1 cochaperone is a negative regulator of p73
Correspondence: Professor G Packham, Somers Cancer Sciences Building (MP824), Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, UK.
Received 24 November 2008; Revised 29 January 2009; Accepted 18 February 2009Advance online publication 17 March 2009
Top of pageAbstractMaterials and methods Results Discussion References Acknowledgements Figures and TablesHigh level expression of Bcl 2 associated athanogene (BAG 1) protects cancer cells from stress induced cell death and growth inhibition. These protective effects of BAG 1 are dependent on interactions with the HSC70 and HSP70 chaperones. However, the key stress response molecules that are regulated by a BAG 1 mechanism have not been identified. In this study, we investigated the effects of BAG 1 overexpression on the function of p53 family proteins, p53, p63 and p73. Overexpression of BAG 1 isoforms interfered with the transactivating activity of p73 and p63, but had modest and variable effects on p53 dependent transcription. p73 and BAG 1 interacted in intact cells and overexpression of BAG 1 decreased the expression of p73. siRNA mediated ablation of endogenous BAG 1 increased the activity of a p73 responsive promoter and this was reversed by knock down of p73. The ability of BAG 1 to modulate p73 activity and expression, and to interact with p73 were dependent on amino acid residues required for the interaction of BAG 1 with HSC70 and HSP70. These results show that BAG 1 inhibits the transactivating functions of p73 and provide new insight into the mechanisms that control the expression of p73. Inhibition of p73 function may be one mechanism that contributes to the pro survival activity of BAG 1.
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Bcl 2 associated athanogene (BAG 1) is a multifunctional protein that interacts with multiple cellular targets and modulates a wide range of cellular processes (Alberti et al, 2003; Townsend et al, 2003a, 2005; Gehring, 2004). Overexpression of BAG 1 protects cells from various apoptotic stimuli, enhances proliferation and metastasis, and modulates the transcriptional activity of a variety of nuclear hormone receptors. BAG 1 is essential for the survival and differentiation of haemopoietic and neuronal cells in mice (Gotz et al, 2005). Functional and expression studies suggest that overexpression of BAG 1 may play an important role in diverse cancer types (Cutress et al, 2002; Tang, 2002; Sharp et al, 2004). For example, BAG 1 is frequently overexpressed in breast cancer and can correlate with important clinical parameters (Tang et al, 1999, 2004; Turner et al, 2001; Townsend et al, 2002; Cutress et al, 2003; Pusztai et al, 2004; Sirvent et al, 2004).
In human cells BAG 1 exists as three major isoforms (BAG 1S, BAG 1M and BAG 1L) derived by alternate translation initiation from a single mRNA (Figure 1). All BAG 1 isoforms contain a C terminal, evolutionary conserved BAG domain (Takayama and Reed, 2001) and a central ubiquitin like domain (ULD), but the larger isoforms have unique N terminal extensions. In general, the functional significance of these variable N terminal regions is poorly understood. However, BAG 1L possesses a nuclear localisation sequence and is a predominantly nuclear protein, whereas the other isoforms partition between the cytoplasm and nucleus (Packham et al, 1997; Takayama et al, 1998; Yang et al, 1998; Brimmell et al, 1999).
Figure 1.
Human Bcl 2 associated athanogene (BAG 1) isoforms. The structures of the three major human BAG 1 isoforms are shown, along with their size (amino acid residues). Translation of BAG 1L initiates at an upstream CUG codon, whereas BAG 1M and BAG 1S are AUG derived. The position of the nuclear localisation sequence (NLS), acidic repeats, ubiquitin like domain (ULD) and BAG domain are shown.
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The C terminal BAG domain is comprised of a bundle of three of which helices 2 and 3 mediate electrostatic interactions with the ATPase domain of the 70 heat shock proteins, HSC70 and HSP70 (Briknarova et al, 2001; Sondermann et al, 2001). BAG 1 acts as a cochaperone and stimulates nucleotide exchange of HSC70 (Hohfeld and Jentsch, 1997; Takayama et al, 1997; Luders et al, 2000b; Brehmer et al, 2001; Nollen et al, 2001). HSC70 and HSP70 play important roles in multiple cell processes, for example, by effects on protein (re)folding and degradation, and on the expression and activity of nuclear hormone receptors (Mayer and Bukau, 2005; Daugaard et al, 2007; Grad and Picard,[url=http://www.canada--goose.co.uk/]@#$%&[/url], 2007). Binding to these multifunctional proteins may explain, at least in part, the multiple effects associated with BAG 1 overexpression. The BAG 1 ULD is required for the interaction of BAG 1 with the proteasome (Luders et al, 2000a),[url=http://www.@#$%&-rosheruns@#$%&/]@#$%& roshe[/url], and substantial evidence shows that BAG 1 can act to coordinate the function of chaperones and the proteasome in the degradation of specific proteins (Arndt et al, 2007). BAG 1 can interact simultaneously with HSC70 and the proteasome (Luders et al, 2000a; Alberti et al, 2002), and its ability to influence chaperone function may facilitate the unloading of chaperone clients in the vicinity of the proteasome to enhance degradation. BAG 1 and CHIP cooperate to target the glucocorticoid receptor for proteasomal degradation (Demand et al, 2001).
