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Previous Article | Next Article 
Molecular and Cellular Biology, May 2003, p. 3477-3486, Vol. 23, No. 10
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.10.3477-3486.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Division of Medical Oncology, Department of Medicine, Veterans Administration Puget Sound Health Care System, Seattle Division, Seattle, Washington 98108,1 Division of Medical Oncology, Department of Medicine, University of Washington Medical Center, Seattle, Washington 98195,2 Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington 981093
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ABSTRACT |
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(eIF2). Recently, we reported the involvement of human SNF5/INI1 (hSNF5/INI1) protein in the functions of GADD34 and showed that hSNF5/INI1 binds GADD34 and stimulates the bound PP1 phosphatase activity. To better understand the regulatory and functional mechanisms of GADD34, we undertook a yeast two-hybrid screen with full-length GADD34 as bait in order to identify additional protein partners of GADD34. We report here that human cochaperone protein BAG-1 interacts with GADD34 in vitro and in SW480 cells treated with the proteasome inhibitor z-LLL-B to induce apoptosis. Two other proteins, Hsp70/Hsc70 and PP1, associate reversibly with the GADD34-BAG-1 complex, and their dissociation is promoted by ATP. BAG-1 negatively modulates GADD34-bound PP1 activity, and the expression of BAG-1 isoforms can also mask GADD34-mediated inhibition of colony formation and suppression of transcription. Our findings suggest that BAG-1 may function to suppress the GADD34-mediated cellular stress response and support a role for BAG-1 in the survival of cells undergoing stress. |
INTRODUCTION |
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The GADD34 protein harbors a highly conserved carboxy-terminal peptide domain homologous to herpes simplex virus type 1 (HSV-1) ICP34.5, a virulence factor that blocks the premature shutoff of protein synthesis in HSV-1-infected neuroblastoma cells and allows HSV-1-infected cells to circumvent apoptosis (6, 10, 11, 19). Both ICP34.5 and mammalian GADD34 engage protein phosphatase-1 (PP1) and target dephosphorylation of the eukaryotic translation elongation factor eIF2 (13, 20). In HSV-1-infected cells, this molecular event enables protein synthesis (19). In mammalian cells, the dephosphorylation attenuates eIF2-mediated induction of stress-responsive genes and has been proposed in feedback regulation of cellular stress response initiated by protein malfolding at the endoplasmic reticulum (35).
The mechanism and regulation of GADD34-mediated growth suppression and apoptosis remain poorly understood. Recently we reported the association of the human SNF5 protein (hSNF5/INI1), a member of the hSWI/SNF chromatin-remodeling complex, with GADD34 (1). We showed that hSNF5/INI1 may function as a regulatory subunit for PP1 (60). hSNF5/INI1, GADD34, and PP1 can coexist in a stable heterotrimeric complex. The disruption of the association between hSNF5/INI1 and GADD34 by the coexpression of Epstein-Barr nuclear antigen 2 interferes with the GADD34-mediated growth-inhibitory function in NIH 3T3 cells harboring a constitutively active12V-Ha Ras. Previously, we also showed that the chimeric leukemic HRX fusion protein, generated as a result of chromatin translocation in acute leukemia involving the 11q23 locus, also inhibits GADD34-mediated apoptosis in p53-defective SW480 cells exposed to UV irradiation (1). GADD34 has also been shown to bind the Src-related tyrosine kinase Lyn (16) and to be phosphorylated by Lyn in response to DNA damage. Although both Lyn and GADD34 have both been implicated in DNA damage response, coexpression of Lyn curiously interferes with GADD34-mediated apoptosis in response to DNA damage. Together, these findings suggest that the GADD34-mediated stress response may require tight cellular control and that viral and leukemic proteins may target GADD34 in order to gain survival advantage.
Bcl-2-associated athanogene protein BAG-1 is a multifunctional protein initially identified to bind Bcl-2 and augments Bcl-2-mediated antiapoptotic function (50). BAG-1 appears to form complexes with several other proteins including a number of nuclear hormone receptors (7, 26, 58, 66), the serine/threonine protein kinase Raf-1 (55), two members of tyrosine kinase growth factor receptor family (3, 29), the p53-indicible negative growth regulator Siah (30), and, most notably, the heat shock-inducible chaperone protein Hsp70/Hsc70 (5, 49). BAG-1 binds to the ATPase domain of Hsp70/Hsc70 (45) and stimulates the ATPase activity. It also modulates the substrate affinity of Hsp70/Hsc70 chaperone complex (5, 48, 49). Human BAG-1 exists in four isoforms (p50, p46, p33, and p29) as result of alternative translations from the BAG-1 mRNA (38, 62). These protein isoforms differ in their amino termini and in their subcellular localization and tissue distribution (62). The largest isoform BAG-1/p50 (also known as BAG-1L) is translated from a noncanonical CUG start site and contains an amino-terminal nuclear localization domain. BAG-1/p50 stimulates the androgen receptor transcription function through its amino terminus, which also associates with Hsp70/Hsc70 (15). The overexpression of BAG-1 isoforms p50, p46, and p33 in C33A cervical carcinoma cells confers resistance of these cells to apoptosis-inducing agents; BAG-1/p29, however, failed to protect these cells from the same stimuli (9). Recently, BAG-1 has been proposed to coordinate cell growth signals as a part of the heat shock response based on the observation that Hsp70/Hsc70 can competitively disrupt the BAG-1-Raf-1 kinase complex, thereby arresting DNA synthesis (46).
