p53-inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth: suppression by BAG-1
1998; Springer Nature; Volume: 17; Issue: 10 Linguagem: Inglês
10.1093/emboj/17.10.2736
ISSN1460-2075
Autores Tópico(s)Cancer-related Molecular Pathways
ResumoArticle15 May 1998free access p53-inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth: suppression by BAG-1 Shu-ichi Matsuzawa Shu-ichi Matsuzawa The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Shinichi Takayama Shinichi Takayama The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Barbara A. Froesch Barbara A. Froesch The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Juan M. Zapata Juan M. Zapata The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author John C. Reed Corresponding Author John C. Reed The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Shu-ichi Matsuzawa Shu-ichi Matsuzawa The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Shinichi Takayama Shinichi Takayama The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Barbara A. Froesch Barbara A. Froesch The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Juan M. Zapata Juan M. Zapata The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author John C. Reed Corresponding Author John C. Reed The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Author Information Shu-ichi Matsuzawa1, Shinichi Takayama1, Barbara A. Froesch1, Juan M. Zapata1 and John C. Reed 1 1The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:2736-2747https://doi.org/10.1093/emboj/17.10.2736 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Drosophila seven in absentia (sina) gene is required for R7 photoreceptor cell formation during Drosophila eye development, where it functions within the Ras/Raf pathway and targets other proteins for degradation via associations with a ubiquitin-conjugating enzyme. Recently, a mammalian sina homologue was reported to be a p53-inducible gene in a myeloid leukemia cell line. To explore the function of human SINA-homologous (Siah) proteins, expression plasmids encoding Siah-1A were transiently transfected into 293 epithelial cells and GM701 fibroblast cells, resulting in growth arrest without induction of apoptosis. We discovered that BAG-1, a ubiquitin-like Hsp70/Hsc70-regulating protein, is a negative regulator of Siah-1A. Siah-1A was identified as a BAG-1-binding protein via yeast two-hybrid methods. Specific interaction of BAG-1 with Siah-1A was also demonstrated by in vitro binding experiments using glutathione S-transferase fusion proteins and co-immunoprecipitation studies. Siah-1A-induced growth arrest in 293 and GM701 cells was abolished by co-transfection of wild-type BAG-1 with Siah-1A but not by a C-terminal deletion mutant of BAG-1 that fails to bind Siah-1A. Over-expression of BAG-1 significantly inhibited p53-induced growth arrest in 293 cells without preventing p53 transactivation of reporter gene plasmids. BAG-1 also prevented growth arrest following UV-irradiation-induced genotoxic injury without interfering with accumulation of p53 protein or p21waf-1 expression. BAG-1 functions downstream of p53-induced gene expression to inhibit p53-mediated suppression of cell growth, presumably by suppressing the actions of Siah-1A. We suggest that Siah-1A may be an important mediator of p53-dependent cell-cycle arrest and demonstrate that Siah-1A is directly inhibited by BAG-1. Introduction Siah is the vertebrate homologue of the protein encoded by the Drosophila seven in absentia (sina) gene, which is required for formation of the R7 photoreceptor cells in the developing eye of the fly (Carthew and Rubin, 1990). The murine homologues of sina (Siah-1A, Siah-1B and Siah-2) are widely expressed in various tissues of the embryo and adult (Della et al., 1993). The murine Siah-1A and -1B genes encode highly homologous proteins that share ≥98% amino acid sequence homology with each other, and >95 and >68% homology with their human and Drosophila counterparts, respectively. In Drosophila, the SINA protein has been shown to interact with a ubiquitin-conjugating enzyme (UBCD1) and assists with the proteasome-dependent degradation of