Research ArticleBiochemistry

Mutant and wild-type p53 form complexes with p73 upon phosphorylation by the kinase JNK

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Science Signaling  03 Apr 2018:
Vol. 11, Issue 524, eaao4170
DOI: 10.1126/scisignal.aao4170

Another look at p53-p73 interactions

The transcription factor p53 critically regulates cell survival or death in response to cellular stress. Mutations in p53 are common in cancer and alter its interactions with other proteins and, consequently, with cell fate–specific genes. Mutant p53 binds to and inhibits its family member p73, thereby promoting cell survival instead of cell death in response to cell stress. It was believed that this interaction with p73 was specific to mutant p53. However, Wolf et al. found that wild-type p53 can bind p73 to promote stress-induced cell death. Phosphorylation of wild-type p53 by the cell stress–responsive kinase JNK caused a conformational change that mirrored the regional structure of the mutant and enabled its binding to p73 but, unlike mutant p53, the wild-type protein could still bind to apoptotic gene targets. These findings refine our understanding of p53 interactions and, specifically, p53-p73 coordination in the cell stress response.


The transcription factors p53 and p73 are critical to the induction of apoptotic cell death, particularly in response to cell stress that activates c-Jun N-terminal kinase (JNK). Mutations in the DNA-binding domain of p53, which are commonly seen in cancers, result in conformational changes that enable p53 to interact with and inhibit p73, thereby suppressing apoptosis. In contrast, wild-type p53 reportedly does not interact with p73. We found that JNK-mediated phosphorylation of Thr81 in the proline-rich domain (PRD) of p53 enabled wild-type p53, as well as mutant p53, to form a complex with p73. Structural algorithms predicted that phosphorylation of Thr81 exposes the DNA-binding domain in p53 to enable its binding to p73. The dimerization of wild-type p53 with p73 facilitated the expression of apoptotic target genes [such as those encoding p53–up-regulated modulator of apoptosis (PUMA) and Bcl-2-associated X protein (BAX)] and, subsequently, the induction of apoptosis in response to JNK activation by cell stress in various cells. Thus, JNK phosphorylation of mutant and wild-type p53 promotes the formation of a p53/p73 complex that determines cell fate: apoptosis in the context of wild-type p53 or cell survival in the context of the mutant. These findings refine our current understanding of both the mechanistic links between p53 and p73 and the functional role for Thr81 phosphorylation.


The transcription factor family comprising p53, p63, and p73 are critical regulators of cell fate, specifically through the induction of apoptotic cell death in response to cellular stress and developmental cues. The three proteins have distinct as well as overlapping and interdependent functions. Mutations in p53 are common in human tumors; gain-of-function p53 mutants have a dominant-negative effect on wild-type p73 by binding it and inhibiting its ability to bind DNA and thereby preventing its transcriptional induction of apoptotic genes (1, 2). In mice, knockout of one allele or both alleles of the individual genes encoding p53, p63, or p73 resulted in reduced cell death in response to DNA-damaging stimuli (3), indicating that all three proteins are necessary to invoke a robust apoptotic response in that context. However, whereas wild-type p63 and p73 form heterodimeric (or higher order) complexes, wild-type p53 reportedly does not (4, 5).

Wild-type p53 has two transactivation domains: TAD1 (amino acids 1 to 40) and TAD2 (amino acids 43 to 54). TAD2 is located proximal to the proline-rich domain (PRD; amino acids 63 to 97) (6, 7), and both TAD2 and the PRD are critical for p53’s induction of apoptotic genes (8, 9), whereas TAD1 is reportedly dispensable for this function (10). Thr81 is a key phosphorylation site within the PRD, as is Ser46 within TAD2 (1115). Several kinases reportedly phosphorylate Ser46 (13, 16, 17), and c-Jun N-terminal kinase (JNK) phosphorylates Thr81 (18), yet precisely how modification of Thr81 regulates p53 activity in the context of apoptosis induction is limited.

The PRD is an important structural domain; prolyl isomerization in this domain regulates the structure of other domains of p53 (19). In addition, the PRD is necessary for growth suppression and apoptosis (20, 21). We rationalized that phosphorylation within this domain may also be sufficient to alter the structure of other domains in p53 and thereby regulate its functional interactions. Our findings support previously published work on genetic knockout mice, the PRD, and mutant p53/p73 complex formation (1, 2) but refine our current understanding of the interaction between wild-type p53 and p73.


