Endoplasmic Reticulum Stress Accelerates p53 Degradation by the Cooperative Actions of Hdm2 and Glycogen Synthase Kinase 3{beta}

Inactivation of the tumor suppressor p53 by degradation is amechanism utilized by cells to adapt to endoplasmic reticulum(ER) stress. However, the mechanisms of p53 destabilizationby ER stress are not known. We demonstrate here that the E3ubiquitin-ligase Hdm2 is essential for the nucleocytoplasmictransport and proteasome-dependent degradation of p53 in ER-stressedcells. We also demonstrate that p53 phosphorylation at S315and S376 is required for its nuclear export and degradationby Hdm2 without interfering with the ubiquitylation process.Furthermore, we show that p53 destabilization in unstressedcells utilizes the cooperative action of Hdm2 and glycogen synthasekinase 3ß, a process that is enhanced in cells exposedto ER stress. In contrast to other stress pathways that stabilizep53, our findings further substantiate a negative role of ERstress in p53 activation with important implications for thefunction of the tumor suppressor in cells with a dysfunctionalER.

INTRODUCTION

Top

Introduction

The p53 gene encodes for a protein whose loss of function isassociated with the majority of human cancers (17). The p53protein primarily functions as a transcription factor and mediatesseveral biological effects including growth arrest, senescence,and apoptosis in response to diverse forms of stress (50). Inthe absence of stress, p53 is a short-lived protein whose activityis maintained at low levels. Upon exposure to a variety of stresses,p53 becomes stabilized and accumulates in the nucleus, whereit resumes its transcriptional function. The levels and localizationof p53 are tightly controlled through several posttranslationalmechanisms, including protein stability, phosphorylation, andsubcellular localization (3, 42, 46). Although several factorsinfluence p53 function, the Hdm2 (human Mdm2) protein playsan essential role in regulating p53 protein levels in unstressedcells. Hdm2 is a nucleoplasmic and nucleolar RING-finger proteinthat promotes p53 nuclear export and degradation through itsspecific E3 ubiquitin ligase activity (2, 40). Other studieshave revealed various Hdm2-independent pathways that impingeon p53 turnover, including c-Jun N-terminal kinase (10), COP1(9), and Pirh2 (32). However, little is known about how andin what cellular context these pathways act.

The current model places both p53 and Hdm2 in an autoregulatoryfeedback loop where p53 induces the transcription of Hdm2 gene.The Hdm2 protein then binds to and ubiquitylates p53 in thenucleus, a process that allows the nuclear export and cytoplasmicproteasome-dependent degradation of the tumor suppressor (40,56). The importance of this autoregulatory loop was demonstratedwhen the lethality of mdm2-null mice was rescued by the deletionof the p53 gene (22, 41). In this model, Hdm2 does not physicallyshuttle p53 out of the nucleus since the nuclear export sequence(NES) of Hdm2 is not necessary for p53 degradation, as opposedto its RING domain, which is important for p53 ubiquitylationand degradation (5, 11, 43). It has also been proposed thatlow Hdm2 levels in unstressed cells promote p53 mono-ubiquitylationin the nucleus, which then facilitates p53 nuclear export (30,35, 65). However, other factors such as p300-CBP can also participatein the polyubiquitylation and degradation of p53 (16). Furthermore,other reports suggested that p53 can also be degraded in thenucleus (54, 64) when Hdm2 levels are elevated (35). The physiologicalsignificance of the nuclear degradation is not yet known, butit could represent an important mechanism for the poststressrecovery of cells from a p53 response. Several other mechanismshave been described to modulate Hdm2 activity and p53 proteinlevels, including phosphorylation of Hdm2 and binding to otherfactors. For example, the tumor suppressor p14^ARF and the humanribosomal protein L11 interact with Hdm2 by relocating Hdm2to the nucleolus, thus reducing p53 ubiquitylation and degradation(38, 47, 67, 68). Although the Hdm2-dependent p53 nuclear exportand degradation are critical for p53 function, the molecularmechanism(s) mediating this process is not fully understoodand still remains a matter of debate.

Stress of the endoplasmic reticulum (ER) induced by physiologicalconditions (such as glucose starvation and hypoxia) or by pharmacologicalagents such as tunicamycin (inhibitor of glycosylation) or thapsigargin(inhibitor of the Ca²⁺-ATPase in the ER) can lead to accumulationof unfolded proteins and protein aggregates that are detrimentalfor cell survival (26). Under ER stress, cells initiate adaptiveresponses by activating specific signaling pathways to limitthe accumulation of unfolded proteins. One of them is calledthe unfolded protein response, in which the transcription ofgenes encoding ER chaperones and folding catalysts is upregulatedto increase protein folding activity (66). Other adaptive responsesinclude the inhibition of protein synthesis as a means to decreasethe protein overload in the ER (49) or the elimination of unfoldedor misfolded proteins by proteasome-dependent proteolysis (28).If adaptation is not possible, then the stressed cell is eliminatedby apoptosis through the activation of the JNK pathway and caspases7, 12, or 3 (27).

We recently demonstrated a novel mechanism of adaptation ofcells to ER stress involving the inactivation of p53 (48). Specifically,we showed that pharmacological or physiological inducers ofER stress prevent the proapoptotic function of p53 by enhancingits nucleocytoplasmic export and degradation (48). This regulationof the tumor suppressor protein requires its phosphorylationwithin the nuclear localization signal (NLS), which is mediatedby the activation of the glycogen kinase 3ß (GSK-3ß),a protein kinase with pleiotropic effects on tumorigenesis,cell differentiation, and apoptosis (8, 14). However, the molecularmechanisms that modulate the cytoplasmic translocation and destabilizationof p53 in ER stressed are poorly understood. We demonstratehere that induction of the cytoplasmic translocation and degradationof p53 by ER stress is mediated by Hdm2 and requires the phosphorylationof the tumor suppressor protein at serine S315 and S376 by GSK-3ß.Significantly, the cooperative action of GSK-3ß andHdm2 also occurs in unstressed cells, but it is enhanced incells subjected to ER stress. Our data reveal a new role forGSK-3ß in the regulation of p53 nucleocytoplasmicshuttling and degradation by Hdm2, with possible important implicationsfor therapies aiming at p53 stabilization through the inhibitionof the p53-Hdm2-GSK-3ß pathway.

