Endoplasmic Reticulum Stress Induces G2 Cell-Cycle Arrest via

Molecular Cell

Article Endoplasmic Reticulum Stress Induces G2 Cell-Cycle Arrest via mRNA Translation of the p53 Isoform p53/47 Karima Bourougaa,1 Nadia Naski,1 Cedric Boularan,2 Coraline Mlynarczyk,1 Marco M. Candeias,1 Stefano Marullo,2 and Robin Fa˚hraeus1,* 1INSERM

Unite´ 716, Institut de Ge´ne´tique Mole´culaire, Universite´ Paris 7, Hoˆpital St. Louis, 27 rue Juliette Dodu, 75010 Paris, France Cochin, Universite´ Paris 5, CNRS U8104, 75014 Paris, France *Correspondence: [email protected] DOI 10.1016/j.molcel.2010.01.041 2Institut

SUMMARY

p53 downstream pathways control G1 and G2 cellcycle arrest, DNA repair, or apoptosis. However, it is still not clear how cells differentiate the cell-biological outcome of p53 activation in response to different types of stresses. The p53/47 isoform lacks the first 39 amino acids of full-length p53 including the Mdm2 binding site and the first trans-activation domain, and tetramers including p53/47 exhibit altered activity and biochemical properties. Here we show that endoplasmic reticulum stress promotes PERK-dependent induction of p53/47 mRNA translation and p53/47 homo-oligomerization. p53/47 induces 14-3-3s and G2 arrest but does not affect G1 progression. This is contrary to p53FL, which promotes G1 arrest but has no effect on the G2. These results show a unique role for p53/47 in the p53 pathway and illustrate how a cellular stress leads to a defined cell-biological outcome through expression of a p53 isoform.

INTRODUCTION Transcriptionally active p53 tetramers bind to a large number of promoter regions and affect the expression of gene products that can prevent cancer development by regulating cell growth, DNA repair, apoptosis, and senescence (Levine and Oren, 2009). p53 is activated in response to a wide spectrum of stresses and damages and is subject to different types of posttranslational modifications and regulatory partners that affect its functions. It is still relatively unknown how p53 activation can lead to the induction of particular sets of gene products that give rise to specific cell-biological effects (Vousden and Prives, 2009). More recently, different p53 isoforms have been identified in human normal and tumor cells that lack different domains but can form homo- or hetero-oligomers and thus have the potential to add a further level of control to p53 activity (Bourdon et al., 2005; Yin et al., 2002). However, the regulation and physiological role of these isoforms are just starting to become known.

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The p53/47 isoform is an alternative p53 mRNA translation product that can be synthesized by cap-independent mechanisms in response to serum deprivation, cell-cycle progression, or endoplasmic reticulum (ER) stress (Candeias et al., 2006; Ray et al., 2006). It lacks the first 39 amino acids of the full-length p53 (p53FL), including the binding site for the E3 ubiquitin ligase Mdm2 and the trans-activation activation domain I (TAI) (Yin et al., 2002). p53/47 retains the DNA-binding and oligomerization capacity and displays a stress-dependent activation pattern of downstream target genes that differs from the full-length p53 (p53FL), including both the induction and suppression of gene products (Ohki et al., 2007; Powell et al., 2008). p53 negatively regulates cell-cycle progression in response to different cellular stresses. Activation following DNA damage leads to trans-activation of the cyclin-dependent kinase inhibitor p21CDKN1A and G1 arrest (el-Deiry et al., 1993). Trans-activation of p21 requires the TA1 domain and p53/47 has been shown to prevent p53-dependent induction of p21 (Courtois et al., 2002). 14-3-3s is upregulated by p53 and causes a G2 arrest by sequestering the Cdc25 phosphatase in the cytosol and thereby preventing dephosphorylation of Cdc2 and activation of the cyclinB/ Cdc2 complex (Hermeking et al., 1997). 14-3-3s has the characteristics of a tumor suppressor and is downregulated in a number of human cancers (Benzinger et al., 2005). More recently, it has been shown to promote cap-independent mRNA translation during the G2 by interacting with the mRNA translation initiation factors eIF4B, eIF2a, and EF1a (Wilker et al., 2007). During the G2/M transition, cap-dependent translation initiation is suppressed at several steps including inhibition of eIF4F assembly by the binding of 4EBP1-2 to eIF4E, phosphorylation of PAP by the cyclinB /Cdc2 complex, eIF2a phosphorylation, or eIF4B sequestering by 14-3-3s (Bonneau and Sonenberg, 1987; Colgan et al., 1998; Datta et al., 1999; Wilker et al., 2007). However, synthesis of certain proteins required for progression into mitosis, like ODC or p58PITSLRE, is maintained via cap-independent initiation of translation (Cornelis et al., 2000; Pyronnet et al., 2000). This type of translation is mediated via internal ribosome entry sites (IRESs) and prevails during the G2 and controls synthesis of proteins involved in growth control and apoptosis during stress (Cornelis et al., 2000; Kieft, 2008; Pyronnet et al., 2000). Exposure to hypoxia or treating cells with drugs that prevent glycosylation (tunicamycin) or target ER calcium influx

Molecular Cell p53/47-Induced G2 Arrest

induces G1 arrest and has no influence on the G2. These data indicate that p53/47 plays a unique role in the p53 response and illustrate how a cell stress pathway, by controlling the translation of the p53 mRNA, differentiates p53 activity and leads to a specific cell-biological outcome. RESULTS