Functional studies show that BAG 1 isoforms promote the survival, proliferation and metastasis of cancer cells. For example, overexpression of BAG 1S or BAG 1L increases breast cancer cell survival in vitro and tumour growth in vivo (Kudoh et al, 2002). Our own studies (Townsend et al, 2003b) showed that overexpression of BAG 1 isoforms provided robust protection from cell death and long term growth inhibition induced by heat shock, and other cellular stress, including hypoxia, radiation and certain cytotoxic agents. RNAi mediated knock down of BAG 1 is also sufficient to promote apoptosis (Sawitzki et al, 2002; Clemo et al, 2008). The survival promoting function of BAG 1 was dependent on the BAG domain as a BAG 1 mutant lacking this region failed to promote cell survival in breast cancer cells (Kudoh et al, 2002; Townsend et al, 2003b). In addition to HSC70 the BAG domain also acts as a docking site for c Raf; BAG 1 activates c Raf independent of Ras (Wang et al, 1996; Song et al,[url=http://www.longchamphandbagsoutlet.us@#$%&/]longchamp handbags[/url], 2001). However, the critical functional requirement for the BAG domain seemed to be chaperone binding as the introduction of mutations that specifically ablated HSC70 interaction interfered with BAG 1 mediated survival in breast cancer cells (Townsend et al, 2003b) and other cell systems (Townsend et al, 2004). Thus, the survival function of BAG 1 is dependent on HSC70 However, the specific molecular regulators of stress induced apoptosis that are targeted by BAG 1 remain to be identified.
Members of the p53 family of transcription factors (p53, p63 and p73 and their splice variants) are critical regulators of stress induced apoptosis (Murray Zmijewski et al, 2006; McKeon and Melino,[url=http://www.nikeair-max.fr/]air max one[/url], 2007; Stiewe, 2007). The function of p53 family proteins is required for normal stress induced apoptosis (Agami et al, 1999; Gong et al, 1999; Yuan et al, 1999; Flores et al,[url=http://www.oakleyit.eu/]oakley[/url], 2002; Gressner et al,[url=http://www.juicycoutureoutlethandbags.in.net/]juicycouture@#$%&[/url], 2005; Stiewe, 2007) and they are frequently inactivated in cancer cells by mutations (p53 in particular) or alternate mechanisms. A number of proteins that have been shown to be overexpressed in cancer cells act, at least in part, by inhibiting the function of p53 family proteins. These proteins may be attractive therapeutic targets, as interfering with their function can lead to a reactivation of tumour suppressor function. For example, Mdm2 is overexpressed in many cancers with wild type p53 and inhibits p53 function by inhibition of transcriptional activity and targeting for degradation (Toledo and Wahl, 2006). Inhibitors of the p53:Mdm2 interaction induce p53 dependent apoptosis and are being developed as anti cancer drugs (Dudkina and Lindsley, 2007).
Our earlier study showed that BAG 1 overexpression suppressed stress induced apoptosis in MCF7 breast cancer cells (Townsend et al, 2003b), despite the presence of wild type p53, p63 and p73 in these cells (Gudas et al, 1995; Toh et al, 2005). This suggested that BAG 1 could interfere with the normal function of these proteins. As transcriptional regulation plays a major role in the function of p53, p63 and p73, we investigated the effects of BAG 1 on transactivation by p53 family proteins. Xin Lu,[url=http://www.saclongchamp-pas-cher.fr/]longchamp @#$%&[/url], (Ludwig Institute for Cancer Research,[url=http://www.michael-kors-outlet-online.us@#$%&/]michael kors handbags clearance[/url], Oxford, UK). The p63, p73 and p73 expression plasmids (De Laurenzi et al, 1998, 2000) were a kind gift of Prof. Gerry Melino (Medical Research Council, Toxicology Unit,[url=http://www.timberland-pas-cher.fr/]timberland @#$%&[/url], Leicester, UK). Human BAG 1S, BAG 1M and BAG 1L isoform specific expression constructs and point mutations have been described earlier (Townsend et al, 2003b, 2004). pcDNA3 plasmid was from Invitrogen Life Technologies (Paisley, UK). pGL2 Basic (Promega, Southampton,[url=http://www.truereligionjean.cc/]wholesale true religion jeans[/url], UK) and the human Bcl X IB promoter construct pBcl XIB (MacCarthy Morrogh et al, 2000) were used as control reporter plasmids.