In the present study, we report the identification of BAG-1 as a protein partner to GADD34. We show that BAG-1 isoforms bind GADD34 through their common carboxy terminus both in vitro and in vivo. Using the proteasome inhibitor sodium benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucyl boronic acid (z-LLL-B) to induce GADD34 in SW480 cells, we demonstrate the association of these two proteins during apoptosis. The GADD34-BAG-1 complex can also bind reversibly to endogenous Hsp70/Hsc70 and PP1. The composition of this multiprotein complex is determined in part by ATP, which reduces the associations of these two proteins with the complex. BAG-1 negatively regulates the GADD34-bound PP1 activity, and the expression of BAG-1 also masks GADD34-mediated growth suppression and reverses GADD34 inhibition of general transcription. These results suggest that BAG-1 may function to suppress the GADD34-mediated cellular stress response and may facilitate cell survival under conditions of stress.
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MATERIALS AND METHODS |
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-Gal (Clontech).
Generation of BAG-1 and GADD34 plasmids.
Generation of the expression vectors for GADD34 was described previously (42). The expression plasmids for BAG-1 isoforms and the 
UBQ mutant were generated by subcloning of appropriate PCR fragments into the KpnI-EcoRI sites of pSG5-FL2 vector.
Cell line transfection and drug exposure. Human 293T, SW480 (American Type Culture Collection), and NIH 3T3-ras cells (a gift from R. Bruce Montgomery) were maintained in Dulbecco modified Eagle medium (Bio-Whittaker) supplemented with 10% fetal bovine serum (Bio-Whittaker) and 1% penicillin-streptomycin. For coimmunoprecipitation analysis, the indicated expression plasmids (10 µg) were transfected by the calcium phosphate method into 293T cells (1, 8, 59). The transfection medium was replaced at 24 h, and the cells were allowed to recover for an additional 24 h. For colony formation assays, the indicated plasmids (5-12.5 µg), along with the marker plasmids, were transfected into NIH 3T3-ras cells with the Superfect reagent (Qiagen) as recommended by the manufacturer. In the colony formation assays, the selective antibiotic puromycin (2 µg/ml) was added 24 h following transfection. z-LLL-B (Affinity Research) and methyl methanesulfonate (MMS; Sigma) in dimethyl sulfoxide were added directly to the growth media.
Flow cytometry and apoptosis ladder assay. Untreated and z-LLL-B-treated SW480 cells were fixed in 1% paraformaldehyde, stored in 70% ethanol, stained with propidium iodide (400 µg/ml), and analyzed on a Beckton-Dickinson FACScan flow cytometer. Total DNA was also harvested from these cells at the indicated times and analyzed on 1.5% agarose gel to detect the presence of the 100-bp DNA ladder.
Coimmunoprecipitation analysis. Cytoplasmic extracts prepared from SW480 cells by the Dignam method (32) or whole-cell lysates from transfected 293T cells described previously (60) were immunoprecipitated with either anti-GADD34 (H193; Santa Cruz) or anti-Myc antibodies (9E10) at a 1:20 concentration for 2 h at 4°C. The immune complexes were captured with protein A-Sepharose beads and washed with 2x lysis buffer (1x lysis buffer is 40 mM HEPES [pH 7.9], 100 mM NaCl, 0.4% NP-40, 2 mM MgCl2, 0.2 mM EDTA, 20% [vol/vol] glycerol, 50 mM NaF, and a cocktail of protease inhibitors [Complete Mini-EDTA free; Roche]) four times, and the bound proteins were eluted with 2x sodium dodecyl sulfate SDS sample buffer (125 mM Tris-HCl [pH 6.8], 1% SDS, 20% glycerol, 0.28 M ß-mercaptoethanol, 0.001% bromphenol blue) at 98°C for 5 min. The proteins were then separated by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide) (PAGE) and transferred onto an Immobilon-P membrane (Millipore). Immunodetection of the proteins was carried out at the indicated antibody concentrations: 1:1,000 for anti-Myc and anti-FLAG M2 (Eastman-Kodak), 1:250 for anti-GADD34, anti-BAG-1 (NeoMarkers), anti-Hsc70 (W-27, Santa Cruz), and anti-PP1 (06-221, Upstate Biotechnology), and 1:10,000 for horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (Sigma) by chemiluminescence (Pierce).