MATERIALS AND METHODS

Top

Materials and Methods

Cell culture and treatments. Mouse embryonic fibroblasts (MEFs), human embryonic kidney HEK293Tcells, and 3T3 cells were maintained in Dulbecco modified Eaglemedium plus 10% calf serum (Invitrogen) and antibiotics. A549,WI38, and LNCaP cells were maintained in F12K medium (Cellgro),minimal essential medium, and RPMI 1640 medium, respectively,supplemented with 10% fetal bovine serum (Invitrogen) and antibiotics.Mouse 70Z/3 cells were maintained in RPMI medium supplementedwith 15% fetal bovine serum, 50 µM ß-mercaptoethanol,and antibiotics. The ER stress inducers tunicamycin (TM; Sigma)and thapsigargin (TG; Sigma), the nucleocytoplasmic transportinhibitor leptomycin B (LMB; Sigma), and the proteasome inhibitorMG132 (Biomol) were dissolved in dimethyl sulfoxide at stockconcentrations of 10 mg/ml, 1 mM, 4 µM, and 10 mM, respectively.Cycloheximide and adriamycin (ADR) were dissolved in ethanolat stock concentrations of 100 mg/ml and 1 mM, respectively.All stock solutions were kept in –20°C. The finalconcentrations of the drugs and the duration of treatments areindicated in the figure legends.

DNA constructs and transfection. The wild-type (WT) Hdm2 cDNA, the NES Hdmd2 mutant cDNA andthe C464A Hdm2 mutant cDNA in pcDNA/3.1 (Neo) vector were previouslydescribed (5). The WT and the NES mutant (L348A/L350A) of p53in the pEGFP/N1 vector were also described previously (5). Thegreen fluorescent protein (GFP)-p53 cDNA bearing the S315A orS376A mutation was generated as described previously (48). WTHA-tagged GSK-3ß or the kinase-dead (KD) GSK-3ßcDNA in pcDNA/3.1 vector was described previously (55). Fortransient transfections, the Lipofectamine Plus reagent (Invitrogen)or FuGENE 6 (Roche) were used as recommended by the manufacturer.

Immunoprecipitation and Western blotting. Whole-cell extracts (WCE) were extracted in ice-cold lysis buffercontaining 20 mM HEPES (pH 7.5), 500 mM NaCl, 0.1% NP-40, 20%glycerol, 0.2 mM EDTA, 1.5 mM MgCl₂, 1 mM dithiothreitol, 100mM NaF, 20 mM ß-glycerophosphate, 50 mM Na₃VO₄, 1mM phenylmethylsulfonyl fluoride, 4 µg of aprotinin/ml,4 µg of pepstatin A/ml, and 4 µg of leupeptin/ml.After incubation on ice for 15 min, the lysates were clearedby centrifugation at 18,000 x g for 15 min. For p53/Hdm2 coimmunoprecipitation,cell extracts ( $~$ 3 mg of protein) were immunoprecipitated withan Hdm2 antibody (Ab-1; Oncogene Science) or p53 Ab (DO-1) conjugatedto protein G-Sepharose beads (Amersham) as described previously(51). For the detection of p53 ubiquitylation, cells were treatedwith 10 µM MG132 for 3 h prior to ER stress treatments,and the immunoprecipitation was performed with a polyclonalp53 Ab (FL393) with 200 µg of protein extract. For immunoblotting,WCE containing 50 µg of protein or immunoprecipitateswere resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand incubated with the indicated Ab. Abs to p53 (FL-393) andCM-5 were purchased from Santa Cruz, Inc., and Novocastra, respectively;DO-1 (Ab-6) and PAb421 (Ab-1) were from Oncogene Science. Themouse monoclonal Ab to actin was from ICN. The anti-Hdm2 (Ab-1)mouse monoclonal Ab was from Oncogene Science. The monoclonalAb to androgen receptor (AnR) and polyclonal Ab to p21 werefrom Santa Cruz, Inc., whereas the anti-GFP rabbit polyclonalAb was from Promega. All Abs were used at dilutions recommendedby the manufacturer. Proteins were visualized by the enhancedchemiluminescence detection method (ECL; Amersham) accordingto the manufacturer's specification.

Immunofluorescence studies. The detection of GFP-p53, Hdm2, or p53 by immunofluorescencewas performed as previously described (48). GFP-positive andlive cells were scored and classified into two groups; the firstgroup with fluorescence predominantly in the nucleus and thesecond with fluorescence in both the nucleus and cytoplasm.Total green fluorescence in the nucleus and whole cells wasquantified by using NIH Image from 200 randomly selected GFP-positiveand live cells. For the detection of Mdm2, cells were stainedwith a 1:100 diluted monoclonal Ab (Ab-4; Oncogene Science),whereas for p53 the cells were stained with a 1:200-dilutedanti-p53 rabbit polyclonal Ab (FL393; Santa Cruz). Cells wereincubated with primary antibodies for 16 h at 4°C, washedwith phosphate-buffered saline, and incubated for 1 h with AlexaFluor 488-conjugated secondary Ab or Alexa Fluor 546-conjugatedsecondary Ab (both from Molecular Probes). The nucleus was visualizedafter staining with 1 µg of DAPI (4',6'-diamidino-2-phenylindole;Sigma)/ml.

Lentivirus infection and Hdm2 knockdown by shRNA. For the Hdm2 knock-down experiments, the lentivirus vectorsexpressing Hdm2 short-hairpin RNA (shRNA) were previously described(24). Lentivirus-containing supernatant was collected 36 h aftertransfection in HEK293T cells, 0.2 µm-filtered, and snap-frozenat –70°C. A549 cells were infected by retrovirus atlow multiplicity (ca. 10% transduction efficiency) by adding4 mg of Polybrene (Sigma)/ml for 12 h prior to incubation withfresh medium. Cells were selected in puromycin, and polyclonalpopulations were expanded and analyzed.

RESULTS

Top

Results

Nuclear export is required for the Hdm2-mediated proteasome-dependent degradation of p53 in ER-stressed cells. We previously showed that ER stress decreases the half-lifeof p53 without decreasing its mRNA levels, suggesting an effectof ER stress on p53 degradation (48). We further investigatedthis phenomenon by verifying the p53 levels in ER-stressed cellsin the presence of proteasome inhibitors. To this end, we usedA549 cells, which carry a WT p53 allele (51) and respond toER stress induced by TM or TG treatment (29). We noticed thattreatment of A549 cells with the inducers of ER stress led tothe downregulation of p53 protein levels, which was blockedwhen cells were subjected to ER stress in the presence of theproteasome inhibitor MG132 (Fig. 1A). These data suggested thatthe proteasome degradation machinery is responsible for thedecrease of p53 protein levels in ER-stressed cells. Becausethe proteasome-dependent degradation of p53 has been suggestedto be mainly a cytoplasmic process, we next determined whethernuclear export of p53 is a prerequisite for its degradationin response to ER stress. When A549 cells were subjected toER stress in the presence of LMB, an inhibitor of p53 nuclearexport (53, 56), we observed a rescue of p53 protein levelsin ER stressed and LMB-treated cells (Fig. 1B). This findingindicated that degradation of p53 in ER-stressed cells requiresthe nucleocytoplasmic shuttling of the tumor suppressor protein.