Transfections and reporter gene assaysFor luciferase assays, SaOs2 cells (5 104) were plated in 24 well tissue culture plate one day before transfection. Cells were transfected using FuGene 6 transfection reagents (Roche Applied Science, Burgess Hill, UK) according to the manufacturer's instructions. Empty vector pcDNA3 was used to maintain equal quantity of total DNA per transfection. In the experiments to determine the effect of BAG 1 on p73 expression levels, H1299 cells were plated in 10 cm tissue culture dishes and co transfected with 1 of p73 expression construct in the presence or absence of 7 of BAG 1S expression construct or pcDNA3. After 24 expression of p73 BAG 1 and PCNA (loading control) were analysed by immunoblotting.
ImmunoblottingImmunoblots were carried out as described earlier (Brimmell et al, 1999) using the following primary antibodies: rabbit polyclonal anti BAG 1 (TB3, (Brimmell et al, 1999)), mouse monoclonal anti BAG 1 (3.10 G3E2; (Brimmell et al, 1999)), mouse monoclonal anti p73 antibody E4 (Santa Cruz Biotechnology,[url=http://www.reebokshoesoutlet.us@#$%&/]reebok shoes[/url], Santa Cruz, CA, USA), rabbit polyclonal anti p73 antibody R26 (generated by immunisation of rabbits with purified GST p73 fusion protein), rabbit polyclonal anti antibody (Sigma, Poole, UK) and mouse monoclonal anti PCNA antibody (Santa Cruz Biotechnology). Horseradish peroxidase conjugated secondary antibodies were from Amersham (GE Healthcare UK, Amersham, UK) and bound immunocomplexes were detected using SuperSignal West Pico Chemiluminescent reagents (Perbioscience UK Ltd, Pierce, Northumberland, UK). To quantify the effects of overexpression of BAG 1 on p73 expression, immunoblots were analysed using Quantity One program (BioRad, Hemel Hempstead, UK). The expression of p73 was normalised to the expression of PCNA and the relative expression of p73 in the absence of BAG 1 overexpression was set at 1.0.
RNA interferenceControl siRNA (control 1), siRNA against Bcl w (control 2), siRNA against human BAG 1 and siRNA against human p73 were obtained from Ambion Ltd (Huntingdon, UK) as annealed double stranded RNA DNA hybrids. After 72 cells were harvested and lysed for western blotting and luciferase assay. cDNA was synthesised using oligo(dT) and MMLV reverse transcriptase (Promega) according to the manufacturer's instructions. Q was carried out in 20 reactions containing 5 cDNA, 10 Universal Taqman PCR master mix (Applied Biosystems, Warrington, UK) and 1 of the Taqman Gene Expression Assay of interest (Applied Biosystems). Expression assays used for this study were p73 (Hs00232088_m1) and (Hs99999903_m1). All reactions were carried out in duplicate using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems) according to the following thermal cycle protocol: 94 for 10 followed by 40 cycles at 94 for 15 and 60 for 1 Control reactions with no cDNA were run on each plate for each Taqman gene Expression Assay used and no amplification was detected in any control reaction. All expression values were normalised using expression of as a control.
Co ImmunoprecipitationsH1299 cells were transfected (FuGene 6) in 4 100 tissue culture plates, each with 5 of p73 expression plasmid together with 5 of BAG 1S expression plasmids, or 5 of pcDNA3 empty vector. A portion (50 of the resultant cell lysate was retained as a whole cell lysate. The remaining sample was pre cleared using protein G Sepharose beads (pre blocked with 5 (w skimmed milk overnight) for 30 at 4 on a Spiramixer. Protein G Sepharose beads were removed by centrifugation. After overnight incubation at 4 the lysate was incubated with protein G Sepharose beads at 4 for 4 and immunocomplexes were removed by centrifugation. The beads were washed four times using HMKEN buffer, re suspended in 50 SDS PAGE sample buffer and heated at 95 for 5 before immunoblot analysis. p53 null SaOs2 cells were selected for this study as they have been widely used for investigations of p53 family protein function (Jost et al, 1997; Mantovani et al, 2004; Melino et al, 2004). SaOs2 cells were transfected with a Bax promoter construct and p53, p63 or p73 expression plasmids,[url=http://www.bottes-uggpascher.fr/]ugg[/url], in the presence or absence of a human BAG 1S expression plasmid (Figure 2A). BAG 1S overexpression did not have a significant effect on the basal expression of the Bax promoter but did interfere with the ability of p53, p63 and p73 to increase promoter expression. Co expression of p53, p63 or p73 did not significantly alter the levels of BAG 1S (Figure 2A).