Figure 1. p53-Dependent G2 Arrest Following ER Stress MLS1765, A549, and HCT116p53+/+ cell lines express endogenous wild-type p53 (p53wt), while H1299, Saos-2, and the HCT116p53/ are p53 null. (A) p53 siRNA (si p53), or control siRNA (si control), was introduced in MLS1764 and A549 cells before 16 hr treatment with 100 nM of thapsigargin, at which the cell-cycle profile (G1 and G2) was determined using FACS analysis. Nonsynchronized HCT116p53+/+ and HCT116p53/ cells were exposed to thapsigargin only. (B) H1299 and Saos-2 cells were transfected with p53wt cDNA and treated as in (A) before the cell-cycle distribution was determined (see also Figure S1). The results are presented in (A) as thapsigargin-dependent changes in the cell-cycle profile relative to DMSO (0.1%)-treated cells (no effect) or (B) as relative to p53wt-transfected cells exposed to 0.1% DMSO. The results show the mean value of at least three independent experiments + SD.

(thapsigargin) result in ER stress and the unfolded protein response (UPR) (Schroder and Kaufman, 2005). During the UPR, global cap-dependent protein synthesis is suppressed via phosphorylation of eIF2a by PERK while the activation of IRE-1 and ATF6 serves to restore the balance between newly synthesized and correctly maturated proteins (Fernandez et al., 2002; Ron and Walter, 2007; Harding et al., 1999). Prolonged ER stress can also lead to the induction of apoptosis via activation of CHOP and caspase-12 or via the p53 pathway and the induction of proapoptotic gene products such as PUMA and Noxa (Li et al., 2006; McCullough et al., 2001; Nakagawa et al., 2000). Here we show that ER stress via PERK leads to the induction of p53/47 mRNA translation, which is followed by an upregulation of 14-3-3s and a G2 arrest. Full-length p53, on the contrary,

ER Stress Causes a p53-Dependent G2 Cell-Cycle Arrest In order to investigate the physiological role of changes in p53 mRNA translation in response to ER stress, we treated three nonsynchronized cell cultures that express endogenous wildtype p53 (p53wt) (MLS1765, A549, and HCT116p53+/+) and the respective counterparts that either lack a functional p53 (HCT116p53/) or in which p53 levels had been reduced by siRNA, with 100 nM thapsigargin for 16 hr. Thapsigargin treatment reduced the average number of cells in the G1 phase of the cell cycle between 5% and 15%. Transfecting A549 or MLS1765 with p53 siRNA prior to thapsigargin exposure, or using the HCT116p53/ cells, resulted in an approximate increase of the number of cells in G1 of 12%, 3%, and 28%, respectively. These results are in line with the notion that there is no specific p53-dependent induction of cells in G1 in response to prolonged ER stress (Pluquet et al., 2005). ER stress resulted in an average 33%, 27%, and 15% increase in G2 in the MLS1765, A549, and HCT116p53+/+ cells. We also observed an actual decrease of 20%, 13%, and 25% in the G2 of MLS1765 and A549 after p53 siRNA treatment and in HCT116p53/ cells, respectively (Figure 1A and see Figure S1 available online). In order to further test if the observed ER stress-dependent increase in G2 is dependent on p53, we tested two nonsynchronized p53 null cells. Exposure of H1299 and Saos-2 cells to thapsigargin resulted in an average 12% and 19% increase in G1 and a 5% and 17% decrease in G2, respectively. When we introduced p53wt, we observed a 5% thapsigargin-dependent increase in the G1 of H1299 cells, whereas the amount of Saos-2 cells in G1 decreased with 12%. However, the p53dependent increase of cells in G2 was approximately 70% for H1299 and 40% for Saos-2 (Figure 1B). Expression of an inactive mutant p53 (p53R175H) showed no changes in G1 or G2. Similar results were also obtained after treatment with 8.5 mM of tunicamycin for 16 hr (data not shown). These results indicate that cells respond with a p53-dependent G2 arrest following prolonged ER stress. p53/47 and Full-Length p53 Have Opposite Effects on G1 and G2 Cell-Cycle Arrest It is known that p53 can induce a G2 arrest (Hermeking et al., 1997), but no stress pathway has been described that specifically causes a p53/47-dependent G2 arrest. p53/47 expression is induced by ER stress, and in order to test if this can account for the observed G2 arrest, we expressed p53FL from an mRNA lacking the p53/47 initiation codon (AUG to GCG), or p53/47 from an mRNA in which the sequence upstream of the p53/47 coding sequence, including the IRES (+1 to +120), was deleted (p53/47DIRES) (Figures 2A and 2B). Translation of the p53wt mRNA results in the expression of both isoforms and in

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Figure 2. p53/47 and p53FL Have Opposite Effects on the G1 and G2 Phases of the Cell Cycle (A) The p53wt construct lacks the 50 UTR. The fulllength p53 (p53FL) construct carries alanine substitution for the p53/47 initiation codon and expresses p53FL only. p53/47DIRES expresses p53/47 only and lacks the IRES sequence located between the p53FL and p53/47 initiation codons (+1 to +120 nt). (B) A representative western blot from one of the FACS analyses (C and D), showing p53 expression levels using the different p53 constructs. (C) H1299 cells expressing indicated p53 constructs, or the inactive p53R175H and p53/ 47R175H mutants, were treated with thapsigargin for 16 hr, and the cell-cycle distribution was determined. Data show relative p53-dependent G1 arrest (left panel). Trans-activation of p21CDKN1A mRNA under the same conditions was measured using qRT-PCR (right panel). (D) The relative change in G2 of H1299 cells expressing indicated p53 constructs after treatment with thapsigargin. The values (C and D) are relative to empty vector (EV)-transfected cells treated with 0.1% DMSO (zero) (Figure S2). (E) The western blot and the corresponding graph show p53/47 dose-dependent G2 arrest. (F) Same as in (D) but using Saos-2 cells. The data represent the average of more than three independent experiments + SD. *p < 0.05 and **p < 0.01.