a transcription factor (Tramtrack) which suppresses R7 cell differentiation (Li et al., 1997; Tang et al., 1997). The human Siah-1 and Siah-2 proteins have also been shown to bind ubiquitin-conjugating enzymes (Hu et al., 1997). Though the function of Siah-family proteins in vertebrates remains unknown, the murine Siah-1B was recently identified by differential cDNA display methods among a group of genes induced during p53-induced apoptosis and G1-arrest in murine M1 myeloid leukemia cells (Amson et al., 1996). The tumor suppressor p53 has multiple functions in cells, including an ability to induce cell-cycle arrest in essentially all types of cells and to trigger apoptosis in some specific cell lineages and cellular contexts (Hartwell and Kastan, 1994; Reed et al., 1996; Hall and Lane, 1997). The cell-cycle arrest induced by p53 can occur at either the G1/S-phase or G2/M-phase boundaries depending on cellular background and other circumstances (el-Deiry et al., 1993; Harper et al., 1993; Agarwal et al., 1995; Guillouf et al., 1995; Stewart et al., 1995; Elledge, 1996; Levine, 1997). p53 has been shown to transactivate or transrepress a wide variety of target genes, including some associated with growth arrest and apoptosis, such as p21waf-1, Bax, Bcl-2, IGF-II, insulin receptor, IGF-I receptor and IGF-BP3 (el-Deiry et al., 1993; Miyashita et al., 1994; Buckbinder et al., 1995; Miyashita and Reed, 1995; Webster et al., 1996; Werner et al., 1996; Zhang et al., 1996; for reviews see Hartwell and Kastan, 1994; Elledge, 1996; Reed et al., 1996; Hall and Lane, 1997; Levine, 1997). To date, investigations of the roles of these p53-regulated genes using targeted gene disruption (knock-out) approaches in mice and cell lines have all suggested that no single gene completely accounts for the ability of p53 to induce cell-cycle arrest and apoptosis (Deng et al., 1995; Brown et al., 1997; Yin et al., 1997). These results have again demonstrated that cellular context plays an important role in determining which specific p53 target genes are relatively more or less required for mediating the actions of this tumor suppressor. BAG-1 is a widely expressed protein that was first discovered by virtue of its ability to bind to and collaborate with Bcl-2 in suppressing cell death (Takayama et al., 1995). The murine and human BAG-1 proteins (also known as RAP46) contain a domain with strong sequence homology to ubiquitin, but otherwise do not share similarity with other known proteins (Takayama et al., 1995, 1996). Since its initial discovery as a Bcl-2-binding protein, however, BAG-1 has been reported to interact with several steroid hormone receptors, the serine/threonine-specific protein kinase Raf-1 and some tyrosine kinase growth factor receptors [hepatocyte growth factor (HGF) receptor and platelet-derived growth factor (PDGF) receptor; Zeiner and Gehring, 1995; Bardelli et al., 1996; Wang et al., 1996]. Recently, we and others have determined that BAG-1 forms tight complexes with Hsp70/Hsc70-family proteins and modulates their chaperone activity (Höhfeld and Jentsch, 1997; Takayama et al., 1997; Zeiner et al., 1997). Thus, BAG-1 is probably a novel component of the chaperone system and presumably exerts its effects on various target proteins by recruiting Hsp70/Hsc70-family proteins to them. We present evidence here that the p53-inducible Siah protein is a negative regulator of cell proliferation. The findings suggest that p53-mediated induction of Siah could represent an alternative to the p21waf-1 pathway for cell-cycle arrest, and therefore suggest a redundant mechanism by which p53 interferes with cell proliferation. In addition, we show that the BAG-1 protein interacts with Siah-1A. BAG-1 abrogates Siah-1A-induced growth inhibition and can interfere with p53-mediated growth arrest. The discovery of a physical and functional connection between Siah-1A and BAG-1 therefore extends the range of potentially oncogenic activities of BAG-1 to suppression of p53-dependent pathways for cell growth inhibition. Results Identification of human Siah-1A as a BAG-1-binding protein During attempts to identify