View larger version (40K):
[in this window]
[in a new window]
FIG. 1. Hdm2-mediated nucleocytoplasmic shuttling and degradation of p53 in ER-stressed cells. (A and B) A549 cells were pretreated with either 10 µM MG132 (A, lanes 4 to 6) or 4 nM LMB (B, lanes 4 to 6) for 4 h prior to treatment with 1 µM TG (lanes 2 and 5) or 10 µg of TM/ml (lanes 3 and 6) for 2 h. Protein extracts were subjected to Western blotting with anti-p53 (DO-1) Ab (top panel) and anti-actin Ab (bottom panel). The p53 protein levels were normalized to actin levels (% p53) by using Scion Image 2.0 software. (C) Subcellular localization of GFP-p53. GFP-p53 WT cDNA (0.25 µg) was transiently transfected in the absence or presence of an equal amount of Hdm2 WT, Hdm2 NES, or Hdm2 C464A cDNA into 2KO cells. After 24 h, the cells were left untreated or treated with either 10 µg of TM/ml or 1 µM TG for 4 h, and GFP-p53 localization was examined by fluorescence microscopy. The nuclei were visualized by DAPI staining. White arrows indicate nuclear localization of GFP-p53 only, whereas orange arrows indicate both cytoplasmic and nuclear localization. The quantification of GFP-p53 localization is described in Materials and Methods. Cells with predominantly nuclear p53 are represented by the black bars, whereas cells with p53 both in the nucleus and in the cytoplasm are represented by the white bars. Values are means ± the standard deviation (SD) from three experiments. (D) Inhibition of Hdm2 expression by shRNA. A549 cells subjected to Hdm2 silencing by shRNA (see Materials and Methods) were treated with either 10 µg of TM/ml or 1 µM TG for 2 h. Protein extracts (50 µg) were immunoblotted with anti-p53 (DO-1) Ab (top panel), anti-Hdm2 Ab (middle panel), or antiactin Ab (bottom panel).

Given that Hdm2 is a major regulator of p53 protein stability(40), we examined its role in nucleocytoplasmic shuttling anddegradation of p53 in response to ER stress. To do so, we utilizedp53^–/– Mdm2^–/– MEFs (23), also knownas double-knockout (2KO) cells, in which the subcellular localizationof GFP-p53 was determined in coexpression assays with Hdm2 (Fig.1C). Because the subcellular localization and degradation ofp53 are controlled by the intracellular concentration of Hdm2,2KO cells were transfected with an amount of Hdm2 sufficientto induce the cytoplasmic translocation and degradation of thetumor suppressor protein as recently described (35) (Fig. 1C).In the absence of Hdm2, we noticed that ER stress failed topromote the cytoplasmic translocation of p53. In the presenceof Hdm2, we observed that a fraction of GFP-p53 became cytoplasmic,a process that was further enhanced in response to ER stress(Fig. 1C). The ability of Hdm2 to promote the cytoplasmic relocationof GFP-p53 was dependent on its ubiquitin ligase activity sinceHdm2 bearing the C464A mutation in the RING finger domain failedto relocate GFP-p53 into the cytoplasm, either in the absenceor presence of ER stress (Fig. 1C). In contrast to this, thecytoplasmic relocation of GFP-p53 was induced in the presenceof an NES mutant of Hdm2, which retains its ubiquitin ligaseactivity (5, 11), indicating that nuclear export of p53 proceedsindependently of the nuclear export of Hdm2 in unstressed, aswell as in ER-stressed cells. Taken together, these findingsindicated that p53 ubiquitylation by Hdm2 is a signal for thenuclear export of the tumor suppressor protein. This process,which does not require the nucleocytoplasmic transport of Hdm2,is enhanced in cells subjected to ER stress. To confirm therequirement of Hdm2 for p53 degradation in response to ER stress,we used a viral vector that express double-stranded shRNA toknockdown endogenous Hdm2 (24). We reasoned that if Hdm2 wasinvolved in p53 degradation in ER stressed cells, then its eliminationby shRNA should rescue p53 from degradation in response to ERstress. Expression of the shRNA in A549 cells led to >80%decrease of the endogenous Hdm2 protein (Fig. 1D, lanes 4 to6). When cells were subjected to ER stress (Fig. 1D, lanes 2,3, 5, and 6), we noticed that downregulation of p53 was notpossible in cells with targeted Hdm2 (lanes 5 and 6) as opposedto control cells (i.e., cells infected with control lentiviruses[24]) in which p53 levels were decreased (Fig. 1D, top panel).In these experiments, we noticed that retrovirus infection ofA549 cells made them less sensitive to ER stress based on thedegree of p53 downregulation (Fig. 1D, lanes 2 and 3) comparedto ER-stressed A549 cells without infection (Fig. 1A and B,lanes 2 and 3). These data strongly supported a major role forHdm2 in p53 degradation in response to ER stress.

Decrease of functional Hdm2 is ER-stressed cells. The data presented above also showed that ER stress leads toHdm2 downregulation (Fig. 1D, middle panel, lanes 2 and 3).To better understand this phenomenon, we first assessed theendogenous Hdm2 protein levels in A549 cells at different timepoints after ER stress. We observed that Hdm2 protein levelswere gradually reduced with the time of the ER stress treatments(Fig. 2A, top panel) concomitant with the downregulation ofp53 protein levels (middle panel). In fact, Hdm2 downregulationwas proportional to the decrease of p53 protein levels as demonstratedby the constant ratio of Hdm2/p53 before and after the ER stresstreatments (Fig. 2A). Furthermore, the half-life of the endogenousHdm2 in A549 cells was not affected by ER stress (Fig. 2B),indicating the lack of regulation of Hdm2 expression at theposttranslational level. Therefore, inhibition of Hdm2 expressionby ER stress could be exerted either at the transcriptional,posttranscriptional or translational level through p53-dependentand/or independent mechanisms. However, ER stress affects theinduction of p53-dependent genes as we have previously reported(48). For example, induction of the cdk inhibitor p21 in humandiploid WI38 cells upon DNA damage by adriamycin (ADR) was inhibitedwhen cells were exposed to ER stress (Fig. 2C). Furthermore,p21 induction in mouse pre-B 70Z/3 cells upon gamma irradiationwas impaired after treatment of cells with TG (data availableupon request). Moreover, upregulation of p21 in NIH 3T3 cellsexposed to ADR was significantly reduced after treatment withinducers of ER stress for long periods of time (data availableupon request). In all of these experiments, p21 expression levelscorrelated with p53 levels, indicating that regulation of p21by ER stress was dependent on p53 response. Taken together,these data provide some evidence for a defective transcriptionalresponse of p53 in ER-stressed cells that could account, atleast in part, for the reduced Hdm2 levels in cells subjectedto ER stress.