H1299 cells without p53. Similar results were also observed in the Saos-2 cells (Figure 2F). These results indicate that p53FL controls the G1 phase of the cell cycle and p53/47 controls G2 progression.

an approximately 30% increase of cells in G1 with an additional non-p53-specific 15% increase after thapsigargin treatment. Expression of p53FL alone resulted in a G1 arrest, as expected, while expression of p53/47 only had no, or even a small negative, effect. The capacity of the different p53 constructs to induce G1 arrest correlates well with p21 mRNA expression levels (Figure 2C, left and right panels). This is in agreement with previous studies showing that the TAI of p53 is required to induce p21 expression under normal conditions. Interestingly, we observed that p53FL had little effect on the G2 phase of the cell cycle, whereas the presence of p53/47 gave a strong G2 arrest (Figures 2D and 2E and Figure S2). The p53wt- and p53/47-induced G2 arrest observed in H1299 cells were increased approximately 2-fold after thapsigargin treatment. This should be compared with the 12% decrease in G2 after thapsigargin treatment in


p53/47 Induces G2 Arrest via Upregulation of 14-3-3s p53/47 retains the TAII and is sufficient to alter the expression of p53 target genes both in a positive and a negative fashion that is different from the activity of the p53FL (Powell et al., 2008). When we analyzed the effect of p53/47 (p53/47DIRES) on a number of gene products linked to p53-dependent cell-cycle control using quantitative RT-PCR (qRT-PCR), we observed an approximately 7-fold induction of 14-3-3s mRNA levels. The increase in 14-3-3s mRNA levels by the p53wt was approximately 3-fold and nonsignificant for the p53FL. There was a near 2-fold further increase after treatment with thapsigargin using either p53wt or p53/47 mRNAs (Figure 3A). 14-3-3s has been reported to induce G2 arrest in response to p53 activation, and to test if the induction of 14-3-3s by p53/47 is important for the G2 arrest we introduced 14-3-3s siRNA. This resulted in an approximately 50% and 70% reduction in p53wt- and p53/47-induced G2 arrest, respectively, as compared with control siRNA (Figures 3B and 3C and Figure S4). To confirm that the induction of 14-3-3s is a direct


Figure 3. p53/47 Induces 14-3-3s Expression (A) The expression of 14-3-3s mRNA levels in H1299 cells following expression of indicated p53 construct was determined using qRT-PCR. (B) Induction of 14-3-3s mRNA levels in the presence of control siRNA or 14-3-3s siRNA in cells expressing the indicated p53 construct or empty vector (EV). (C) p53-dependent G2 arrest after thapsigargin treatment was estimated in the presence of 14-3-3s siRNA. The amount of cells in G2 after expression of indicated p53 construct and treated with siRNA control was set to 100% (Figure S4). (D) The binding of p53 isoforms to the p53 response element in the 14-3-3s promoter was estimated in H1299 cells by ChIP assays using the anti-p53 polyclonal CM-1 sera. (E) ChIP assays of endogenous p53 isoforms binding to the 14-3-3s promoter in MLS1765 cells following thapsigargin treatment using the CM-1 pAb or the DO-1 mAb. The latter recognizes p53FL only. The ChIP data show values subtracted by a nonspecific IgG control. (F) Induction of 14-3-3s in cells expressing endogenous p53wt in response to ER stress. The data show values from at least three independent experiments + SD. *p < 0.05, **p < 0.01, and ***p < 0.001.

effect of p53/47, we performed chromatin immunoprecipitation (ChIP) assays on the p53 response element in the 14-3-3s promoter (Hermeking et al., 1997). Expression of p53/47 in H1299 cells resulted in a greater than 20-fold increase in the amount of 14-3-3s promoter that coimmunoprecipitated using the polyclonal anti-p53 CM-1 sera, as compared with a 5-fold increase with the p53wt mRNA (Figure 3D and Figure S3). The amount of 14-3-3s promoter that coimmunoprecipitated with p53 products derived from the p53wt or the p53DIRES mRNAs is increased a further 2-fold following treatment with thapsigargin, whereas the amount of promoter that coimmunoprecipitated with p53FL instead decreased by an average 50%. The larger amount of 14-3-3s promoter that coimmunoprecipitated with p53/47 and induced 14-3-3s expression using the p53/47DIRES construct correlates with the higher levels of p53/47 protein expressed from this mRNA, as compared with p53/47 expressed from the p53wt mRNA (compare Figures 2B and 4). However, the total amount of p53 proteins expressed from the p53 constructs is similar, indicating that the levels of p53/47 expression, and not