The phosphorylation of p53 by JNK results in the induction of apoptotic protein PUMA

Because JNK is a key inducer of p53-mediated apoptosis and JNK phosphorylates p53 at Thr81, we were first curious whether Thr81 was phosphorylated in mutant p53 in cells. In MDA-MB-468 cells, which have a missense mutation in p53 (R273H), TMD231 cells, which have a missense mutation in p53 (R280K), and MDA-MB-157 cells, which have a truncation mutation in p53 (A88fs*52), JNK was activated, as determined by immunoblotting with antibodies against phospho-Tyr183/Tyr185 JNK (Fig. 1A). In addition, mutant p53 was phosphorylated at Thr81 in cells cultured in growth-supporting conditions. Treatment with the DNA topoisomerase inhibitor camptothecin or the protein synthesis inhibitor anisomycin increased phosphorylation of JNK and phospo-Thr81. Various cells that have wild-type p53, namely, untransformed cell lines NIH3T3 (mouse fibroblast) and HME1 (human mammary epithelial), as well as human foreskin fibroblast BJ cells and glioblastoma cell lines U87 and SF767, exhibited limited activation of JNK and phosphorylation of p53-Thr81 under growth conditions, but robust induction of both upon camptothecin or anisomycin exposure (Fig. 1B and fig. S1). Thus, in “normal” and tumor cells, JNK is activated and p53 (wild-type or mutant) is phosphorylated at Thr81 in response to cell stress.

Fig. 1 Phosphorylation of Thr81 of p53 in response to JNK activation.

(A) Western blot of phosphorylated c-Jun N-terminal kinase (JNK), phosphorylated Thr81 of p53, and actin in cellular extracts from TMD231, MDA-MB-468, and MDA-MB-157 cells treated with either camptothecin or anisomycin for the indicated times. Blots are representative of three independent experiments. (B) Western blot of phosphorylated JNK or Thr81 of p53 and actin from cellular extracts from 3T3, SF767, HME-1, BJ, and U87 treated with anisomycin or camptothecin for the indicated times. Blots are representative of three independent experiments (quantified in fig. S1). (C) U87 or SF767 cellular extracts from DMSO, SP600125, anisomycin, or pretreatment with SP600125 and then anisomycin and Western-blotted for p53–up-regulated modulator of apoptosis (PUMA) and actin. Blots are representative of two independent experiments.

We then examined the dependence of JNK activation on the induction of PUMA, the apoptotic downstream target of p53. p53–up-regulated modulator of apoptosis (PUMA) abundance was increased by anisomycin treatment in cells that had wild-type p53 (Fig. 1C), but this induction was blocked by pretreatment with the small-molecule JNK inhibitor SP600125, thereby confirming that activation of JNK was important for induction of the p53 target PUMA. Alongside this increase in PUMA abundance was a significant increase in cell death, thereby confirming the functional outcome of the pathway (fig. S2).

The predicted structure of a Thr81 phosphomimic shows a potential mode of binding to p73

To gain insight into the structural implications of phosphorylation at Thr81 of p53, we took advantage of a protein structure prediction algorithm, I-TASSER (Iterative Threading ASSEmbly Refinement) (2224). Using I-TASSER, we generated predicted protein structure models for wild-type p53, R248W mutant p53 (a common DNA binding domain mutant), and a Thr81 phosphomimic p53 (T81E) (Fig. 2 and data files S1 to S5). Analysis of wild-type and mutant p53 showed distinct structural differences in the N and C termini. The Thr81 phosphomimic mutation (T81E) substantially changed the structure of the tetramerization/regulatory (TET/REG) domain compared to that of the unphosphorylated wild-type p53. The TET/REG domain of the T81E model more closely resembled the R248W mutant p53 model than the wild-type p53 model. The R248W mutation of p53 is one of the mutants that binds to p73 (1). It is probable that it is this structural change in the TET/REG domain that enables p53 to interact with p73, which explains the original finding that wild-type p53 was unable to bind to p73. The results of our structural analysis implies that phosphorylation at Thr81 of p53 is permissive of binding to p73. We also produced in silico structural models of the S46D phosphomimic p53 and an R248W/T81E mutant p53 phosphomimic (Fig. 2) to examine whether there were any structural changes in these constructs. The TAD2 of the S46D phosphomimic was structurally similar to the T81E phosphomimic but distinct from either of the R248W mutant p53 models. Given that phosphorylation at Ser46 of p53 is needed for a robust apoptotic response, and phosphorylation at Thr81 of p53 causes a structural change similar to that of phosphorylation at Ser46, these data suggest that TAD2 and PRD are integrated into important structural changes that are associated with induction of apoptotic genes. Compared to mutant p53 alone (R248W), the Thr81 phosphomimic of R248W mutant p53 (R248W/T81E) causes an additional structural change in almost every single functional domain. This finding would suggest that the use of a JNK inhibitor may reduce the binding of mutant p53 to p73 without completely ablating the interaction.