View larger version (27K):
[in this window]
[in a new window]
FIG. 2. Inhibition of Hdm2 expression in ER-stressed cells. (A) Downregulation of p53 and Hdm2 proteins by ER stress. A549 cells were left untreated or treated with either 10 µg of TM/ml (lanes 2 and 3) or 1 µM TG (lanes 4 and 5) for the indicated times. Cells were also treated for 4 h with 10 µM MG132 (lane 6) or 1 µM ADR (lane 7) to stabilize p53 (controls). Protein extracts (50 µg) were subjected to immunoblotting with anti-p53 (DO-1) Ab (top panel), anti-Hdm2 Ab (middle panel), or antiactin Ab (bottom panel). Hdm2 and p53 protein levels were normalized to actin by using Scion Image 2.0 software. (B) Hdm2 stability in ER-stressed cells. A549 cells were untreated or treated with 1 µM TG for 4 h, followed by a 40-µg/ml cycloheximide chase for the indicated time. Protein extracts (50 µg) were subjected to Western blotting with anti-Hdm2 Ab (top panel) and antiactin Ab (bottom panel). The exposures shown are different so that the p53 levels in control (CON) and TG-treated cells for time zero (lanes 1 and 5) are equal. Hdm2 levels were normalized to actin levels by using Scion Image 2.0 software, and the ratio in unstressed (control) and ER-stressed cells (TG) plotted against the time is shown. (C) WI-38 cells were treated with 1 µM (adriamycin) ADR alone or with either 10 µg of TM/ml or 1 µM TG for the indicated times. Protein extracts (50 µg) were used for immunoblotting with an anti-p53 rabbit polyclonal Ab (top panel) or with an anti-p21 polyclonal Ab (middle panel). A nonspecific (NS) band was used as a loading control (bottom panel).

Control of p53/Hdm2 interaction and p53 ubiquitylation in ER-stressed cells. We further investigated the mechanisms of p53 degradation byHdm2 in ER-stressed cells. First, we examined the interactionbetween the two proteins in A549 cells subjected to ER stress(Fig. 3A). The formation of Hdm2/p53 complex was detected byimmunoprecipitation with either anti-Hdm2 Ab (lanes 1 to 7)or anti-p53 Ab (lanes 8 to 10), followed by immunoblotting witheither anti-p53 Ab or anti-Hdm2 Ab, respectively. Using bothapproaches we measured a partial ( $~$ 50%) inhibition of Hdm2/p53complex formation in cells subjected to ER stress compared tocontrol untreated cells (top panel, compare lanes 2 and 3 withlane 1 and lanes 9 and 10 with lane 8). However, the total proteinlevels of both Hdm2 and p53 were diminished in cells subjectedto ER stress (Fig. 3A, WCE), and as such, this partial inhibitionof Hdm2/p53 complex might have been resulted from the differentamounts of the proteins subjected to coimmunoprecipitation.To bypass this limitation, the A549 cells were treated withthe proteasome inhibitor MG132 to stabilize p53 and preventthe downregulation of Hdm2 prior to exposure to ER stress (lanes4 to 6). Upon these conditions, coimmunoprecipitation of Hdm2/p53with anti-Hdm2 Ab showed no differences in the amount of complexformation before and after ER stress (top panel, lanes 4 to6), indicating that ER stress does not interfere with the intermolecularinteraction of the two proteins. On the other hand, DNA damageof A549 cells with ADR led to the dissociation of the Hdm2.p53complex (lane 7), indicating that these cells are not refractoryto signals that modulate the interaction of the two proteins.

View larger version (44K):
[in this window]
[in a new window]
FIG. 3. Physical and functional interactions between p53 and Hdm2 in ER-stressed cells. (A) Physical interaction between Hdm2 and p53 in ER-stressed cells. A549 cells were left untreated (lanes 1, 2, 3, 8, 9, and 10) or pretreated with 10 µM MG132 for 2 h (lanes 4, 5, and 6), followed by treatment with 10 µg of TM/ml (lanes 2, 5, and 9) or 1 µM TG for 4 h (lanes 3, 6, and 10). Cells were also exposed to DNA damage with 1 µM ADR (lane 7), which was used as a control. WCE (50 µg of protein) were used for immunoblotting with anti-p53 (DO-1) Ab (second from the top panel), anti-Hdm2 Ab (third from the top panel), or antiactin Ab (bottom panel). The same protein extracts (3 mg) were subjected to immunoprecipitation with either anti-Hdm2 Ab (lanes 1 to 7) or anti-p53 (DO-1) Ab (lanes 8 to 10) and subsequently to immunoblotting with anti-p53 (DO-1) Ab or anti-Hdm2 Ab, respectively. (B) ER stress enhances the ubiquitylation of p53 by Hdm2. GFP-p53 WT cDNA (0.5 µg) was transiently transfected in the absence or presence of Hdm2 WT cDNA (0.5 µg) and/or HA-Ub cDNA (0.5 µg) into 2KO cells. Cells were treated with 10 µM MG132 for 3 h, prior to ER stress treatment with 10 µg of TM/ml or 1 µM TG for 3 h. Protein extracts (200 µg) were subjected to immunoprecipitation with anti-53 rabbit polyclonal Ab, followed by immunoblotting with anti-p53 (DO-1) Ab. WCE (50 µg of protein) were used for immunoblotting to detect the transfected Hdm2. (C) Specificity of p53 degradation ER stress. LNCaP cells were exposed to ER stress with 10 µg of TM/ml (lane 2) or 1 µM TG (lane 3) or subjected to DNA damage with 1 µM ADR (lane 4) for 2 h. Protein extracts (50 µg) were used for immunoblotting with anti-AnR Ab (top panel), anti-Hdm2 Ab (second from the top), anti-Hdm2 Ab (third from the top), or antiactin Ab (bottom panel).