proteins that associate with the BAG-1 by yeast two-hybrid cDNA library screening, we discovered a human SINA homologue. From a pool of 23 candidate clones, four corresponded to overlapping clones encoding polypeptides of 276 (clone 21) and 262 (clones 9, 17 and 26) amino acids in length which shared >95% homology at the nucleotide level with murine cDNA sequences encoding Siah-1A and Siah-1B (Della et al., 1993). Subsequently, a human homologue of Siah-1 was reported which is essentially identical to the cDNAs identified by two-hybrid screening with BAG-1 as a bait (Nemani et al., 1996). The longest cDNAs cloned during two-hybrid screening encompasses residues 22–298 of the predicted 298 amino acid human Siah-1 protein, whereas the shortest encoded residues 36–298 (Figure 1C). Like its murine and fly counterparts, the predicted human Siah protein contains a putative RING-finger domain, cysteine-rich region and nuclear localization signal sequence (Figure 1C). In the region corresponding to residues 17–298, the human Siah-1 protein differs from the murine Siah-1A homologue by only one amino acid, at position 132 where a glutamic acid to aspartic acid substitution occurs. In this same region, the human Siah-1 protein differs from murine Siah-1B at six residues, prompting us to designate the BAG-1-binding protein obtained from two-hybrid screening as Siah-1A as opposed to Siah-1B. Figure 1.In vitro and in vivo interaction between BAG-1 and Siah. (A) GST fusion proteins (10 μg) were immobilized on glutathione–Sepharose and incubated with 10 μl of 35S-labeled, in vitro translated (IVT) BAG-1 (left) or Siah (right). After extensive washing, beads were boiled in Laemmli sample buffer and eluted proteins were analyzed by SDS–PAGE (12% gels) followed by detection by fluorography. In the last lanes, 1 μl of in vitro translated (IVT) proteins were run directly in the gel as a control. The control GST fusion protein represents the cytosolic domain of CD40 (Sato et al., 1995). (B) 293 cells (5×106) were transiently transfected with the indicated combinations of expression vectors encoding FLAG-tagged BAG-1, HA-tagged Siah or HA-Bax (lane 1–4). After 48 h, lysates were prepared and immunoprecipitated with anti-FLAG monoclonal antibody M2-conjugated to agarose. Immune complexes were analyzed by SDS–PAGE/immunoblotting using anti-HA monoclonal antibody. In the last lanes, 10 μl of lysate was also loaded directly in the gel without immunoprecipitation as a control. (C) Diagram of the putative domain structure of human Siah-1A. Shown are the positions of the conserved RING-finger domain, cysteine-rich region and predicted nuclear localization signal sequence. The regions encoded by the partial cDNAs that were obtained by yeast two-hybrid screening are indicated below the diagram. Download figure Download PowerPoint Specific interaction of Siah-1A with BAG-1 Siah-1A exhibited specific interactions with the BAG-1 protein in two-hybrid assays (Table I). Interestingly, Siah also appeared to interact with itself, suggesting that it can homodimerize or homo-oligomerize. To further confirm the interaction of Siah-1A with BAG-1, in vitro binding assays were performed using in vitro translated, 35S-labeled BAG-1 and affinity-purified glutathione S-transferase (GST)–Siah-1A fusion protein. 35S-BAG-1 bound to GST–Siah-1A but exhibited little or no tendency to bind to GST–CD40 (Figure 1A) and several other GST control proteins including GST–Bax, GST–TNF-RI and GST–XIAP (data not shown). Furthermore, a GST–Siah-1A fusion protein interacted with in vitro translated, 35S-labeled Siah, implying that Siah can indeed form homodimers or homo-oligomers. In contrast, GST–Siah-1A did not bind in vitro translated 35S-Bax, indicating that the homodimerization detected for Siah-1A is specific (data not shown). Recently, the Drosophila SINA protein was also reported to homodimerize (Li et al., 1997; Tang et al., 1997). Table 1. Specific interactions of Siah with BAG-1 LexA B42 LEU β-Gal BAG-1 Siah + + Bax Siah − − Fas Siah − − Ras Siah − − Lamin-C Siah − − empty Siah − − Siah BAG-1 + + Siah Bax − − Siah Ras − − Siah Siah + + Three micrograms of plasmids producing LexA DNA-binding domain fusion proteins (listed at left) were co-transformed with 3 μg of pJG4-5 plasmid producing B42 transactivation domain fusion proteins (listed at right) into EGY48 strain yeast. Transformed cells were grown on semi-solid media lacking leucine, or containing leucine as a control which resulted in equivalent amounts of growth for all transformants (data not shown). Plasmid combinations that resulted in growth on leucine-deficient media within 4 days were scored as positive (+). β-galactosidase activity of each colony was tested by filter assay and scored as blue (+) or white (−) after 60 min. To examine whether the interaction of Siah-1A with BAG-1 occurs in cells, we performed co-immunoprecipitation experiments. An expression plasmid encoding FLAG epitope-tagged BAG-1 was transfected into 293 cells alone or in combination with plasmids encoding either hemagglutinin (HA) epitope-tagged Siah-1A, or HA-Bax as a negative control. The resulting cell lysates were immunoprecipitated using a monoclonal antibody specific for the FLAG epitope (M2) and associated Siah-1A-HA protein was detected by immunoblotting using an anti-HA monoclonal antibody (12CA5). Siah-1A-HA co-immunoprecipitated with FLAG-BAG-1 whereas HA-Bax did not (Figure 1B). Analysis of BAG-1/Siah-1A interaction To explore the regions of BAG-1 responsible for interaction with Siah-1A, we prepared a series of BAG-1 deletion mutants that were expressed in yeast as fusion proteins with a LexA DNA-binding domain (Figure 2A). Western blotting using an anti-LexA antiserum confirm production of all BAG-1 proteins at comparable levels (data not shown). An N-terminal deletion mutant of BAG-1 (amino acids 90–219) which removed the ubiquitin-like domain retained Siah-binding activity. In contrast, a C-terminal deletion mutant of BAG-1 (amino acids 1–172) failed to interact with Siah-1A, as did a double-truncation mutant of BAG-1 (90–172) which lacked both the N- and C-terminal regions. Similar results were obtained by in vitro binding assay, using GST–BAG-1, GST–BAG-1 (ΔN) and GST–BAG-1 (ΔC) fusion proteins (Figure 2B). These observations demonstrate that the C-terminal portion of BAG-1 (residues 173–219) is required for binding to Siah-1A. It is known that this same C-terminal region of BAG-1 is also essential for association with Hsp70/Hsc70 (Takayama et al., 1997), steroid hormone receptors (Zeiner and Gehring, 1995), Raf-1 (Wang et al., 1996) and the HGF receptor (Bardelli et al., 1996). The observation that a C-terminal truncation mutant of BAG-1 failed to bind Siah provides further evidence of the specificity of these results. Figure 2.Mapping of BAG-1–Siah and Siah–Siah interaction domains. (A) Expression plasmids encoding wild-type BAG-1 and the indicated BAG-1 deletion mutants fused to the LexA DNA-binding domain were co-transformed into yeast EGY48 cells with a plasmid encoding B42 transactivation domain–Siah (22–298) fusion protein. Transformed cells were grown on semi-solid media lacking leucine or containing leucine as a control. Plasmid combinations that resulted in growth on leucine-deficient media within 4 days were scored as positive (+). β-galactosidase activity for each colony was tested by filter assay and scored as blue (+) versus white (−) (β-gal) based on 1 h of color development. (B) GST fusion proteins (10 μg) containing wild-type BAG-1 and the indicated BAG-1 deletion mutants were immobilized on glutathione–Sepharose and incubated with 35S-labeled Siah. After extensive washing, beads were boiled in Laemmli buffer and eluted proteins were analyzed by SDS–PAGE (12% gels) and detected by fluorography. In vitro translated (IVT) Siah protein (1 μl) was run directory in the gel as a control. A GST fusion protein representing the cytosolic domain of CD40 protein was included as a control. Production of mostly intact GST–BAG-1 wild type and mutant fusion proteins was verified by Coomassie staining (data not shown). (C) Expression plasmids encoding the indicated Siah-1A deletion mutants fused to the B42 transactivation domain were co-transformed into yeast EGY48 