Fig. 2 Predicted structure for a Thr81 phosphomimic reveals a possible mode of binding to p73.

(A and B) Structures (A) and amino acid coverage (B) of domains in wild-type and mutant p53. Whole protein represents amino acids 1 to 393 of p53. Transactivation domains 1 and 2 (TAD1/TAD2) represent amino acids 1 to 40 and 43 to 54. The proline-rich domain (PRD) represents amino acids 63 to 97. The tetramerization and regulatory domains (TET/REG) represent amino acids 323 to 393. T81, Thr81; E, glutamic acid in the T81E phosphomimic mutant domain. Arrows in the TET/REG domain structures indicate the region of a conformational change that is permissive to binding.

Phosphorylation of Thr81 of p53 promotes a p53-p73 complex

Our structural data (Fig. 2) combined with previous reports that mutant p53 binds wild-type p73 and the observation of high basal Thr81 phosphorylation of mutant p53 (Fig. 1) led us to examine whether blocking JNK with the selective small-molecule inhibitor SP600125 could alter the mutant p53-p73 interaction endogenously. Mutant p53 was immunoprecipitated from lysates from TMD231 cells treated with dimethyl sulfoxide (DMSO) or SP600125 and blotted for coincident pulldown of p73. We found that p73 copurified with mutant p53 and that this binding was decreased by 40% when JNK activity was blocked with SP600125 (Fig. 3A). This data suggests that JNK has only a modest effect on the interaction between mutant p53 and p73, which was predicted in the structural data (Fig. 2). We next tested whether wild-type p53 and p73 were affected by JNK-mediated phosphorylation. To do this, we transiently transfected H1299 cells with wild-type p53 and HA-tagged p73 and then treated the transfected cells with DMSO or anisomycin. Immunoprecipitation of wild-type p53 from cellular extracts shows that p73 copurified only when JNK was activated with anisomycin (Fig. 3B). To provide evidence that phosphorylation of Thr81 was necessary for wild-type p53 to copurify with p73, we transiently transfected H1299 cells with wild-type p53 or the T81E phosphomimic. Immunoprecipitation of exogenous p53 revealed that T81E, but not wild-type p53, copurified with endogenous p73 (Fig. 3C). Likewise, immunoprecipitation of endogenous p73 from extracts from U87 cells treated with DMSO, anisomycin, SP600125, or pretreated with SP600125 before anisomycin showed that endogenous wild-type p53 copurifed with p73 only when Thr81 was phosphorylated (Fig. 3D). These data substantially suggest that Thr81 phosphorylation in p53 is critical for the formation of a wild-type p53-p73 complex. To definitively show this, we used various p53 phosphomimics (T81E, S46D, and T81E-S46D) and p73 produced in bacteria and performed Far-Western analysis to examine the requirement of Thr81 phosphorylation. We fixed recombinant wild-type p53, the phosphomimics, and two positive controls (MDM2 and MDMX) to nitrocellulose and then incubated the membrane with recombinant p73 and blotted it for p73. In support of our cellular data presented above, this in vitro approach confirmed that p73 bound only to T81E and not wild-type p53 or S46D p53 (Fig. 3E). We also probed for p53 in a separate lane to show the relative amounts of p53 loaded per well (Fig. 3E). Collectively, through overexpression, endogenous, and recombinant approaches, we conclude that p73 will bind to wild-type p53 when p53 is phosphorylated at Thr81.