Given that ER stress does not significantly affect Hdm2/p53interaction, we next examined whether p53 ubiquitylation byHdm2 is modulated in ER-stressed cells. To this end, 2KO cellswere cotransfected with GFP-p53 and HA-tagged ubiquitin (HA-Ub)in the absence or presence of Hdm2, followed by treatment withinducers of ER stress (Fig. 3B). In the absence of Hdm2, theubiquitylation of GFP-p53 was undetectable, but it was significantlyinduced by the presence of Hdm2 (compare lanes 2 and 5). Incells subjected to ER stress, the ubiquitylation of GFP-p53in the absence of Hdm2 remained undetectable (lanes 3 and 4).However, when Hdm2 was present, GFP-p53 ubiquitylation was enhancedupon treatment with either TM (lane 6) or TG (lane 7) comparedto unstressed cells (lane 5). In similar experiments, we foundthat GFP-p53 ubiquitylation was possible with Hdm2 NES and furtherenhanced in response to ER stress as opposed to Hdm2 C464A,which was unable to act on GFP-p53 (data not shown). We furtherwanted to determine whether the induction of Hdm2 activity inER-stressed cells was specific for p53 or degradation of otherHdm2 substrates was also enhanced in response to ER stress.To this end, we examined the effect of ER stress on AnR proteinlevels, which is a substrate of Hdm2 (37). As shown in Fig.3C, treatment of LNCaP cells with ER stress did not induce thedegradation of AnR as opposed to p53, which was downregulatedby this treatment. These results suggested a specific effectof Hdm2 on p53.

Degradation of p53 by Hdm2 requires phosphorylation of p53 at S315 and S376. We recently demonstrated that phosphorylation of p53 at S315and S376 is required for its cytoplasmic relocation in ER-stressedcells (48). Specifically, we showed that both serine residuesare constitutively phosphorylated within cells and their phosphorylationis enhanced in response to ER stress (48). To determine therole of these serine residues in the regulation of p53 stabilityby Hdm2, we assessed the Hdm2-mediated degradation of GFP-p53bearing either the S315A or S376A mutation. Using transient-expressionassays in 2KO cells, we found that GFP-p53-S315A and GFP-p53-S376Awere more resistant to Hdm2-mediated degradation compared toGFP-p53 WT (Fig. 4A). This indicated that phosphorylation ofS315 and S376 is critical for efficient degradation of p53 byHdm2. To gain a better mechanistic insight into this process,we next determined the ubiquitylation of GFP-p53-S315A and GFP-p53-S376Aby Hdm2 in 2KO cells. We found that ubiquitylation of eitherGFP-p53-S315A or GFP-p53-S376A was undetectable in the absenceof Hdm2 (Fig. 4B, top panel, lanes 2 and 11), but it was inducedwhen Hdm2 was coexpressed (Fig. 4B, top panel, lanes 5 and 14).In the absence of ER stress, ubiquitylation of both GFP-p53mutants was equal to GFP-p53 WT ubiquitylation (compare lanes5 and 14 with lanes 9 and 18, respectively). However, treatmentwith ER stress did not further enhance the ubiquitylation ofthe GFP-p53 mutants by Hdm2 (Fig. 4B, lanes 6, 7, 15, and 16),as opposed to GFP-p53 WT, whose ubiquitylation was increased(Fig. 3B). These data suggested that S315 and S376 phosphorylationdoes not interfere with p53 ubiquitylation in unstressed cellsbut is required for the induction of ubiquitylation of p53 inER-stressed cells. Since phosphorylation at S315 and S376 isrequired for the nucleocytoplasmic shuttling of p53 in bothunstressed and ER-stressed cells (48), we next examined theeffects of Hdm2 on the nuclear export of the GFP-p53 phosphorylationmutants in 2KO cells (Fig. 4C). We found that the distributionof GFP-p53 mutants remained mainly nuclear when expressed aloneor together with Hdm2. Furthermore, the GFP-p53 mutants remainednuclear in cells subjected to ER stress. This was differentfrom the regulation of the nucleocytoplasmic regulation of GFP-p53WT, which was enhanced by the coexpression of Hdm2 and treatmentwith ER stress inducers (Fig. 2A). Collectively, these datashowed that ubiquitylation of p53 is necessary but not sufficientfor the nuclear export of the tumor suppressor in unstressedand ER-stressed cells. Furthermore, nuclear export of p53 byHdm2 requires phosphorylation of p53 at S315 and S376.

View larger version (38K):
[in this window]
[in a new window]
FIG. 4. Role of S315 and S376 phosphorylation in subcellular localization and degradation of p53 in ER-stressed cells. (A) Hdm2-dependent degradation of GFP-p53 and its phosphorylation mutants. GFP-p53 WT cDNA or the indicated GFP-p53 mutant cDNAs (0.5 µg) were transiently transfected in 2KO cells in the presence of increasing amounts of Hdm2 WT cDNA (0.5 to 1.5 µg). The GFP cDNA (0.1 µg) was used as an internal control. After 24 h, protein extracts (50 µg) were used for immunoblotting with anti-p53 (DO-1) Ab (top panel) or anti-GFP Ab (bottom panel). The GFP-p53 proteins were normalized to GFP, and their degradation profiles with increasing amounts of Hdm2 are shown. The values in the graphs represent means ± the SD from three independent experiments. (B) Control of ubiquitylation of GFP-p53 phosphorylation mutants by Hdm2 in ER-stressed cells. GFP-p53 cDNA (0.5 µg) bearing the S315A or S376A mutation was transiently transfected in 2KO cells in the absence or presence of Hdm2 WT cDNA (0.5 µg) and HA-Ub cDNA (0.5 µg). The cells were treated with 10 µM MG132 for 3 h, followed by treatment with 10 µg of TM/ml or 1 µM TG for 3 h. The immunoprecipitation of GFP-p53 and immunoblotting were performed as in Fig. 3B. (C) Control of localization of GFP-p53 phosphorylation mutants by Hdm2. GFP-p53 mutant cDNAs (0.25 µg) were transiently transfected in 2KO cells in the absence or presence of Hdm2 WT cDNA (0.25 µg). After 24 h, cells were left untreated or treated with either 10 µg of TM/ml or 1 µM TG for 4 h, followed by the examination of GFP-p53 localization by fluorescence. Cell nuclei were visualized by staining with DAPI. Transfection of GFP cDNA alone was used as a control (right panel). White arrows indicate nuclear localization of GFP-p53 only. Quantification of GFP-p53 localization was performed as described for Fig. 2A and in Materials and Methods.

