples were incubated for 1 h at 16°C and were subsequently subjected to immunoprecipitation to isolate ubiquitylated CFTR. To this end, samples were split into two equal aliquots, diluted 50-fold in buffer D (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 10% glycerol, 2 mM EDTA, Complete protease inhibitor) and mixed with either 5 µg/ml control IgG or 5 µg/ml anti-ubiquitin antibody (FK2; Affiniti Research Products, Biomol International, Plymouth Meeting, PA). The samples were incubated for 2 h at 4°C with gentle agitation, followed by addition of protein G-Sepharose and an additional incubation for 1 h at 4°C. Sepharose beads were collected by centrifugation and washed three times with buffer D. Sepharose-bound proteins were eluted by addition of SDS-PAGE sample buffer.
Luciferase Aggregation Assay
To assay luciferase aggregation, 3 µM Hsc70, 2 µM Hsp40, 3 µM His-HspBP1, and 3 µM CHIP were preincubated in indicated combinations for 5 min at room temperature in 20 mM MOPS, pH 7.2, 50 mM KCl, 2 mM MgCl2, 1 mM DTT, and 2 mM ATP. Subsequently, luciferase (0.6 µM) was added, and samples were incubated at 42°C for 15 min. The reaction was stopped by addition EDTA to a concentration of 5 mM, and samples were centrifuged at 120,000 x g for 20 min at 4°C. Supernatant and pellet fractions were separated and analyzed by SDS-PAGE and immunoblotting with an anti-luciferase antibody, followed by densitometric quantification.
CFTR Turnover and Maturation
To determine the steady-state levels of CFTR in the presence of overexpressed HspBP1 and CHIP, human embryonic kidney (HEK)293 cells were seeded in six-well plates and transfected with 0.38 µg of pcDNA3.1-CFTR or pcDNA3-CFTR F508 by using Effectene transfection reagent (QIAGEN, Valencia, CA). When indicated, 0.38 µg of pcDNA3.1-chip and 0.38 or 0.75 µg of pcDNA3.1-hspBP1 was cotransfected. The total amount of added DNA was kept constant at 1.5 µg by addition of pcDNA3.1. Twenty-four hours after transfection the cells were washed once with phosphate-buffered saline (PBS) and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, and 0.2% sodium dodecyl sulfate) containing Complete protease inhibitor. The lysate was centrifuged at 20,000 x g for 20 min at 4°C, and the supernatant was used as a soluble extract. For immunoblotting, 60 µg of total protein was loaded onto an SDS-PAGE gel, and extracts were analyzed for CFTR, Hsc70, and cochaperone expression.
To analyze the degradation kinetics of CFTR, HEK293 cells were seeded in six-well plates and were transfected with 0.25 µg of pcDNA3.1-CFTR and 0.375 µg of pcDNA3.1-CHIP or the same amount of pcDNA3.1-hspBP1 as indicated. The total amount of added DNA was kept constant at 1 µg/well by addition of pcDNA3.1. After 24 h, cells were shifted to methionine-free medium for 1 h, followed by metabolic labeling for 30 min in the presence of [35S]methionine at a concentration of 150 µCi/ml. Cells were washed once with PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 and lysed immediately in lysis buffer containing Complete protease inhibitor (time point 0) or incubated in medium supplemented with 1 mM methionine and 0.5 mM cycloheximide for the indicated chase periods. The lysates were centrifuged at 20,000 x g for 20 min at 4°C, and the resultant supernatants were precleared by addition of protein G-Sepharose and incubation for 30 min at 4°C. After removal of the Sepharose by centrifugation, supernatant fractions were mixed with 0.4 µg of anti-CFTR antibody (24-1; R&D Systems, Minneapolis, MN). Samples were incubated for 1 h at 4°C, protein G-Sepharose was added, and samples were further incubated for 1 h. Immunocomplexes were washed three times with lysis buffer. Isolated CFTR was deglycosylated for 2 h at 25°C with 250 U of PNGase F (New England BioLabs) in digestion buffer (50 mM sodium phosphate, pH 7.5, 1% Nonidet P-40, 10% glycerol, 0.5% SDS, and 100 mM 
-mercaptoethanol). Deglycosylation was stopped by addition of SDS-PAGE loading buffer, and samples were analyzed by SDS-PAGE. Quantification of CFTR was carried out by densitometric analysis using a PhosphorImager (PerkinElmer Life and Analytical Sciences, Boston, MA).