p53FL, are important for the induction of 14-3-3s. To support this notion we observed an approximately 50% increase in p53 products binding to the 14-3-3s promoter in MLS1765 cells using the polyclonal CM-1 sera and an actual decrease using the DO-1 mAb, which only recognizes p53FL after thapsigargin treatment (Figure 3E). Interestingly, p53FL interacts with the 14-3-3s promoter in untreated cells, indicating that p53FL interacts with the 14-3-3s promoter in an inactive form but is displaced following the UPR. In addition, qRT-PCR data revealed an approximately 8- and 4-fold induction of 14-3-3s mRNA levels in MLS1765 and A549 cells, respectively, under the same conditions (Figure 3F). PERK Controls p53/47 mRNA Translation We next investigated the signaling pathway that mediates ER stress-dependent induction of p53/47. The mRNA sequence located between the initiation codons for p53FL and p53/47 (+1 to +120) controls cap-independent translation of p53/47 in response to ER stress (Candeias et al., 2006). The PERK kinase plays an important role in the UPR by phosphorylating the translation initiation factor eIF2a (Fernandez et al., 2002; Harding et al., 1999). Expression of p53/47 from an p53 mRNA that carries the 50 UTR (p53wt+50 UTR) is less as compared to one lacking the 50 UTR (p53wt) (Figures 4A and 4B). Treatment with thapsigargin for 16 hr resulted in an approximately 5- and 3-fold increase in p53/47 expression from respective mRNAs



Figure 4. PERK Controls p53/47 mRNA Translation (A) Cartoons illustrating p53 mRNA constructs (see also Figure 2A). p53wt+50 UTR carries the p53 coding sequence plus the 50 UTR. The p53/ 47 construct has an alanine (GCG) substitution for the p53FL initiation codon and expresses p53/47 only. p53Bi-cis initiates cap-independent p53/47 mRNA translation from a bicistronic setting with a hairpin structure inserted in the 30 UTR of the GFP to minimize cap-dependent ribosomal readthrough. (B) Western blots show the effect of the dominantnegative PERKDC on the expression of p53/47 from the p53wt+50 UTR and p53wt constructs. The 421 mAb is directed toward the p53 C terminus. (C) The graph shows the relative change in expression of p53 isoforms in cells expressing the p53wt mRNA after treatment with thapsigargin or tunicamycin over 16 hr. (D) Western blots show the effect of PERKDC on p53/47 expression in MLS1765 cells or H1299 cells expressing indicated p53 constructs. Similar results were obtained in Saos-2 cells (Figure S6). (E) PERK siRNA prevents induction of p53/47 after thapsigargin treatment (Figure S5). (F) Western blot showing the effects of PERKDC and PERK siRNA on the phosphorylation of eIF2a at Ser51 using a phosphospecific mAb (P- eIF2a), as compared with total eIF2a levels. (G) Cobalt chloride (CoCl2) mimics the effect of hypoxia-induced ER stress. Treatment with 10 mM for 16 hr results in an increase in p53/47 expression. The presented results (B–G) show one of at least three independent experiments. The numbers represent relative p53/47 expression and are derived from ECL detection using the Bio1D software. Data are from transfected H1299 cells except for the MLS1765 cells.

and with little effect on the expression of p53FL. Coexpression of the dominant-negative PERKDC (Harding et al., 1999) suppressed thapsigargin-induced p53/47 expression from either mRNA with approximately 70%. A similar PERK-dependent control of p53 mRNA translation was also observed in Saos-2 cells (Figure S6). This indicates that PERK activity is required for ER stress-induced expression of p53/47 independently of the 50 UTR of the p53 mRNA. To study the kinetics of ER stress-dependent induction of p53/47, we followed the expression levels during 16 hr after treatment with tunicamycin or thapsigargin. The results show a gradual induction of p53/47 with a peak around 10–12 hr, which is in line with induction of the UPR (Schroder and Kaufman, 2005). The levels of p53FL changed only marginally during the same time frame (Figure 4C). The p53/47 construct lacks the p53FL initiation codon but retains the IRES, and PERKDC prevented expression of p53/47 from this mRNA, indicating that translation of p53/47 is PERK


dependent, independently of p53FL expression. However, deletion of the first 120 nt of the p53 encoding sequence harboring the IRES sequence (p53/47DIRES) prevented thapsigarginand PERKDC-dependent control of p53/47 expression (Figure 4D and Figure S5). The presence of PERKDC suppressed the expression of p53/47 also when the p53/47 mRNA sequence was inserted downstream of GFP and a hairpin structure (p53Bicis), indicating that PERK can control cap-independent translation of p53/47. When we expressed the PERKDC in MLS1765 cells treated with thapsigargin, we also observed a similar reduction in endogenous p53/47 levels. In further support of PERK controlling p53/47 expression, we found that PERK siRNA resulted in a reduction in p53/47 levels (Figure 4E and Figure S5). The efficacy of the PERKDC and the PERK siRNA to suppress PERK activity was confirmed by testing the phosphorylation on Ser51 of eIF2a and PERK mRNA levels, respectively (Figure 4F). In addition to changes in the Ca-flux or glycosylation, UPR can


Figure 5. PERK-Mediated Induction of p53/47 Is Required for ER Stress-Dependent G2 Arrest (A) Expression of dominant-negative PERK (PERKDC) prevents p53wt- and p53/47-induced G2 arrest in H1299 cells following treatment with thapsigargin. Deletion of the IRES sequence from the p53 mRNA (p53/47DIRES) renders p53/47induced G2 arrest PERK independent. The relative amount of thapsigargin-treated cells arrested in G2 in the presence of indicated p53 construct is set at 100%. Similar results were obtained using Saos-2 cells (S6). (B) Relative induction of 14-3-3s mRNA (qRTPCR) by indicated p53 constructs after thapsigargin treatment (set to 100%) in the presence of PERKDC or empty vector (EV). (C) FACS analysis shows the effect of PERKDC on thapsigargin-induced G2 arrest in MLS1765 and A549 cells expressing endogenous p53wt. The value for EV-transfected and thapsigargin-treated cells was set to zero. (D) PERKDC prevents the binding of endogenous p53 products to the 14-3-3s promoter in MLS1765 cells. The value for EV-transfected cells treated with DMSO was set to zero. (E) MCF-7 cells express a nonfunctional 14-3-3s protein. The effects of PERKDC on endogenous p53/47 expression in MCF-7 cells (left panel) and on G2 progression (right graph) are shown. The relative amount of MCF-7 cells in G2 after transfection with EV and treatment with DMSO was set to 100%. The data represent the average of three independent experiments + SD. *p < 0.05.