GSK-3ß is involved in Hdm2-mediated p53 cytoplasmic shuttling in ER-stressed cells. Since p53 phosphorylation at S315 and S376 in ER-stressed cellsis mediated by GSK-3ß (48), we sought to determinethe role of GSK-3ß in Hdm2-mediated nuclear exportand degradation of the tumor suppressor. To do so, we examinedthe localization of GFP-p53 in spontaneously immortalized GSK-3ß^–/–MEFs (20), which were devoid of endogenous p53 (48). In thesecells, GFP-p53 exhibited a nuclear localization either in theabsence or presence of Hdm2, and it remained nuclear even aftertreatment with ER stress (Fig. 5A). When the GSK-3ß^–/–MEFs were transfected with GSK-3ß WT and Hdm2, weobserved that a fraction of GFP-p53 became cytoplasmic, whichwas further enhanced in response to ER stress (Fig. 5A). Incontrast to GSK-3ß WT, the cytoplasmic localizationof GFP-p53 by Hdm2 was not induced when the cells were reconstitutedwith a KD GSK-3ß (Fig. 5A). Similar results were obtainedwhen the localization of GFP-p53 was examined in 2KO cells transfectedwith GSK-3ß and Hdm2 (Fig. 5B). Quantification ofthe subcellular distribution of GFP-p53 showed that cytoplasmicrelocation of GFP-p53 was enhanced by GSK-3ß WT andimpaired by GSK-3ß KD. Collectively, these data suggestedthat GSK-3ß is required for the Hdm2-mediated nucleocytoplasmicshuttling of p53.

View larger version (32K):
[in this window]
[in a new window]
FIG. 5. GSK-3ß is required for Hdm2-mediated cytoplasmic shuttling of p53 in ER-stressed cells. (A) Subcellular localization of GFP-p53 in GSK-3ß^–/– MEFs. Cells were transfected with 0.05 µg of GFP-p53 WT cDNA, 0.05 µg of Hdm2 cDNA, and 0.1 µg of either GSK-3ß WT or GSK-3ß KD cDNA. After 24 h, cells were treated with either 10 µg of TM/ml or 1 µM TG for 4 h and then examined for GFP-p53 localization by fluorescence microscopy. The cell nuclei were visualized by DAPI staining. Quantification of GFP-p53 localization was performed as described in Fig. 2A and Materials and Methods. (B) Subcellular localization of GFP-p53 in 2KO MEFs. Cells were transiently transfected with 0.25 µg of GFP-p53 cDNA in the absence or presence of 0.25 µg of Hdm2 of WT cDNA and 0.5 µg of either GSK-3ß WT cDNA or GSK-3ß KD cDNA. After 24 h, cells were left untreated or treated with either 10 µg of TM/ml or 1 µM TG for 4 h, followed by examination with fluorescence microscopy. Quantification of the subcellular localization of GFP-p53 was performed as described above.

GSK-3ß controls Hdm2-mediated p53 degradation in unstressed and ER-stressed cells. We next wanted to determine the role of GSK-3ß inHdm2-mediated p53 degradation. To this end, spontaneously immortalizedGSK-3ß^+/+ and GSK-3ß^–/– MEFswere transfected with GFP-p53 WT cDNA and increasing amountsof Hdm2 cDNA, followed by immunoblot analysis for GFP-p53. Wefound that GFP-p53 WT was more resistant to Hdm2-mediated degradationin GSK-3ß^–/– MEFs than in GSK-3ß^+/+MEFs (Fig. 6A), indicating that p53 degradation by Hdm2 requiresGSK-3ß. On the other hand, GFP-p53-S315A and GFP-p53-S376Awere more resistant to Hdm2-mediated degradation in GSK-3ß^–/–MEFs and in GSK-3ß^+/+ MEFs than GFP-p53 WT (Fig. 6B).To confirm the role of GSK-3ß in p53 nuclear exportand degradation, we determined the localization of GFP-p53 inimmortalized GSK-3ß^+/+ and GSK-3ß^–/–MEFs transfected with increasing amounts of Hdm2 cDNA (Fig.6C). We observed that GFP-p53 was mainly nuclear in both GSK-3ß^+/+and GSK-3ß^–/– MEFs. Transfection of cellswith equal amounts of Hdm2 and GFP-p53 cDNAs (ratio, 1:1) resultedin the cytoplasmic relocation of GFP-p53 in GSK-3ß^+/+MEFs but not in GSK-3ß^–/– MEFs in whichGFP-53 remained nuclear. When the Hdm2/GFP-53 ratio was increasedto 5:1, we noticed that the GFP-p53 signal was significantlydecreased in both MEF types although GFP-p53 was more resistantto Hdm2-mediated degradation in GSK-3ß^–/–than in GSK-3ß^+/+ MEFs (Fig. 6C). From these data,we concluded that the nucleocytoplasmic relocation and subsequentdegradation of GFP-p53 by Hdm2 requires GSK-3ß.

View larger version (48K):
[in this window]
[in a new window]
FIG. 6. GSK-3ß is necessary for p53 degradation in ER-stressed cells. (A) Control of GFP-p53 stability by GSK-3ß. GFP-p53 WT cDNA (0.25 µg) was transiently transfected with increasing amounts of Hdm2 cDNA (0.25 to 1.25 µg) into immortalized GSK-3ß^–/– and GSK-3ß^+/+ MEFs. The GFP cDNA (0.25 µg) was used as an internal control. The GFP-p53, Hdm2, and GFP protein levels were detected by immunoblotting of 50 µg of protein extracts with antibodies against each protein. The bands were quantified with the Scion Image 2.0 software, and the ratio of GFP-p53 to GFP in each lane is indicated. (B) Hdm2-dependent degradation of GFP-p53 and its phosphorylation mutants. GFP-p53 WT cDNA or the indicated GFP-p53 phosphorylation mutants were cotransfected with Hdm2 WT cDNAs in immortalized GSK-3ß^–/– and GSK-3ß^+/+ MEFs as described in panel A. Immunoblot analyses of GFP-p53 and GFP were performed as in panel A. The bands were quantified, and the graphs representing the degradation rates of the GFP-p53 forms plotted against Hdm2/GFP-p53 ratios are shown. (C) Immortalized GSK-3ß^+/+ and GSK-3ß^–/– MEFs were transfected with different ratios of GFP-p53 WT to Hdm2 cDNAs as indicated. At 24 h posttransfection, cells were examined for GFP-p53 fluorescence. Cell nuclei were visualized by DAPI staining. The quantification of the subcellular distribution of GFP-p53 was performed as described for Fig. 2A and Materials and Methods. NS, not significant. (D) Regulation of p53 in primary GSK-3ß^–/– MEFs. MEFs (passage 3) were treated with 1 µM TG for 2 and 4 h, followed by immunoblotting of 50 µg of protein extracts for endogenous p53 (top panel), GSK-3ß (middle panel), and actin (bottom panel). Immunostaining of endogenous Mdm2 (green) or p53 (red) in the primary MEFs was performed as described in Materials and Methods. The cell nuclei were detected by DAPI staining.