cells with LexA DNA-binding domain–BAG-1 or LexA–Siah (22–298) expression plasmids. Yeast two-hybrid assays were performed as in (A). Download figure Download PowerPoint We examined the regions in Siah-1A which are required for binding to BAG-1 and for homodimerization using yeast two-hybrid assays. All Siah-1A deletion mutants tested failed to interact with BAG-1 (Figure 2C), suggesting that two or more separate sites on Siah-1C may be required for BAG-1-binding or that these mutants fail to fold properly for presentation of BAG-1 binding structures. In contrast, Siah-1A mutants lacking the RING domain or most of the C-terminal unique domain retained the ability to bind to Siah-1A in yeast two-hybrid assays (Figure 2C). These results imply that the region of Siah-1A corresponding to amino acids 102–193 where the cysteine-rich domain resides is involved in Siah-1A homodimerization. Siah-1A alters the subcellular location of BAG-1 in cells If BAG-1 and Siah-1A associate in intact cells, we reasoned that their co-expression might affect the intracellular locations of these proteins in cells. To test this hypothesis, FLAG-tagged BAG-1 and Siah-1A proteins was co-expressed in GM701 cells and their distribution patterns were determined by indirect immunofluorescence. When a cDNA encoding nearly full-length Siah-1A (22–298) with a FLAG epitope-tag was transfected into GM701 cells by itself and then detected with an antibody directed against the FLAG epitope by immunofluorescence microscopy, the Siah-1A protein was predominantly present in the nucleus, though some weak cytosolic immunofluorescence was also seen (Figure 3). Consistent with these results, the Drosophila SINA protein was reported to display a very similar subcellular distribution (Li et al., 1997; Tang et al., 1997). Moreover, the immunolocalization of Siah-1A in the nucleus suggested a punctate pattern, implying association with unidentified substructures in the nucleus. When a FLAG-BAG-1 protein was expressed in these cells by itself, immunofluorescence was found in both the cytosol and nucleus, but was greater in the cytosol (Figure 3B). In contrast, cells co-transfected with FLAG-BAG-1 and untagged Siah-1A expression plasmids displayed a predominantly nuclear localization of the FLAG-BAG-1 protein (Figure 3C). In reciprocal experiments where FLAG-tagged Siah-1A was expressed with untagged BAG-1, the intracellular pattern of Siah-1A distribution was not substantially different from that seen in cells expressing FLAG-Siah-1A alone (data not shown). The specificity of these immunofluorescence data was verified by use of irrelevant control primary antibodies as well as by immunostaining with secondary antibody alone (data not shown). Taken together, these data suggest that Siah-1A can alter the intracellular location of BAG-1, targeting a portion of the BAG-1 molecules to the nucleus. As with Siah-1A, the BAG-1 immunofluorescence within the nuclei of cells that had been co-transfected with BAG-1 and Siah-1A was distributed in a punctate pattern (Figure 3C). The significance of this speckled nuclear immunolocalization pattern is unclear. Figure 3.Immunofluorescence microscopic evaluation of locations of BAG-1 and Siah-1A proteins. 293 cells were transiently transfected with pCI–FLAG-Siah (A), pCI–FLAG-BAG-1 (B) and pCI–FLAG-BAG-1 in combination with pCI–Siah (C). After 48 h, cells were immunostained with anti–FLAG monoclonal antibody M2 and FITC-conjugated goat anti-mouse immunoglobulin and analyzed by fluorescence microscopy (∼800× original magnification). Download figure Download PowerPoint Siah-1A suppresses cell growth Because Siah expression has been reported to be induced by p53, we investigated whether Siah-1A could induce either growth arrest or apoptosis. To explore this possibility, an expression plasmid encoding FLAG-Siah-1A was transiently transfected into 293 cells. These cells have been used extensively for studying apoptosis-inducing proteins because of their high efficiency of transient transfection (>90%). Two days after transfection, the numbers of viable and dead cells were determined by trypan blue dye