be triggered by hypoxia, and treatment of cells with cobalt chloride (CoCl2), which mimics hypoxia (Koumenis et al., 2002), resulted in an increase in p53/47 expression (Figure 4G), underlining that p53/47 is induced by a wide variety of different types of damages to the ER known to trigger the UPR. PERK Is Required for p53/47-Induced G2 Arrest in Response to ER Stress We next set out to test if PERK-dependent control of p53/47 expression is responsible for ER stress-induced G2 arrest. By expressing the dominant-negative PERKDC together with the p53wt or the p53/47 mRNAs, we observed a reduction in p53/ 47-dependent G2 arrest in response to ER stress with approximately 60% and 70%, respectively (Figure 5A). Similar results were also observed in Saos-2 cells (Figure S6). Interestingly, when p53/47 was expressed from the non-PERK-responsive p53/47DIRES mRNA, PERKDC had little effect on G2 arrest. In addition, PERKDC affects 14-3-3s expression when p53/47 is expressed from the p53wt mRNA, but not when expressed

from the p53/47DIRES message (Figure 5B). Both the MLS1765 and the A549 cells showed an approximately 17% reduction in thapsigargin-induced G2 arrest after transfection with PERKDC (Figure 5C). This can be compared to the 20% reduction in p53-induced G1/S arrest after DNA damage following ATM exclusion (Westphal et al., 1997). In addition, the induction of endogenous p53 products bound to the 14-3-3s promoter in MLS1765 cells following thapsigargin treatment was reduced from 50% to less than 20% in the presence of PERKDC (Figure 5D). To further test the role of 14-3-3s in PERKdependent G2 arrest, we used MCF-7 cells which have a nonfunctional 14-3-3s due to an excess of the E3 ubiquitin ligase Efp (Urano et al., 2002). Expression of PERKDC resulted in a decrease in p53/47 expression without any effect on the G2 phase (Figure 5E, left and right panels). Taken together, these results indicate that ER stress-induced G2 arrest is dependent on the capacity of PERK to control p53/47 mRNA translation and the subsequent induction of 14-3-3s. ER Stress Promotes the Formation of p53/47 Oligomer Complexes The observation that p53FL and p53/47 have the opposite effects on the G1 and G2 phases of the cell cycle shows that



Figure 6. ER Stress Induces p53/47 HomoOligomerization Bioluminescence resonance energy transfer (BRET) assays were carried out to monitor p53 oligomerization in live cells. RLuc (donor) or YFP (acceptor) was fused to the N terminus of the p53FL construct or to the N terminus of p53/47 (p53DIRES mRNA). Rluc was activated by coelenterazine H, which leads to emission from YFP molecules within 100 A˚. The normalized mBRET values correspond to RLuc-dependent YFP activation. YFP corresponds to background fluorescence in cells expressing the BRET donor alone. (A) Rluc-p53/47 gives higher BRET emission with YFP-p53/47 than withYFP-p53FL. BRET50 corresponds to half of the maximal BRET value and reflects the relative affinity of the two partners. Treatment with thapsigargin reduced the BRET50 for p53/47 homo-oligomers from approximately 10 to 1 but had no significant impact on the affinity of p53/47-p53FL hetero-oligomers. (B) Cells expressing RLuc-p53/47 and YFP-p53/47 in the presence of the p53/47 oligomerizationdeficient mutant (I332V, F342L and L344I). The data show the average values of three independent experiments (Figure S7). (C) MLS1765 cells expressing endogenous p53wt were treated with thapsigargin, and p53 complexes were crosslinked using formaldehyde (1%) before cell lysis. The relative amount of both p53 isoforms in the monomer, dimmer, and tetramer complexes was estimated following immunoblotting using the CM-1 sera (left panel). The DO-1 mAb was used (right panel) in a parallel blot to estimate the relative amount of p53FL. In order to clearly visualize p53 monomers, dimmers, and tetramers using CM-1, two exposures of 10 and 30 s are shown. (D) H1299 cells expressing indicated p53 constructs were subjected to formaldehyde crosslinking and the p53 complexes. The graphs show the relative changes in p53 complexes in response to thapsigargin treatment (C and D). The data show one of three similar experiments, and the estimated relative values of p53 products in each complex are derived from ECL detection using Bio1D software.