To further substantiate the role of GSK-3ß in p53degradation, we looked at the endogenous WT p53 in primary GSK-3ß^+/+and GSK-3ß^–/– MEFs. Specifically, we assessedthe protein levels and subcellular localization of p53 beforeand after ER stress treatment with TG (Fig. 6D). We found thatp53 protein was more resistant to the inhibitory effects ofER stress in GSK-3ß^–/– MEFs than in GSK-3ß^+/+MEFs demonstrating that GSK-3ß is involved in thedownregulation of p53 in ER-stressed cells. It is of interestthat primary MEFs are more sensitive to downregulation of p53by ER stress than tumor cells (Fig. 1), indicating differencesin ER stress responses between normal and transformed cells.The immunostaining data showed a significant loss of nuclearp53 staining in TG-treated GSK-3ß^+/+ MEFs as opposedto GSK-3ß^–/– MEFs. Furthermore, we noticedthat in untreated GSK-3ß^+/+ MEFs a fraction of p53was cytoplasmic, as opposed to GSK-3ß^–/–MEFs in which p53 was predominantly nuclear. This result indicatedthat GSK-3ß also regulates the nucleocytoplasmic shuttlingof p53 in unstressed cells. On the other hand, Mdm2 localizationwas both nuclear and cytoplasmic in both MEF types (Fig. 6D).However, treatment with TG decreased the Mdm2 signal GSK-3ß^+/+MEFs concomitant with the decrease of p53 signal in these cells.The Mdm2 signal did not significantly change in TG-treated GSK-3ß^–/–MEFs consistent with the resistance of p53 to ER stress-mediateddegradation in these cells. Collectively, these data demonstratedthe important role of GSK-3ß in localization and degradationof p53.

DISCUSSION

Top

Discussion

Our findings provide a mechanistic explanation for the higherdegradation rates of p53 in ER-stressed cells (48). Specifically,we show that Hdm2-dependent ubiquitylation, nuclear export,and degradation of p53 are all enhanced in ER-stressed cells.At the molecular level, Hdm2 interaction with p53 was not significantlyaffected by ER stress (Fig. 2), indicating that their interactionis necessary but not sufficient for the enhanced ubiquitylationof the tumor suppressor protein. Our data support the notionthat enhanced ubiquitylation of p53 is likely to be mediatedby posttranslational modifications of p53 rather than Hdm2.Indeed, our observations showed that the auto-ubiquitylationactivity of Hdm2 was not affected in cells subjected to ER stress(data not shown) and that degradation of AnR, which is anothersubstrate of Hdm2 (37), was not induced in response to ER stress(Fig. 3). In fact, our findings support the notion that phosphorylationof p53 is necessary for its enhanced ubiquitylation by Hdm2.That is, ER stress induces p53 phosphorylation at S315 and S376(48), modifications that are important for the induction ofp53 ubiquitylation (Fig. 3) and subsequent degradation (Fig.4) in response to ER stress. It is possible then that phosphorylationof p53 upon ER stress results in conformational changes thatfurther facilitate its ubiquitylation process by Hdm2. Thisis reminiscent of the regulation of p53 degradation by phosphorylationat threonine (T) 55, which is mediated by TAF1 and facilitatesp53 oligomerization (33), a process required for efficient Hdm2-mediatedubiquitylation and degradation of the tumor suppressor protein(39). The role of S315 in p53 destabilization is further supportedby recent findings demonstrating that p53 phosphorylation atthis site by Aurora A kinase facilitates Hdm2-mediated ubiquitylationand degradation of p53 (25). Concerning S376, our data are supportedby other findings for a negative effect of its phosphorylationon p53 stabilization (6). Interestingly, S376 of p53 was foundto be phosphorylated in unstressed cells and its dephosphorylationto be induced upon DNA damage by gamma irradiation, resultingin a higher capacity of p53 to bind DNA (62). Our data showthat S315 and S376 phosphorylations are equally important forp53 ubiquitylation, nucleocytoplasmic shuttling, and degradationin response to ER stress (Fig. 4B and C). Since the functionalstatus of p53 is usually determined by its overall phosphorylationstatus rather than phosphorylation at a single residue (51),it remains possible that phosphorylation at S315 or S376 affectsphosphorylation at other sites that also contribute to the inactivationof p53 in ER stress-treated cells.

It is of interest that degradation of either transfected orendogenous p53 still takes place in GSK-3ß^–/–cells but to a much lesser extent than in GSK-3ß^+/+cells (Fig. 6), providing evidence that GSK-3ß maynot be the only determinant of p53 degradation. One possibilityis that GSK-3 ${alpha}$ also exerts a similar effect on p53 degradationin ER-stressed cells. Also, in A549 cells ER stress had a partial( $~$ 50%) inhibitory effect on p53 protein levels (Fig. 1), whichraises the interesting question what of makes the other 50%of p53 resistant to ER stress. It is possible that upon ER stressonly a fraction of p53 is modified by phosphorylation from GSK-3ß,thus leading to the partial destabilization of p53. Anotherinterpretation may have to do with the differential sensitivityof cells to ER stress since destabilization of p53 is greaterin primary MEFs (Fig. 6D) than in the A549 tumor cells (Fig.1). Furthermore, ER stress may modulate p53 through recentlyidentified pathways that control its stability. For example,recent findings demonstrated that p53 degradation is inhibitedby the interaction of Hdm2 with the ribosomal proteins L11 andL23 (7, 21, 38). Although ER stress signals to the translationalribosomal machinery through the activation of the eIF2 ${alpha}$ kinasePERK (49), it is not presently known whether Hdm2 interactionwith either L11 and/or L23 is affected by ER stress and whetherthis type of stress can cause perturbations in ribosomal biogenesisthat signal to Hdm2 and p53. Other control mechanisms mightimplicate the Yin Yang 1 (YY1) protein, which has recently beenshown to facilitate Hdm2-mediated ubiquitylation of p53 (15,57). A link between YY1 and ER stress might be implied by theability of YY1 to induce the expression of grp78 gene, whichis also transcriptionally induced in response to ER stress (36).Moreover, YY1 can enhance the transactivation capacity of ATF6,a protein involved in the transcriptional induction of manygenes in response to ER stress (34). Another tentative linkbetween p53 degradation and ER stress may be provided by thefunction of Rad23, a protein implicated in the stimulation ofER-associated protein degradation pathway (59). Specifically,human Rad23 (HR23) exhibits a dual function in p53 degradationby preventing the deubiquitylation of p53 and delivering theubiquitylated protein to the proteasome machinery for degradation(12). Furthermore, HR23 interacts with p300/CBP resulting inthe inhibition of the protein stability and transcriptionalactivity of p53 (69). In addition to ubiquitylation, conjugationof both p53 and Hdm2 by the ubiquitin-like protein NEDD8 (neddylation)has recently been shown to interfere with the biological functionsof both proteins (63). Nevertheless, neddylation of p53 is notaffected by treatment of cells with inducers of ER stress (datanot shown), thus excluding the possibility for a regulatoryrole of this modification in p53 function in response to ERstress.