Biotinylation of glycosylated CFTR exposed on the surface of intact cells was performed as described by Lisanti et al. (1989). Twenty-four hours after transfection, cells were washed twice with ice-cold PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBS-c/m) and were incubated with 10 mM NaIO4 in PBS-c/m for 30 min at 4°C in the dark to oxidize the carbohydrate moieties of surface glycoproteins. Cells were washed twice with PBS-c/m and once with acetate buffer (100 mM sodium acetate, pH 5.5, 0.1 mM CaCl2, and 1 mM MgCl2) and were biotinylated using 2 mM biotin-LC-hydrazide (Pierce Chemical, Rockford, IL) in acetate buffer for 30 min at 4°C. Unbound biotin was removed by washing three times with ice-cold PBS-c/m followed by cell lysis in lysis buffer containing Complete protease inhibitor. Lysates were centrifuged for 20 min at 20,000 x g, and the supernatant (1 mg of protein in 1 ml of lysis buffer) was incubated with 100 µl of Avidin beads (UltraLink NeutrAvidin; Pierce Chemical) at 4°C for 1 h. Beads were collected by centrifugation and washed three times with lysis buffer followed by elution of Avidin-bound proteins in SDS-PAGE sample buffer.
HspBP1 levels of HEK293 cells were reduced using a small hairpin RNA (shRNA) expression plasmid, which was generated according to the pSUPER RNAi system (Oligoengine, Seattle, WA). The siRNA produced in pSUPERHspBP1-transfected cells targets the sequence CAT GGA CAA TGC CGC AGA C of the human HspBP1 gene. HEK293 cells were seeded in six-well plates and were transfected with 0.38 µg of pcDNA3.1-CFTR by using the Effectene transfection reagent (QIAGEN). When indicated 0.56 µg of pSUPERHspBP1, the same amount of pSUPER, 0.56 µg of pcDNA3.1 and pcDNA3.1-hspBP1 were transfected. Cells were harvested 72 h after transfection and processed as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Because HspBP1 and CHIP bind to different domains of Hsc70, it was conceivable that the chaperone provides a physical link between the two cochaperones. Therefore, His-tagged HspBP1 was immobilized on an affinity resin and incubated with purified Hsc70 and CHIP in different combinations. An interaction between immobilized HspBP1 and CHIP was indeed largely dependent on the presence of Hsc70 (Figure 1B). However, a low amount of CHIP also interacted with HspBP1 in the absence of Hsc70, suggesting that a low-affinity direct interaction between the two cochaperones may contribute to the formation of the ternary complex (Figure 1B). The formed complex resembles the previously described BAG-1/Hsc70/CHIP complex (Demand et al., 2001). We therefore investigated whether HspBP1 and BAG-1 compete in binding to Hsc70. Addition of purified BAG-1 to binding reactions indeed strongly reduced the amount of Hsc70 retained on immobilized HspBP1 (Figure 1C). Similarly, HspBP1 interfered with the binding of Hsc70 to BAG-1, when the latter cochaperone was immobilized on an anti-BAG-1 antibody resin (Figure 1D). The data identify HspBP1 as a novel component of Hsc70/CHIP complexes, which competes with the degradation-stimulating cochaperone BAG-1 in complex binding. In contrast to the findings for BAG-1, addition of CHIP to binding reactions stimulated the interaction between HspBP1 and Hsc70 (Figure 1C). In this regard, it is also noteworthy that overexpression of HspBP1 in HeLa cells increased the amount of Hsc70 detectable in CHIP complexes (Figure 1E). HspBP1 and CHIP apparently interact with Hsc70 in a cooperative manner.