p53/47 harbors unique properties that are not carried by p53FL. But how the p53/47-dependent G2 arrest can be dominant in the presence of higher levels of p53FL during prolonged ER stress was puzzling. p53 complexes become transcriptionally active, as tetramers and previous studies have shown that p53/47 can form both hetero- and homo-oligomers and that the ratio of p53FL:p53/47 has important consequences for p53 activity (Joerger and Fersht, 2008; Powell et al., 2008; Weinberg et al., 2004). To investigate the formation of p53 isoform complexes in response to ER stress in live cells, we used the bioluminescence resonance energy transfer (BRET) technology (Bacart et al., 2008). Renilla luciferase (RLuc) or yellow fluorescent protein (YFP) was fused directly to the N terminus of the coding sequences of p53FL and p53/47. Cells expressing indicated


constructs were treated with coelenterazine H, which activates the luciferase donor and promotes emission from YFP acceptor molecules located less than 100 A˚ apart. Altering donoracceptor pairs; dose response; and the normalization to YFPp53/47, YFP-p53, and YFP ensures for specificity. Fusion of p53/47 to the RLuc and to YFP and p53FL to YFP revealed that oligomer complexes including p53/47 result in a higher BRET. This was observed using either p53FL or p53/47 as the donor (Figure 6A and Figure S7). The highest emission was obtained using p53/47 as donor and acceptor. However, the affinity for p53FL and p53/47 homo- and hetero-oligomers is similar, as the BRET50 is unchanged (Marullo and Bouvier, 2007). Interestingly, treatment with thapsigargin resulted in a strong increase in the affinity of p53/47 homo-oligomers as the BRET50 shifts from approximately 11 to 1. This is not observed between


hetero-oligomers and indicates that ER stress promotes the specific formation of p53/47 homo-oligomers. When we introduced a ‘‘cold’’ p53/47 carrying neither RLuc nor YFP but with point mutations that prevent oligomerization (I332V, F341L, and L344I), we observed no disruption of the p53/47-p53/47 BRET, indicating that the observed BRET profiles are due to the capacity of the p53 fusion constructs to form specific oligomers (Figure 6B). The observation that p53/47 more easily forms oligomers is in line with what we and others have observed previously (Ghosh et al., 2004; Powell et al., 2008). But what is more surprising was the selective formation of p53/47 homo-oligomer complexes after ER stress. The BRET assay does not distinguish between dimers and tetramers, and to see what happens with p53 tetramer complexes under similar conditions, we chemically crosslinked p53 complexes in MLS1765 cells. In the presence of thapsigargin, we could observe an increase in tetramers recognized by the CM-1 polyclonal sera which recognizes both p53 isoforms. By blotting the same samples against the DO-1 mAb, which is specific for the p53FL, we instead observed a decrease in the amount of p53FL in tetramers complexes. Thus, the increase in tetramers observed using the CM-1 sera following thapsigargin treatment is due to an increase of p53/47 molecules (Figure 6C). In agreement with this, we also observed an increase in p53/47 tetramers in H1299 cells following ER stress, while tetramers including p53FL were reduced (Figure 6D). These results indicate that ER stress, in addition to increasing the expression of p53/47, also promotes the formation p53/47 homotetramers. p53/47 Is Sufficient to Induce Apoptosis after Prolonged ER Stress Prolonged or severe cell damage tips the balance of the cellbiological response of p53 activation from cell-cycle arrest and repair to apoptosis, and it is known that prolonged stress to the ER also triggers a p53-dependent apoptotic program via upregulation of the proapoptotic genes noxa and puma (Li et al., 2006). p53/47 has previously been shown to induce apoptosis (Ohki et al., 2007; Yin et al., 2002), and we were interested to know if activation of p53/47 following ER stress is sufficient to also support ER stress-induced apoptosis or, alternatively, if this requires p53FL. Expression of p53wt, p53FL, or p53/47 mRNAs in H1299 cells all resulted in an increase in apoptosis with approximately 30%–60%. Treatment with thapsigargin for 16 hr further increased apoptosis in cells expressing the p53wt or p53/47 mRNAs with an average 2- to 3-fold, while it had no significant additional effect on the amount of cells undergoing apoptosis expressing p53FL only (Figure 7A). Interestingly, transfection of cells with 14-3-3s siRNA, which prevents p53/ 47-induced G2 arrest, resulted in a 6-fold increase in apoptosis after thapsigargin treatment in cells expressing p53/47 (Figure 7B). Analysis of Noxa and PUMA mRNA levels using qRTPCR revealed an increase in expression of both gene products using either p53 construct. However, the relative induction of Noxa and PUMA in response to each p53 isoform varied after thapsigargin treatment (Figure 7C, left and right panels). This is in line with a previous report showing that p53FL, p53/47, or the combination of both isoforms differently activates down-

stream target genes in a stress-responsive fashion (Powell et al., 2008). Neither Noxa nor PUMA levels were further increased in the presence of p53FL following thapsigargin treatment. Thus, the expression of p53/47 alone is sufficient to trigger a p53-dependent apoptosis in response to prolonged ER stress. DISCUSSION p53-Mediated G2 Arrest Following ER Stress Many different types of cellular stresses and damages lead to p53 activation, and one of the more puzzling questions is how the cells can differentiate p53 activation in order to respond with a specific and suitable cell-biological outcome such as G1 or G2 cell-cycle arrest, senescence, repair, or apoptosis. Here we report that in addition to the previously reported induction of apoptosis (Li et al., 2006), ER stress also leads to a p53dependent G2 arrest. ER stress triggered by changes in the calcium homeostasis or glycosylation, or via the oxygen stress pathway result in an induction of p53/47 expression. This is caused by PERK-mediated stimulation of p53/47 mRNA translation via a sequence located within the first 120 nt of the p53-encoded mRNA (Candeias et al., 2006; Ray et al., 2006). Blocking PERK activity by expressing a dominant-negative PERK or by using PERK siRNA, as well as by removing the p53/47 initiation codon from the p53 mRNA, all suppress ER stress-induced G2 arrest. p53/47-dependent G2 arrest is mediated by p53/47 binding to the 14-3-3s promoter and the induction of 14-3-3s expression. Downregulation of 14-3-3s expression reduces ER stress-induced G2 arrest. Once p53/47 is expressed, PERK no longer has any effect on 14-3-3s expression or on G2 progression. p53FL binds to the 14-3-3s promoter, but without any effect on its expression, in line with the idea that p53 products can bind promoters in active or inactive forms depending on cellular conditions (Espinosa et al., 2003; Llanos et al., 2009; Schumm et al., 2006). At the same time, p53/47 suppresses p53-induced expression of p21 and G1 arrest. These results suggest a signaling pathway in which ER stress leads to the activation of PERK and p53/47 mRNA translation, which leads to the induction of 14-3-3s and the consequent G2 arrest (Figure 7D). The fact that p53/47 efficiently induces 14-3-3s and G2 arrest in the absence of ER stress shows that alternative ER-dependent response pathways are not required. Diversifying the p53 Pathway by Selective Activation of p53/47 It has been known for some time that human p53 isoforms can be expressed either through alternative translation, promoter usage, or splicing, but the physiological role of these isoforms has been obscure (Bourdon et al., 2005; Yin et al., 2002). These results show that p53/47 exhibits a unique ability to induce G2 arrest that cannot be mimicked by the p53FL and, vice versa, that p53FL can induce a G1 arrest that is not mimicked by p53/47. One of the puzzling questions, however, was how relatively small changes in the p53FL:p53/47 ratio following ER stress can give rise to a p53/47-dependent G2 arrest when it might have been expected that the activity of the p53FL would be dominant, considering it is expressed at higher levels. This can be explained by the observation that ER stress not only