GST-binding assays. In vitro-transcribed-translated 35S-labeled full-length GADD34 was generated as described previously (1, 59). Glutathione S-transferase (GST)-BAG1/p46 was prepared from bacterial lysates as recommended by the manufacturer (Pharmacia) and adsorbed onto glutathione-agarose beads (Sigma).35S-labeled GADD34 was then mixed with glutathione-agarose, GST-bound agarose, or GST-BAG1/p46-bound agarose in 1x binding buffer (40 mM HEPES [pH 8.0], 10% glycerol, 1 mM MgCl2, 0.1 mM EDTA, 50 mM NaCl, 0.5% NP-50) for 1 h at 4°C. The agarose-bound proteins were then washed three times with 1x binding buffer and eluted with 2x SDS sample buffer. The eluents were resolved by SDS-PAGE (10% polyacrylamide) and autoradiography carried out overnight at 25°C.
Colony formation assays. A total of 2 x 104 to 5 x 104 cells were seeded onto 60-mm-diameter plates 16 to 24 h prior to transfection. At 24 h following plasmid transfection, the medium from each plate was changed and puromycin was added at the indicated concentration. The cells were maintained for 10 to 14 days, with fresh medium and drug replacement every 3 to 4 days. The number of colonies formed per plate was quantitatively determined following staining with crystal violet. Duplicate transfections were performed for each condition.
Protein phosphatase assays. The substrate 32P-labeled histone (type III-SS; Sigma) was generated as previously described (28, 60) with the substitution of porcine heart protein kinase catalytic subunit (5 U/ml) (Sigma), precipitated twice with 20% trichloroacetic acid, and washed extensively with cold acetone. The pellet was resuspended in 1 ml of 50 mM Tris-HCl (pH 7.5). In vitro protein phosphatase assays were carried out in phosphatase buffer (20 mM Tris-HCl [pH 7.5], 1 mg of bovine serum albumin per ml, 0.1 mM EGTA, 0.1% ß-mercaptoethanol) with 20 µg of 32P-labeled histone per reaction, in the presence of the indicated amount of anti-Myc immunoprecipitates at 30°C for 1 to 2 h in 100 µl of reaction mixture. Okadaic acid (Calbiochem) or a nuclear inhibitor of PP1 (Calbiochem) was included in indicated samples. The immune complexes immobilized on protein A-Sepharose were first washed twice with 1x immunoprecipitation buffer and then once with the phosphatase buffer before the phosphatase assay (28). The reactions were terminated with 200 µl of 20% trichloroacetic acid, the mixtures were centrifuged for 5 min, and 200-µl volumes of the supernatant were counted by Cerenkov counting.
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RESULTS |
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134.5 encoding amino acids 537 to 630 of GADD34, a domain homologous to HSV-1 ICP34.5.
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GADD34 associates with BAG-1 in proteasome inhibitor z-LLL-B-treated SW480 cells undergoing apoptosis. Proteasome inhibitor z-LLL-B is a cell-permeable leupeptin analogue that inhibits the "chymotrypsin-like" and "post-glutamyl" hydrolase activities of the human 20S proteasome (36). z-LLL-B induces apoptosis in MOLT-4 and L5178Y cells via a p53-dependent pathway (44). In the p53-defective SW480 colon carcinoma cells, z-LLL-B at 50 to 250 nM arrested the cell proliferation primarily at G2/M 8 h following exposure (Fig. 2A) and induced detectable apoptosis as early as 16 to 24 h. The apoptosis was confirmed by a rising sub-G1 fraction on the propidium iodide DNA histogram (Fig. 2A) and the presence of the apoptotic DNA ladder (Fig. 2B). Coincidentally with apoptosis, GADD34, whose level is normally barely above the threshold of detection, accumulated in the treated SW480 cells in a z-LLL-B concentration-dependent manner (Fig. 2C). The addition of an alkylating agent, MMS, known to increase GADD34 transcripts in SW480 cells at an optimal concentration of 0.1 µM had only a minimal effect on the z-LLL-B induction of the GADD34 protein at 24 h (Fig. 2C). Utilizing z-LLL-B to induce a high level of endogenous GADD34, we tested the associations of BAG-1 and GADD34 in SW480 cells by a coimmunoprecipitation assay (Fig. 2D). BAG-1/p50, BAG-1/p46, and BAG-1/p33 isoforms were found in SW480 cell lysates, with BAG-1/p33 being the predominant form. Their levels were unaltered by z-LLL-B. All three were found to coprecipitate to various extents with GADD34 in the cytoplasmic extracts from z-LLL-B-treated cells, with BAG-1/p50 being found preferentially in complex with GADD34. These results suggest that a portion of the BAG-1 proteins can complex with GADD34 in mammalian cells undergoing apoptosis.