exclusion assay. Siah-1A suppressed growth of 293 cells, without stimulating an increase in cell death (Figure 4A). Attempts to observe effects of Siah over-expression on cell death using other methods for detecting apoptotic cells such as DAPI fluorochrome staining and DNA-content analysis by flow cytometry also failed to demonstrate increased amounts of apoptosis in cultures of Siah-transfected cells. In contrast, over-expression of Bax, which is a p53-inducible pro-apoptotic gene (Miyashita and Reed, 1995), induced an ∼5-fold increase in the number of dead cells as determined by trypan blue dye exclusion. The Siah-1A-mediated growth suppression was reversed by co-expression with BAG-1, but not by the C-terminal deletion mutant of BAG-1 which fails to bind Siah-1A. Immunoblot analysis confirmed that the expected ∼34 kDa FLAG-Siah-1A (22–298), ∼32 kDa FLAG-BAG-1 and ∼27 kDa FLAG-BAG-1 (ΔC) (retains only amino acids 1–172) proteins were all produced at readily detectable levels in transiently transfected 293 cells (Figure 4B). Figure 4.Cell growth inhibition by Siah-1A in 293 cells. (A) 293 cells were transiently transfected with 10 μg of pCI–Neo (1), 5 μg of pCI–neo + 5 μg of pCI–Bax (2), 5 μg of pCI–Neo + 5 μg of pCI–FLAG-Siah (22–298) (3), 5 μg of pCI–FLAG-Siah (22–298) + 5 μg of pCI-FLAG–BAG (1–219) (4), or 5 μg of pCI–FLAG-Siah (22–298) + 5 μg of pCI–FLAG-BAG (1–172) (5). After 48 h, both floating and attached cells were collected and the numbers of viable and dead cells were estimated by trypan blue dye exclusion. Data (mean ± SE) are shown as the absolute number of living cells (open bar) and dead cells (closed bar) per 60 mm dish for three independent transfection experiments. (B) Immunoblot analysis confirmed production of expected FLAG-Siah (22–298) protein of ∼34 kDa, FLAG-BAG-1 (1–219) protein of ∼32 kDa and FLAG-BAG-1 (1–172) protein of ∼27 kDa. Cell lysates were prepared from duplicated dishes of each transfection, normalized for total protein content (20 μg per lane), and analyzed by SDS–PAGE or immunoblotting using anti-FLAG monoclonal antibody with ECL-based detection. Download figure Download PowerPoint Siah-1A inhibits DNA synthesis in replicating cells To further examine the ability of Siah-1A to inhibit cell growth, GM701 cells were transfected with plasmids encoding FLAG-tagged Siah and the relative amounts of DNA synthesis (3H-thymidine incorporation into DNA) and the approximate percentage of S-phase cells [bromo-deoxyuridine (BrdU)-labeling] were determined. As shown in Figure 5, Siah-1A expression reduced by over half on average: (i) the number of cells present in cultures 2 days after transfection; (ii) the relative amount of DNA synthesis (3H-TdR incorporation); and (iii) the percentage of cells that were either in S-phase or had passed through S-phase (BrdU-positive). Propidium iodide-staining followed by flow-cytometric DNA content analysis also confirmed marked reductions in S-phase cells (58 ± 3% versus 39 ± 4%), with accumulations of the Siah-1A-expressing cells in both G0/G1 and G2/M (data not shown). In contrast, these inhibitory effects of Siah-1A were largely reversed by co-expression of BAG-1 (Figure 5). Immunoblotting confirmed production of the Siah-1A and BAG-1 proteins and verified that BAG-1 did not inhibit production of the Siah-1A protein (data not shown). Figure 5.Siah inhibits DNA synthesis in replicating GM701 cells. GM701 cells in DMEM containing 10% FCS were transiently transfected with pCI–neo (neo), pCI–FLAG-Siah or pCI-FLAG-Siah together with pRc/CMV–mBAG-1. (A) After 48 h, both floating and attached cells were recovered and viable cell numbers were estimated by trypan blue dye exclusion. As a control for growth inhibition, a duplicate culture of pCI–neo-transfected cells was switched to medium containing 0.1% FCS after 24 h. Alternatively, 24 h after transfection, 3H-thymidine (B) or BrdU (C) was added and cultures were continued for 24 h. Data (mean ± SE) represent results from three independent transfection experiments. Transfecting BAG-1 expression plasmids by itself had no significant effects on cell growth or proliferation (data