HspBP1 Inhibits the Ubiquitin Ligase Activity of CHIP When Complexed with Hsc70
To elucidate the functional consequences of the observed interaction, effects of HspBP1 on CHIP-mediated ubiquitylation were investigated using a previously established in vitro assay (Demand et al., 2001; Alberti et al., 2002). In the presence of the ubiquitin-activating enzyme E1 and the ubiquitin-conjugating enzyme UbcH5b, CHIP mediated the ubiquitylation of the protein kinase Raf-1 when bound to Hsc70 (Figure 2A). Addition of HspBP1 strongly inhibited the ubiquitylation of the protein kinase. Similarly, the CHIP-mediated ubiquitylation of firefly luciferase, heat denatured in the presence of Hsc70, was attenuated by HspBP1 (Figure 2B). The amount of high molecular mass polyubiquitylated forms of luciferase was strongly reduced in the presence of HspBP1 and at the same time the level of unmodified luciferase increased. Remarkably, HspBP1 itself is not recognized as a substrate protein by the CHIP ubiquitin ligase (Figure 2A). A competition of HspBP1 and a substrate for limited resources during ubiquitylation can thus be excluded as the molecular basis for inhibition. An alternative explanation may invoke an only inefficient recognition of substrate proteins by Hsc70 in the presence of HspBP1. However, HspBP1 did not interfere with the Hsc70-mediated stabilization of luciferase during heat treatment (Figure 3), arguing against such a hypothesis. Furthermore, HspBP1 also inhibited the CHIP-induced ubiquitylation of Hsc70 (Figure 2A), which is itself a cellular target of the CHIP ubiquitin ligase (Jiang et al., 2001). Because HspBP1 and CHIP bind to Hsc70 in a cooperative manner (see above), HspBP1 apparently attenuates the ubiquitin ligase activity of CHIP when the two cochaperones are simultaneously associated with Hsc70. In contrast to the inhibitory activity that HspBP1 displayed in conjunction with Hsc70, HspBP1 did not significantly affect the CHIP-mediated ubiquitylation of luciferase bound to Hsp90 (Figure 2B). Complex formation between HspBP1, Hsc70 and CHIP is apparently necessary for HspBP1 to exert its CHIP-inhibiting activity.
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HspBP1 Interferes with the Chaperone-assisted Degradation of CFTR
Immunofluorescence analysis revealed that a fraction of HspBP1 is localized at the ER membrane in mammalian cells (unpublished data). We therefore investigated whether HspBP1 participates in the ER-associated folding and maturation of the CFTR ion channel, which was previously identified as a substrate of CHIP (Meacham et al., 2001). In fact, elevating the cellular concentration of HspBP1 significantly increased the steady-state levels of coexpressed CFTR in HEK293 cells (Figure 4A). The increase was observed for the immature ER-localized B form of CFTR as well as for the maturely glycosylated C form that is located in the plasma membrane. HspBP1 apparently stimulates the maturation of CFTR. In contrast, overexpression of CHIP resulted in a strong decrease of CFTR levels (Figure 4A), which reflects the degradation-inducing activity of the ubiquitin ligase (Meacham et al., 2001). Notably, coexpression of HspBP1 counteracted the destructive activity of CHIP during CFTR biogenesis. This is apparently in agreement with a function of HspBP1 as an inhibitor of the CHIP ubiquitin ligase. Similar findings were obtained for CFTRF508, a form of the ion channel that is largely unable to proceed to the C form and is expressed in 70% of patients suffering from cystic fibrosis (Kopito, 1999). Overexpression of HspBP1 led to a significant increase of the cellular levels of CFTRF508 (Figure 4B). To verify that HspBP1 affects the degradation of CFTR, pulse-chase experiments were performed. HspBP1 slowed down CFTR turnover and attenuated the degradation-inducing activity of CHIP (Figure 5). Furthermore, we confirmed that HspBP1 inhibits the CHIP-mediated ubiquitylation of CFTR. For this purpose, CFTR was cotranslationally translocated into ER microsomes, followed by addition of the CHIP ubiquitin conjugation machinery and increasing amounts of HspBP1. HspBP1 significantly interfered with the CHIP-induced ubiquitylation of CFTR (Figure 6). Apparently, HspBP1 stabilizes CFTR by inhibiting chaperone-assisted degradation pathways. This results in a more efficient sorting of the ion channel to the plasma membrane as revealed by the increased detection of surface-biotinylated CFTR after overexpression of HspBP1 in HEK293 cells (Figure 7). Finally, we investigated how a decrease of the endogenous HspBP1 levels affected CFTR maturation. For this purpose, HEK293 cells were transfected with a pSUPER construct for expression of a HspBP1 shRNA, which is subsequently cleaved into siRNA in transfected cells. Transient transfection of this plasmid led to a considerable reduction of HspBP1 levels (Figure 8). At the same time, CFTR levels were significantly reduced, consistent with an essential role of HspBP1 in CFTR maturation. When HspBP1 was overexpressed, shRNA coexpression no longer affected cellular CFTR levels. This demonstrates that the HspBP1 shRNA does not unspecifically decrease CFTR expression. Together, the data identify HspBP1 as a cochaperone of Hsc70 and inhibitor of the CHIP ubiquitin ligase, which plays an essential role in the maturation of CFTR.