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UBQ by the same antibodies. The ubiquitin-like region on BAG-1 was found to be unnecessary for this association. Of note, the preferential association of BAG-1/p50 with GADD34 in SW480 cells (shown in Fig. 2D) suggests that there may also be other unique sequences in the amino terminus of BAG-1/p50 that can mediate its direct or indirect association with GADD34.
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BAG-1 isoforms negatively modulate GADD34-associated phosphatase activity. We proceeded to examine whether BAG-1 affects the GADD34-bound PP1 phosphatase activity. In the experiments in Fig. 4, we isolated GADD34 complexes immunoprecipitated with anti-Myc antibodies from 293T cells transfected with a Myc-GADD34 expression plasmid. These GADD34 complexes were previously found to be associated with phosphatase activities that can be differentiated based on their sensitivity to either okadaic acid (OA) or to NIPP1, a selective inhibitor for PP1 (18, 25, 53). We cotransfected increasing amounts of BAG1/p46 or BAG-1/p29 to determine if the BAG1 association will influence GADD34-bound PP1 activity. In the experiment in Fig. 4A, the PP1-specific phosphatase activity associated with the GADD34 immune complex was determined by measuring the fraction of the total phosphatase activity that could be inhibited by 50 nM OA. Both OA-inhibited and -uninhibited phosphatase activities have been found, and the results show that increasing levels of either BAG-1/p46 or BAG-1/p29 reduced GADD34-associated PP1 activities. In contrast, the OA-uninhibited "nonspecific" phosphatase activities remained unaffected by BAG-1. Although 50 nM OA inhibits the phosphatase activities of both PP1 and PP2A, we have shown previously that only PP1 is bound to the overexpressed GADD34 (60). These results were further confirmed with the PP1 selective inhibitor NIPP1 (data not shown). The immunoblots shown in Fig. 4B depict the levels of BAG-1 and GADD34 expression and the level of coprecipitated BAG-1 and endogenous PP1. Both BAG-1 isoforms failed to displace endogenous PP1 that was bound to GADD34, since this level (lanes 3 to 5, 7 and 8) remained significantly above the level of PP1 nonspecifically bound to the Sepharose beads (lanes 1, 2, and 6). The reduction of GADD34-bound PP1 activity is therefore probably a result of either direct modulation by BAG-1 or displacement of an unidentified activation factor but is not a reflection of decreased levels of PP1 bound to GADD34.
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DISCUSSIONS |
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Mammalian BAG-1 has antiapoptotic and growth-regulatory functions in a number of settings. BAG-1 augments the antiapoptotic function of Bcl-2 since the enforced expression of the mouse BAG-1 protein in Jurkat cells stably expressing Bcl-2 confers resistance to Fas ligand; staurosporin, and cytotoxic T-lymphocyte-mediated cell lysis in these cells (50). Similarly, the expression of BAG-1 either with heparin-bound epidermal growth factor in CHO cells or alone in cervical carcinoma cells has been reported to reduce the sensitivity of these cells to a variety of apoptotic stimuli (29, 64). Conversely, decreased levels of BAG-1 were found to precede olfactory neuronal apoptosis and may be related to ubiquitination of the BAG-1 protein (47). Since GADD34 facilitates apoptosis to these stimuli, the observed BAG-1 effect may be explained in part by its ability to interfere with GADD34 pathways that facilitate apoptosis. BAG-1 has been reported to reverse both Siah-1A- and p53-induced growth arrest in 293 cells without affecting p53-mediated transcription activity (30). Although the related protein murine Siah-1B is induced early after activation of p53 in leukemia cell lines (23), there is currently no direct evidence to suggest that Siah-1A plays a role in mediating cellular stress response.