Fig. 3 Formation and activity of a p53-p73 complex.

(A) Immunoprecipitation of p53 or control immunoglobulin G (IgG) from TMD231 cells treated with DMSO or SP600125 and Western-blotted for p73 and p53. Blots are representative of four independent experiments. (B) Transient transfection of H1299 cells with p53 and HA-p73 and treated with either DMSO or anisomycin. Western blot of p53 and HA from p53 immunoprecipitated from cellular extracts. Blots are representative of two independent experiments. (C) Immunoprecipitation of p53 or control IgG and Western blot of p53 and p73 from H1299 cells transfected with wild-type p53 or T81Ep53. WCL is the whole-cell lysate from H1299 cells. Blots are representative of two independent experiments. (D) Immunoprecipitation of endogenous p73 from U87 cells treated with SP600125, anisomycin, or both and Western blot of phospho-Thr81, p53, and p73. Blots are representative of two independent experiments. (E) Far-Western analysis of p73 binding to p53. Lane 1 corresponds to p53 and the phosphomimics of p53; lane 2 is MDM2 and MDMX. Right, blotting for p73 to detect binding to p53, MDM2, and MDMX. Left, the relative amount of p53 on the membrane. Blots are representative of two independent experiments.

To gain insight into where p53 and p73 interact, we performed confocal immunofluorescence microscopy analysis on MDA157, HME, or TMD231 cells grown on coverslips and treated with anisomycin, SP600125, or DMSO. Anisomycin treatment caused a significant increase in colocalization between p53 and p73 in the nucleus of p53–wild-type HME cells but not p53-mutant MDA157 cells (Fig. 4, A and B, and table S1). Blocking JNK activity with SP600125 in TMD231 cells, which have mutant p53 and high basal activation of JNK, caused a significant decrease in the colocalization of mutant p53 and p73 in the nucleus (Fig. 4C and table S1), which is in accordance with the decrease in binding of mutant p53 and p73 (Fig. 3). To ensure that anisomycin was indeed activating JNK and increasing phosphorylation at Thr81 and that SP600125 was inhibiting JNK and decreasing phosphorylation at Thr81 of p53, the same cells were treated as described in the confocal experiments, and lysates were Western-blotted for active JNK and phosphorylated Thr81 p53 (fig. S3).

Fig. 4 Colocalization of p53 with p73.

(A to C) Immunofluorescence assessing nuclear colocalization of p53 (green) and p73 (red) in MDA157 (A) and HME cells (B) treated with anisomycin (Aniso) or DMSO and in TMD231 cells (C) treated with SP600125 or DMSO. Arrows indicate the cell magnified in the inset (bottom right) of each image. Scale bars, 25 μm (MDA157 and TMD231) or 50 μm (HME). Data are mean ± SE from three independent experiments; P is determined by two-tailed unpaired t test.

The JNK-initiated p53-p73 pathway is important for apoptosis

Above, we showed that phosphorylation of Thr81 of p53 resulted in its interaction with p73 (Fig. 3) and an increased abundance of PUMA (Fig. 1C). Therefore, we examined whether p53, p73, and JNK activation was necessary for the induction of apoptosis. We showed that the removal of p53 in p53–wild-type BJ cells resulted in resistance to JNK-activated cell death by anisomycin treatment (Fig. 5A). Specifically, treatment with anisomycin for 9 hours initiated a steady increase in the abundance of BAX and PUMA, which was prevented by short hairpin RNA (shRNA)–mediated knockdown of p53. In contrast, the abundance of BAX increased after 6 hours of anisomycin treatment; this may be due to the cells overcoming the shRNA knockdown at 6 hours in response to prolonged anisomycin-induced stress (Fig. 5A).

Fig. 5 Downstream effects of the p53-p73 pathway.