Our data demonstrate that p53 phosphorylation at S315 and S376mediated by GSK-3ß (49) serves as a signal that iscrucial not only for the ubiquitylation but also for the cytoplasmicrelocation and degradation of p53. These effects of GSK-3ßon p53 take place in unstressed cells but are accelerated incells subjected to ER stress (for a model of p53 regulationby ER stress, see Fig. 7). Indeed, the loss of GSK-3ßimpeded p53 nuclear export (Fig. 5) and increased p53 stability(Fig. 6) in the absence of ER stress. Moreover, endogenous WTp53 is more stable in primary GSK-3ß^–/–than GSK-3ß^+/+ MEFs in response to ER stress, a findingconsistent with a higher nuclear presence of p53 in GSK-3ß^–/–MEFs exposed to ER stress (Fig. 6D). The immunostaining datashow a decrease in endogenous Mdm2 levels concomitant with thedownregulation of endogenous p53 in response to ER stress. Thisregulation is more apparent in GSK-3ß^+/+ MEFs thanin GSK-3ß^–/– MEFs. These data providestrong evidence that GSK-3ß plays a major role inp53 nucleocytoplasmic transport and degradation. The molecularevent(s) that lead to activation of GSK-3ß upon ERstress are currently under investigation. One possibility isthat GSK-3ß becomes activated by phosphorylation byanother kinase(s) that is induced in response to ER stress (18).Such phosphorylation events may be important in determiningthe substrate specificity of GSK-3ß. Given the pleiotropiceffects of GSK-3ß in cell signaling (45), it is conceivableto speculate that the ability of the kinase to regulate p53function may tightly be dependent on signaling induced by aspecific type of stress. For example, induction of GSK-3ßby ER stress leads to the downregulation of p53 protein (48;this study), whereas activation of GSK-3ß by a specifictype of genotoxic stress is associated with p53 activation (58,60, 61).

View larger version (13K):
[in this window]
[in a new window]
FIG. 7. Schematic model of p53 regulation by Hdm2 in unstressed and ER-stressed cells. In unstressed cells, the nucleocytoplasmic transport of p53 is mainly mediated by Hdm2. We show here that phosphorylation of p53 at S315 and S376 by GSK-3ß does not interfere with the ubiquitylation process but is required for the cytoplasmic relocation of p53 by Hdm2. Cytoplasmic p53 is then prone to degradation by the 26S proteasome pathway. When cells are exposed to ER stress, the nuclear fraction of GSK-3ß becomes activated leading to the induction of p53 phosphorylation at S315 and S376 (49). As shown in the present study, these phosphorylation events enhance the ubiquitylation of p53 by Hdm2 and the cytoplasmic relocation of the tumor suppressor. As a result, p53 degradation rates in the cytoplasm are induced.

The role of ER stress in tumor development is strongly impliedby the transcriptional induction and expression of glucose-regulatedproteins (Grps) or ER chaperones that confer a survival advantageto the stressed cell. Whereas in normal cells the inductionof Grps is associated with tissue preservation or organ protection,in cancer cells the induction of Grps can facilitate tumor progressionand drug resistance (31). Given the apoptotic functions of p53,its inactivation in ER-stressed cells could certainly providecells with a growth advantage and promote the transformationprocess. In fact, our previous findings demonstrated an inhibitoryeffect of ER stress on p53-depenent apoptosis (49). A physiologicalinducer of ER stress is hypoxia (29), a condition that is commonin solid tumors. Although some reports have shown hypoxia toinduce p53 (1, 13), other reports have provided evidence tothe contrary (45, 53). This discrepancy can be explained notonly by the differences in experimental conditions but alsoby the pleiotropic effects that are associated with hypoxia,including nutrient deprivation or low pH (acidosis) (19). Thelatter interpretation is supported by findings demonstratingthe downregulation of p53 in hypoxic cells that are free fromacidosis (52). Furthermore, recent data also showed the lackof p53 accumulation in hypoxic cells upon conditions that preventedacidosis and maintained nutrient levels (44). These and ourdata are consistent with a model where induction of the ER stressresponse in hypoxic tumors is associated with the downregulationof p53. Such an induction of ER stress in the microenvironmentsof solid tumors may have detrimental effects on therapies aimingat the activation of endogenous p53 and may provide an explanationfor tumor resistance to radio/chemotherapies. Understandingthe mechanisms of downregulation of p53 by Hdm2 in ER-stressedcells may be useful for the design of strategies leading tothe activation of p53 in tumors with deregulated ER (i.e., hypoxictumors). Tumors that retain WT p53 could be sensitized to p53-mediatedapoptosis by inhibiting the expression of Hdm2 or blocking p53/Hdm2interaction with synthetic peptides. Such approaches alone orin combination with novel strategies that utilize membrane-associatedGrps for the destruction of tumors with increased levels ofER stress (4) may prove promising for therapeutic intervention.

ACKNOWLEDGMENTS

We thank J. Woodgett for GSK-3ß WT cDNA, KD GSK-3ßcDNA, and GSK-3ß knockout MEFs; S. Boyd and T. Jacksfor the GFP-p53 WT, Hdm2 WT, Hdm2 NES, and Hdm2 C464A plasmids;M. Treier for HA-ubiquitin vector; S. Jones for 2KO MEFs; R.Iggo for Hdm2 shRNA vectors; and M. Triffiro for LNCaP cellsand androgen receptor Ab.

D.B. is a research student of the Terry Fox Foundation throughan award from the National Cancer Institute of Canada (NCIC)and a recipient of the CIHR Cancer Consortium Training GrantStudentship Award. This study was supported by a grant fromthe NCIC to A.E.K.

FOOTNOTES

* Corresponding author. Mailing address: Lady Davis Institute for Medical Research, McGill University, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote-Ste-Catherine St. Montreal, Quebec H3T 1E2, Canada. Phone: (514) 340-8260, ext. 3697. Fax: (514) 340-7576. E-mail: antonis.koromilas{at}mcgill.ca.

REFERENCES

Top