The aberrant overexpression of BAG-1 has been reported in a number of naturally occurring human cancers and in cancer cell lines. BAG-1 overexpression has been estimated to occur in 50% of breast carcinomas (51, 54), 73% of non-small cell lung carcinomas (41), 
80% of laryngeal carcinomas (61), 59% of oral squamous cell carcinomas (43), and 25 to 75% of cervical carcinomas (63, 64). The pattern of subcellular BAG-1 expression and BAG-1 isoform distribution in these cases requires further investigation. Using BAG-1 overexpression as a biomarker, prognostic stratification can be demonstrated for subsets of breast, cervical, head and neck, and lung cancers (41, 43, 51, 54, 61, 64). A consistent clinical effect of BAG-1 overexpression on prognosis has not been demonstrated, suggesting that BAG-1 may have pleiotropic effects in tumor cells depending on the environment, specific BAG-1 isoform expression pattern, and subcellular localization of the overexpressed proteins.
The cellular ATP concentration (and energy charge) has long been known to regulate a multitude of cellular physiological pathways, most notably involving metabolic, mitochondrial, contractile protein, and neuronal functions (17, 40, 56, 57). In stressed cells, the intracellular ATP concentration can drop precipitously from the normal concentration of 5 to 10 mM. A fall in the intracellular ATP level, commonly observed in dying cells, may also determine the mode of cell death (14, 27). ATP, which influences the function of a number of heat shock proteins, is known to induce molecular conformational change in Hsp70/Hsc70 (31, 37, 52). Here, we show that ATP reduces the affinity of both Hsp70/Hsc70 and PP1 for the BAG-1-GADD34 complex without disrupting the association of BAG-1 and GADD34. The association of BAG-1 with Bcl-2 has been previously shown to be stabilized by the presence of ATP (49). The interaction of the carboxy-terminal "BAG" domain on BAG-1 with the ATPase domain on Hsp70/Hsc70 results in a conformation change in both proteins such that the latter becomes incompatible to nucleotide binding (45). These ATP-dependent reversible protein associations raise the possibility that both the composition and function of the BAG-1-GADD34 complex may be regulated by ATP. Of note, subunits of PP1 have been reported to be in a phosphoprotein complex with Hsc70/Hsp70 and Hsp90 in resting platelets (39).
In addition to GADD34 and Hsp70/Hsc70, a number of other proteins interact with the carboxy terminus of BAG-1, including Bcl-2, Raf-1 kinase, and Siah-1A (30, 50, 55). Raf-1 and Hsp70/Hsc70 compete for binding to BAG-1 such that elevated levels of Hsp70/Hsc70 sequesters BAG-1 and down-regulates the BAG-1-mediated activation of Raf-1 kinase and its downstream mitogen-activated protein kinase effector pathways (46). BAG-1 has therefore been proposed to coordinate cell growth signals and mitogenesis, with Hsp70/Hsc70 functioning as a sensor in stress signaling. Our findings here suggest that GADD34 may also serve as a transducer of negative growth signals by binding to BAG-1 at the same carboxy terminus. The effect of GADD34 on BAG-1 functions remains to be determined; however, it is predicted to have negative regulatory consequences.
The proteasome is the central cellular enzyme complex that catalyzes the ATP-dependent proteolytic degradation of many rate-limiting enzymes, transcriptional regulators, and critical regulatory proteins (12, 33). Although the GADD34 transcript is induced by a variety of apoptosis-inducing agents, the GADD34 protein level in both unstressed and stressed cells is rarely above the level of detection, probably reflecting a tight regulation, in part by the proteasome system. Overexpression of GADD34 in eukaryotic cells results in growth arrest and facilitates apoptosis independent of functional p53 (22). We show here that the potent proteasome inhibitor z-LLL-B can elicit apoptosis in the p53-defective SW480 cells and cause a rapid accumulation of GADD34 protein. The mechanism underlying GADD34 accumulation by z-LLL-B probably involves both induction of GADD34 and interference of GADD34 degradation through inhibition of proteasome systems. BAG-1 has recently been shown to undergo polyubiquitylation in a ternary complex with Hsp70/Hsc70 and the ubiquitin ligase CHIP, resulting in the stimulation of a degradation-independent association of the BAG-1 with the proteasome (2). It is possible that GADD34 may be a target for the proteasome through its association with BAG-1. Proteasome inhibitors such as z-LLL-B may therefore potentiate the effect of other anticancer agents by up-regulating GADD34-mediated growth suppression and apoptosis, particularly in tumors with a high prevalence of p53 mutation. Proteasome inhibitors should also prove to be useful in the study of GADD34, whose low endogenous levels have precluded in vivo investigations.
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ACKNOWLEDGMENTS |
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This work was supported by the Department of Veteran Affairs (VA Merit Review) and a grant from the NIH (5K08CA71928-01) to D.Y.W.
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FOOTNOTES |
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