(A) Western blot of BAX, PUMA, and p53 in cellular extracts from BJ cells transfected with empty vector control (E.V.) or p53–short hairpin RNA (shp53) and treated with anisomycin for the indicated time. Blots are representative of three independent experiments; E.V. and shp53 samples were developed on the same membrane. (B) Methylene blue staining cell viability assay in E.V.- and shp53-transfected BJ cells treated with DMSO or anisomycin for 9 hours. Data are representative of three independent experiments. (C) Relative green fluorescent protein (GFP) fluorescence was measured in GFP shp53 and LVTHM empty vector BJ cells. Cells were treated with DMSO or anisomycin for 48 hours. Relative fluorescence was derived from DMSO controls. Data are representative of four independent experiments. Significance was determined by a two-tailed unpaired t test (***P < 0.005). (D) Green fluorescence was measured in H1299 cells transfected with GFP alone or GFP and either wild-type or T81A p53 and then treated with DMSO or anisomycin. Fold-change survival was calculated as the change in survival of anisomycin-treated cells relative to DMSO-treated cells and normalized to the GFP-alone control. Data are means ± SE from three independent experiments.

The apoptotic resistance conferred to BJ cells transfected with p53-shRNA was further investigated using a methylene blue cell death assay (Fig. 5B) and by assessing cell number as a factor of green fluorescent protein (GFP) (shRNA vector) signal (Fig. 5C). Loss of p53 improved cell viability through anisomycin treatment (Fig. 5, B and C), suggesting that the lack of the p53-p73 complex results in resistance to apoptosis. To test the impact of specifically Thr81 phosphorylation on cell death, we coexpressed GFP and either wild-type p53 or a p53 mutant that cannot be phosphorylated at Thr81 (T81A) in H1299 cells and examined survival after anisomycin treatment by GFP fluorescence. The data revealed an increase in survival in cells expressing T81A point mutation compared to wild-type p53 (Fig. 5D), confirming previously published observations with this mutant (18), and that Thr81 phosphorylation in p53 is critical to cell stress–induced apoptosis.

Fig. 6 Cell fate is dictated by the formation of a p53-p73 complex.

Schematic of our model. Apoptotic stimuli (for example, DNA damage) results in the phosphorylation and activation of JNK. JNK phosphorylates wild-type p53 (green hexagons) at Thr81, causing a structural change in p53 and the subsequent binding of p73 (red diamonds). This complex drives the transcription of PUMA and BAX, leading to apoptosis. Mutant p53 (blue hexagons) does not require phosphorylation by JNK to form a complex with p73. Phosphorylation of mutant p53 instead serves to enhance p53 binding with p73, hence increasing total complex formation. However, because of the dysfunctional DNA binding domain in mutant p53, as well as its sequestering of p73 by complex formation, PUMA and BAX are not transcribed, thus decreasing the apoptotic signal and promoting tumor cell survival.


The tumor suppressor p53 is highly studied, given its role in regulating many cellular activities and responses, including cell cycle arrest, DNA repair, cell death, and metabolism. Its family member p73 has some described overlapping functions; yet, unlike p53, p73 is rarely mutated in human cancer. Whereas p73 is rarely mutated, mutation of p53 may function as a dominant negative to ablate p73 activity (1). The regulation of p53 is extremely important given that its differential regulation can dictate distinct binding partners and, consequently, distinct outcomes through transcriptional targets, such as PTEN to promote apoptosis versus MDM2 to promote re-entry into the cell cycle and survival (25, 26). Increased abundance of PTEN, a protein/lipid phosphatase that inhibits the PI3K (phosphatidylinositol 3-kinase)–AKT (protein kinase B) pathway, can protect p53 from MDM2-mediated degradation induced by AKT (11, 2730). Thus, p53 can selectively induce PTEN expression and programmed cell death instead of the autoregulatory feedback loop with MDM2 to promote cell survival (11, 31). Moreover, PTEN can form a complex with p73 and bind to the PUMA promoter to increase its expression, leading to the induction of apoptosis (32). These molecular and biochemical studies are supported by genetic studies in mice where the loss of p53, p73, or PTEN confers resistance to apoptotic stimuli and an increase in neoplastic development (3, 3336). Thus, an effective response to apoptotic stimuli requires the integration of numerous intact tumor suppressors, yet how these tumor suppressors are signaled to form a complex has not previously been clearly defined.