Figure 7. p53/47 Induces Following ER Stress

Apoptosis

(A) The relative p53-dependent increase in H1299 cells undergoing apoptosis following thapsigargin treatment for 16 hr was estimated by measuring the sub-G1 population using FACS analysis. The value for EV-transfected and DMSO-treated cells was set to zero. (B) p53/47-induced apoptosis was estimated in H1299 cells expressing indicated siRNA. (C) The fold induction of the proapoptotic genes Noxa and PUMA was estimated using qRT-PCR (left and right panels). The results show the mean value of three independent experiments + SD. *p < 0.05. (D) ER stress activates p53/47 via PERK-mediated p53/47 mRNA translation and by stimulating p53/47 homo-oligomerization. ER stress- and PERK-regulated G2 arrest depend on an intact p53 mRNA . p53/47 has no effect on the G1 while promoting 14-3-3s expression and G2 arrest. p53/47 upregulates expression of Noxa and PUMA and induces apoptosis in response to ER stress. Global cap-dependent translation is suppressed during the G2/M transition and, thus, the p53/47-induced G2 arrest will act together with PERK to repress protein synthesis during the UPR and thereby facilitate ER repair. Hence, p53/47 provides a link between ER stress, G2 arrest, and ER damage repair.

tion is regulated. The use of the BRET approach described here should open up for further studies aimed at understanding the regulation of p53 complex formation.

increases p53/47 expression levels but also promotes the formation of active p53/47 homo-oligomers. This selective formation of p53/47 complexes is reflected in an increase in p53/47 binding to the 14-3-3s promoter, in the induction of 14-3-3s expression, in the increase in G2 arrest, and in p53/47-induced apoptosis. By using antibodies specific for the full-length p53 protein, or that recognize both forms, we can conclude that binding of p53/47 to the 14-3-3s promoter following ER stress is selective also in cells expressing endogenous p53wt mRNA. p53/47 is more easily incorporated in oligomer complexes, and it has previously been shown that the N terminus of p53 is important for p53 folding (Hansen et al., 1998; Powell et al., 2008). But the molecular mechanism underlying the increase in the affinity of p53/47 oligomers following ER stress is not known. It was more recently reported that binding of 14-3-3 proteins to the C terminus of p53 promotes its oligomerization, indicating that trans-acting factors might play a role (Rajagopalan et al., 2008). p53 is active as a tetramer, but relatively little is yet known how the oligomeriza-


PERK-Dependent Control of p53/47 Translation The key aspect of the selective p53mediated G2 arrest following ER stress is the PERK-dependent induction of p53/47 mRNA translation. We and others have previously shown that the +1 to +120 sequence of the p53 mRNA includes an IRES that can promote cap-independent translation initiation of p53/47 (Candeias et al., 2006; Ray et al., 2006). These results show that this sequence is required for ER stress-induced G2 arrest. As ER stress-induced expression of p53/47 from a bicistronic mRNA is sufficient to induce G2 arrest (data not shown), it is possible that cap-independent translation of the p53 mRNA can promote G2 cell-cycle arrest. Like many eukaryotic IRESs, the p53/47 IRES is stress responsive, but it is atypical in some other aspects. It constitutes the encoding sequence for the N terminus of the full-length p53 and can initiate the synthesis of an alternative translation product and not, like most IRESs, ensure the synthesis of the same protein that is produced by cap-dependent translation. This is unusual, but the cell-cycleregulated IRES that mediates p58PITSLRE expression is also located within the coding sequence of the p110PITSLRE mRNA (Cornelis et al., 2000).