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) (Figure 9). The characterization of HspBP1, which competes with BAG-1 in binding to the Hsc70/CHIP complex, now points to an unprecedented regulatory mechanism that confines the destructive activity of CHIP. HspBP1 inhibits CHIP-mediated ubiquitylation within an assembled Hsc70 chaperone complex (Figure 9). This inhibition similarly affects a client protein bound to Hsc70 as well as Hsc70 itself, which is a physiological substrate of the CHIP ubiquitin ligase (Jiang et al., 2001). Association of HspBP1 with the chaperone complex seems to shield Hsc70 and the bound client against CHIP-mediated ubiquitylation or may induce conformational changes of the chaperone complex that remove ubiquitin attachment sites from the reach of the CHIP ubiquitin ligase. In this way, HspBP1 directly modulates Hsc70-mediated protein quality control, as is evident from the role of the cochaperone in CFTR maturation. A main cause for misprocessing of CFTR is the inefficient folding of the first of two cytoplasmically exposed nucleotide binding domains (NBD1) of the membrane protein (Sato et al., 1996; Qu et al., 1997). The disease-causing F508 mutation localizes to NBD1 and further decreases the folding propensity of this domain. During the cotranslational insertion of CFTR into the ER membrane, Hsc70 and its cochaperone Hdj-2 bind to NBD1 and facilitate intramolecular interactions between the domain and another cytoplasmic region of CFTR, the regulatory R-domain (Strickland et al., 1997; Meacham et al., 1999). When CHIP exerts its proteasomal targeting activity at this stage, immature CFTR is diverted onto the degradation pathway (Meacham et al., 2001). In this regard, HspBP1 fulfills a key regulatory function during CFTR biogenesis. By inhibiting the CHIP-mediated ubiquitylation of CFTR, HspBP1 strongly increased the cellular concentration of wild-type and mutant CFTR and stimulated the sorting of the ion channel to the plasma membrane. It was previously noted that other cochaperones of Hsc70 such as BAG-1 and Hip do not affect CFTR maturation (Meacham et al., 2001). Thus, HspBP1 seems to play a unique role within the cellular surveillance system that monitors the folding state of CFTR and apparently represents an essential CFTR assembly factor of limited availability.
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Notably, CHIP participates in the regulation of the ATPase cycle of Hsc70 (Ballinger et al., 1999). The HspBP1-mediated inhibition of the ubiquitin ligase activity may therefore enable CHIP to modulate the Hsc70 chaperone cycle without exerting its destructive activity. In fact, degradation-independent functions of CHIP recently emerged (Cardozo et al., 2003; Jiang et al., 2003; Kampinga et al., 2003; Dai et al., 2003). CHIP was shown to regulate the chaperone-assisted folding and sorting of the androgen receptor and of endothelial nitric-oxide synthase without inducing degradation (Cardozo et al., 2003; Jiang et al., 2003). Moreover, CHIP fulfills an essential role in the chaperone-mediated regulation of the heat shock transcription factor, independent of its degradation-inducing activity (Dai et al., 2003). It remains to be seen, however, whether HspBP1 cooperates with CHIP in the regulation of the heat shock response. Although HspBP1 interfered with the degradation of CFTR, we noted that elevating the cellular levels of HspBP1 did not influence the CHIP-mediated turnover of the glucocorticoid hormone receptor (unpublished observation). Such a client specificity may arise in part from the fact that HspBP1 inhibits the ubiquitin ligase activity of CHIP in a complex with Hsc70, but it leaves Hsp90-associated ubiquitylation unaffected. In addition, direct interactions between HspBP1 and a subset of chaperone substrates may contribute to substrate selection. This is suggested by the finding that HspBP1 on its own partially stabilized luciferase during heat treatment and may therefore possess the ability to recognize certain chaperone substrates.
Modulating chaperone-assisted folding and degradation pathways has recently emerged as a promising therapeutic strategy. For example, antitumor agents, which inhibit Hsp90, induce the proteasomal degradation of signaling proteins, such as ErbB2, that play a key role in oncogenic transformation (Xu et al., 2002; deCandia et al., 2003; Kamal et al., 2003). CHIP participates on such pharmacologically induced degradation pathways (Xu et al., 2002). Consequently, the identification of HspBP1 as a cellular inhibitor of CHIP may open new roads to modulate pathologically relevant processes that involve chaperone action.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Corresponding author. E-mail address: hoehfeld{at}uni-bonn.de.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-ubiquitin IP). Control reactions received unrelated immunoglobulins (IgG). `IVT' represents 20% of the sample used for immunoprecipitation. (B) Densitometric quantification of ubiquitylated CFTR [ub(n)-CFTR] immunoprecipitated under A. Values were averaged from two independent experiments.