not shown). Download figure Download PowerPoint BAG-1 interferes with p53-mediated cell growth suppression Since Siah is a potential downstream mediator of the effects of p53 in cells, we examined the possibility that BAG-1 might prevent p53-induced cell-cycle arrest. For these experiments, an expression plasmid encoding wild-type p53 protein was transiently transfected into 293 cells, with or without a BAG-1-encoding plasmid. Over-expression of p53 in 293 cells (i) induced increases in Siah-1A mRNA levels, as determined by RNase protection assay (Figure 6A), and (ii) produced a significant reduction in cell growth (Figure 6B), but did not induce apoptosis, as determined by several criteria including DAPI-staining and trypan blue dye exclusion assays (data not shown). Co-transfection of BAG-1 and p53 expression plasmids revealed that BAG-1 can partially reverse the inhibitory effects of p53 on 293 cell growth in a dose-dependent fashion. The C-terminal deletion mutant of BAG-1, however, did not abrogate p53-mediated growth arrest. Immunoblot analysis verified production of the p53, BAG-1 and BAG-1 (ΔC) proteins (data not shown). Thus, BAG-1 was capable of interfering with the p53-induced inhibition of cell growth in 293 cells. Figure 6.(A) RNase protection analysis of Siah-1A RNA after p53 over-expression. 293 cells were transiently transfected with 10 μg of pCMV–Neo (Neo) of 10 μg of pCMVp53wt (p53). Total RNA was extracted from cells at 0, 12, 24 and 36 h after transfection and Siah-1A RNA expression was measured with the use of an RNase protection assay utilizing a probe containing 324 bp of Siah-1A cDNA. A probe containing 218 bp fragment of the human keratin 18 cDNA was used as internal control. (B) Effect of BAG-1 expression on p53-induced growth arrest. 293 cells were transiently transfected with expression vectors [1, 10 μg pCI–Neo; 2, 1 μg pCMVp53wt; 3, 2.5 μg pCMVp53wt; 4, 5 μg pCMVp53wt; 5, 5 μg pCMVp53179; 6, 5 μg pCMVp53wt + 0.5 μg pCI–FLAG-BAG-1; 7, 5 μg pCMVp53wt + 1 μg pCI–FLAG-BAG-1; 8, 5 μg pCMVp53wt + 5 μg pCI–FLAG-BAG-1; 9, 1 μg pCMVp53wt + 5 μg pCI–FLAG-BAG-1; or 10, 5 μg pCMVp53wt + 5 μg pCI–FLAG-BAG-1 (1–172)]. After 48 h, viable cell numbers were estimated by trypan blue dye exclusion. Data (mean ± SE) are expressed as a percentage relative to pcI–Neo transfected cells. Download figure Download PowerPoint BAG-1 does not interfere with p53 protein induction or transactivation of target genes Though we suspected that BAG-1 blocked p53-induced growth arrest indirectly by nullifying the actions of Siah-1A, it was possible that BAG-1 interfered directly with some aspect of p53 function. No interaction was detected between BAG-1 and p53 by co-immunoprecipitation or in vitro binding studies, indicating that BAG-1 does not bind directly to p53 (data not shown). Moreover, immunofluorescence microscopy analysis of p53 protein demonstrated that BAG-1 over-expression does not preclude entry of p53 into the nucleus of 293 cells (unpublished data). The effects of BAG-1 on p53 protein levels were explored using several stable transfectants, representing human tumor cell lines that contain wild-type (MCF7, ZR75-1) or mutant (293, Jurkat) p53 (Figure 7A). Over-expression of BAG-1 did not alter the basal levels of p53 protein in these cell lines, as demonstrated by immunoblotting with anti-p53 antibodies. Furthermore, BAG-1 over-expression did not interfere with the upregulation of p53 protein levels induced by γ-radiation and UV-radiation in MCF7 cells which express wild-type p53 (Figure 7B). In addition, BAG-1 did not prevent p53 from transactivating a reporter gene plasmid that contains a p53-response element (p53-RE) derived from the BAX gene promoter (Figure 7C). Taken together, these data argue that BAG-1 interferes indirectly with p53-mediated growth suppression by acting at a step distal to p53-inducible gene expression, as expected if BAG-1 operates as an inhibitor of Siah-1A. Figure 7.BAG-1 does not interfere with p53 induction or p53-mediated gene transactivation. (A) Tumor cell lines [MCF-7, ZR 75.1, flag-tagged
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