A previous study has shown that competition between capdependent and cap-independent translation of the p53 mRNA is an important factor in controlling p53/47 translation (Candeias et al., 2006), and thus it is possible that phosphorylation of eIF2a forms part of PERK’s capacity to stimulate p53/47 synthesis. This shares similarities with ER stress-mediated induction of ATF4 in which PERK-dependent phosphorylation of eIF2a endorses initiation of the main ORF by preventing initiation of short upstream ORFs (Vattem and Wek, 2004). However, suppressing initiation of p53FL is unlikely to be the single mechanism by which PERK promotes p53/47 translation and expression of p53FL remains relatively unchanged during UPR. Furthermore, initiation of p53/47 is PERK dependent even in the absence of p53FL initiation. P53/47 Activity in Cell-Cycle Checkpoints and Apoptosis One of the more intriguing conclusions of this study is that cells appear to favor a G2 arrest in response to ER stress. The reason for this might be explained by the observation that cap-dependent translation initiation is actively suppressed during the G2/M transition (Sivan and Elroy-Stein, 2008). Thus, it is conceivable that p53/47 serves to position the cell in G2 and facilitates the ER stress damage repair by acting in conjunction with PERK to repress protein synthesis while restoring the protein maturation capacity (Schroder and Kaufman, 2005). This can be compared with the p53-dependent G1/S arrest following DNA damage, which allows the cells to repair the DNA before entering replication (Kuerbitz et al., 1992). As with DNA damage, failure to repair ER damage and to restore protein folding can trigger a p53-dependent apoptosis (Li et al., 2006). In this context it is interesting that suppression of 14-3-3s expression leads to an increase in p53/47-dependent apoptosis in response to ER stress, indicating that a proapoptotic response is more likely if the cell fails to arrest in G2. The fact that p53/47 upregulates expression of proapoptotic genes such as PUMA and Noxa and that it supports apoptosis indicates that p53/47 activity in response to ER stress is sufficient to switch the cell from a G2 arrest to a proapoptotic state without the need of p53FL. Taken together, these results illustrate how cells can differentiate p53 activity in response to a defined cellular stress pathway in order to trigger a specific and suitable cell-biological outcome.

were resuspended with a lysis buffer in the presence of Complete Protease inhibitor cocktail (Roche). After sonication, lysates were separated on a 4%–12% Bis-Tris gel (Invitrogen). BRET Assay H1299 cells were seeded on 12-well plates and transfected with 50 ng per well of the DNA construct coding for BRET donor and increasing (0–1.45 mg per well) amounts of the BRET acceptor plasmid (or control YFP). Twenty-four hours after transfection, cells were treated for 16 hr with thapsigargin, and the luciferase substrate, coelenterazine H (Molecular Probes, Leiden), was added at a final concentration of 5 mM to 1 3 105 cells. Luminescence and fluorescence were measured simultaneously by using the Mithras fluorescence-luminescence detector (Berthold). Cells expressing BRET donors alone were used to determine background. Filter sets were 485 ± 10 nm for luciferase emission and 530 ± 12.5 nm for YFP emission. BRET ratios were calculated as described previously (Storez et al., 2005). RNA Preparation and Quantitative RT-PCR RNA extraction techniques and primer sequences used for specific amplification are described in the Supplemental Experimental Procedures. Chromatin Immunoprecipitation H1299 cells, transfected with different p53 constructs, and MLS1765 cells, transfected with PERKDC, were treated with thapsigargin. ChIP was performed as described in the Supplemental Experimental Procedures. Small Interfering RNA Transfection Cells were transfected with p53 small interfering RNA (siRNA) (forward, 50 -GCAUGAACCGGAGGCCCAU-30 ; and reverse, 50 -AUGGGCCUCCGGUU CAUGC- 30 ) at a final concentration of 1 mM using the HiPerFect Transfection Reagent (QIAGEN) following the manufacturer’s instructions. Cells were transfected with 14-3-3s and PERK-specific and control siRNAs (FlexiTube siRNA, QIAGEN) at a final concentration of 10 nM. Cell-Cycle Analysis Sixteen hours after treatment with thapsigargin, cells were harvested and fixed with ice-cold 70% ethanol. After 30 min incubation with RNase at 37 C, cells were stained with propidium iodide (50 mg/ml, Sigma-Aldrich) and analyzed with a LSR flow cytometer (Becton Dickinson). The values obtained from empty vector-transfected and DMSO-treated cells are subtracted from p53transfected and thapsigargin-treated cells to calculate relative changes (%). SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures and Supplemental Experimental Procedures and can be found with this article at doi:10.1016/j. molcel.2010.01.041. ACKNOWLEDGMENTS

EXPERIMENTAL PROCEDURES Cells, Constructs, and Drug Treatment Human cells HCT116p53/ (colon cancer cells), H1299 (non-small cells lung cancer), and Saos-2 (osteosarcoma cells) are p53-negative cells. Human cells HCT116 p53+/+, MLS1765 (fibrosarcoma cells), MCF-7 (breast carcinoma cells), and A549 (lung carcinoma cells) express endogenous wild-type p53. Cells were cultured, transfected, and treated as described in the Supplemental Experimental Procedures. Immunoblotting Antibodies and immunoblotting techniques are described in the Supplemental Experimental Procedures. Crosslinking Cells were crosslinked with formaldehyde (1%) for 10 min at 37 C, and the reaction was quenched which glycine (1 M). After washing with PBS, pellets

This work was supported by La Ligue Nationale Contre le Cancer and the INSERM. Anti-p53 antibodies and the PERKDC cDNA construct were kind gifts from Dr. Borek Vojtesek and Dr. David Ron, respectively. Flow cytometry experiments were performed at the Imagery Department at the Institut Universitaire d’He´matologie-IFR105. Received: May 20, 2009 Revised: September 30, 2009 Accepted: January 29, 2010 Published: April 8, 2010 REFERENCES Bacart, J., Corbel, C., Jockers, R., Bach, S., and Couturier, C. (2008). The BRET technology and its application to screening assays. Biotechnol. J. 3, 311–324.



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