Figure 2.
Effect of Bcl 2 associated athanogene (BAG 1)S overexpression on transcriptional regulation by p53 family proteins. (A) SaOs2 cells were transfected with 100 of Bax luc reporter construct and 100 of p53, 100 of p63 or 50 of p73 expression plasmids, respectively, in the presence or absence of BAG 1S expression construct (2500 Transfected cells were analysed for luciferase activity (top) and BAG 1S expression (by immunoblotting (IB); bottom) 48 after transfection. Data shown are the mean luciferase activity ( of duplicate transfections normalised to cells transfected with Bax luc only (set to 1.0). (B) SaOs2 cells were transfected with 100 of Bax luc reporter construct in the absence or presence of p73 expression plasmid (50 and increasing amount of BAG 1S expression construct (0, 500, 1000 Transfected cells were analysed for luciferase activity (top) and BAG 1S expression (bottom) 48 after transfection. Data shown are the mean luciferase activity ( of duplicate transfections normalised to cells transfected with Bax luc only (set to 1.0). (C) SaOs2 cells were transfected with 200 of pGL Basic or Bcl XIB reporter constructs in the absence or presence of 1000 of BAG 1S expression plasmid and luciferase activity (top), and BAG 1S expression (by immunoblotting (IB); bottom) was measured after 48 Data shown are the mean luciferase activity ( of duplicate transfections normalised to cells without BAG 1S overexpression (set to 1.0). The experiments shown in (A are representative of two similar experiments.
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As the effects of BAG 1S on p73 function were most dramatic, we focused our analysis on this interaction. Plasmid titration experiments showed that the effects of BAG 1S overexpression were concentration dependent (Figure 2B) and were specific, because BAG 1S overexpression did not interfere with the activity of control promoters not regulated by p73 (Figure 2C). The inhibitory effects of BAG 1S were also observed in all the cell lines tested (HEK293, NIH3T3 and H1299; Figure 3A) and using a range of p73 responsive promoter constructs (IGFBP3, GADD45,[url=http://www.prom-weddingdresses@#$%&/]evening dresses[/url], Pig3 and MDM 2; Figure 3B). All three BAG 1 isoforms inhibited p73 transcription when overexpressed in SaOs2 cells (Figure 3C).
Regulation of p73 transcriptional activity by Bcl 2 associated athanogene (BAG 1) isoforms. (A) Human embryonic kidney (HEK)293, NIH3T3 and H1299 cells were transfected with the Bax luc reporter (100 and the indicated amounts (ng) of p73 and BAG 1S expression plasmids. Luciferase activity (top) and BAG 1S expression (by immunoblotting (IB); bottom) was measured after 48 Data shown are the mean luciferase activity ( of duplicate transfections normalised to cells transfected with Bax luc only (set to 1.0). Experiments shown are representative of at least three similar experiments. (B) SaOs2 cells were transfected with various promoter constructs with or without p73 (25 for transfections with the Pig3 promoter, 50 for transfections with the Bax promoter, IGFBP3 or MDM 2 promoter or 100 for transfections with the GADD45 promoter) and in the presence (closed bars) or absence (open bars) of BAG 1S (1000 The following amounts of reporter constructs were used in each transfection; Bax, 100 GADD45 200 IGFBP3, 50 MDM 2, 50 or Pig3, 100 Luciferase activity (top) and BAG 1S expression (by immunoblotting (IB); bottom) was measured after 48 Data shown are the mean luciferase activity ( of duplicate transfections normalised to cells transfected with each reporter construct in the absence of BAG 1S or p73 (set to 1.0). Experiments shown are representative of at least two similar experiments. (C) SaOs2 cells were transfected with the Bax luc reporter construct (100 and the p73 expression plasmid (50 and BAG 1S, BAG 1M or BAG 1L expression plasmids (1000 as indicated. Luciferase activity (top panel) was measured after 48 Data shown are the mean luciferase activity ( of duplicate transfections normalised to cells transfected with the Bax reporter construct in the absence of BAG 1 or p73 (set to 1.0). Experiment shown is representative of four similar experiments. The bottom panel shows BAG 1 protein expression in transfected cells analysed by immunoblotting. Arrows indicate the BAG 1L, BAG 1M and BAG 1S isoforms. Note that some faster migrating BAG 1 forms are detected, especially in cells transfected with the BAG 1M expression plasmid. These may derive from internal translation initiation or degradation (Lee et al, 2007).

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