There are well-characterized kinase signaling pathways that phosphorylate p53 and p73 in response to DNA damage, but some phosphorylation sites appear to be dispensable for the induction of apoptosis. It has been reported by several groups that Ser46 and Thr81 are important for the induction of apoptosis (11, 13, 15, 17). Homeodomain-interacting protein kinase 2 and the mitogen-activated protein kinase p38 reportedly phosphorylate Ser46 of p53, whereas JNK, which plays dual roles in cellular proliferation and cell death (37), phosphorylates Thr81 of p53 (11, 13, 15, 17). One of JNK’s substrates is p73, which mediates JNK-induced apoptosis (38), but it was unclear how. Analysis of apoptosis by the JNK-p73 axis in that previous study (38) was performed in cells that maintained expression of wild-type p53, and notably, p53 abundance does not directly correlate with its activity; for example, neddylation of p53 in response to growth factor signaling renders p53 stable but inactive (39). Untransformed cell lines have an intact apoptotic pathway and deneddylating enzymes, tumor suppressors, active kinases, and posttranslational modification enzymes that are activated in response to genotoxic stress that provide a robust induction of apoptosis.

There is renewed interest in p53 family member interactions, and several studies have highlighted the importance of the interactions between different family members (4042). Our studies have linked the JNK-p53-p73 proteins showing that phosphorylation of p53 at Thr81 by JNK is permissive of a p53-p73 interaction. Notably, our structural modeling analysis revealed that the structure of phosphorylated Thr81 is nearly identical to that of R248W. Therefore, activated JNK can convert wild-type p53 to mimic mutant p53 with regard to binding to p73 by exposing the DNA binding domain. Ser46 is proximal to the PRD in the second transactivation domain, which may be important for additional structural changes to recruit other factors for gene expression. However, we also found that phosphorylation of Ser46 has no effect on binding to p73, supporting that this phosphorylation may instead be required for recruiting additional coactivators. The JNK-mediated interaction between wild-type p53 and p73 provides biochemical evidence in support of the existing animal models and gene dose sensitivity of p53 and p73 to apoptosis (33). We also show that in mutant p53 cells, JNK activity is increased, resulting in a mutant p53/wild-type p73 complex, with mutant p53 acting as a dominant-negative partner of p73. These data are consistent with previous reports showing that mutant p53/p73 form a complex (2). This complex mediates apoptosis in cells that express wild-type p53 but survival in cells that express mutant p53 (Fig. 6). Thus, our findings refine our understanding of how cells promote or, in certain contexts, circumvent the p53 apoptotic signaling axis.


Cell culture and transfection

H1299, TMD231, U87, HME1, SF767, 3T3, hTert BJ, MDA-MB-468, and MDA-MB-157 were cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum supplemented with penicillin and streptomycin at a 37°C humidified incubator in 5% CO2. Anisomycin, SP600125, and camptothecin were purchased and suspended as described by the manufacturer (Sigma-Aldrich). Anisomycin was used at 30 μM, camptothecin was used at 30 μM, and SP600125 was used at 25 μM unless otherwise noted. Transient transfections were performed using Lipofectamine per the manufacturer’s instructions (Life Technologies). Annexin V and Dead Cell Assay kit from Millipore was used to stain cells that were analyzed on a Muse cytometer.

Plasmids, protein purification, and Western blotting

We generated mutant p53 at Thr81 with a site-directed mutagenesis kit (Life Technologies). Recombinant p73 and p53 proteins were produced in BL21DE3 cells and purified as previously described (30, 32). Far-Western analysis was completed using the 96-well dot blot system, and recombinant proteins were placed in wells and fixed to nitrocellulose by drying. Blot was blocked with 5% nonfat dry milk and incubated with recombinant p73. p73 was then detected by the p73 antibody followed by a secondary antibody and an enhanced chemiluminescence reagent. Western blot analysis used whole-cell lysates lysed under denaturing conditions (6 M urea buffer) or immunoprecipitated proteins extracted with lysis buffer, as described previously (32). Antibody for p73 (OP1008) was purchased from Millipore; antibodies for phos-T81, phos-Y183/Y185 JNK, and PUMA were purchased from Cell Signaling Technology; and antibodies for p53 (D0-1), actin, and glyceraldehyde-3-phosphate dehydrogenase were purchased from Santa Cruz Biotechnology.

I-TASSER protein structure prediction

Amino acid sequences for wild-type p53, R248W p53, T81E p53, S46D, and R248W/T81E p53 were input to the I-TASSER web server as FASTA sequences. Predicted protein structures were generated using a previously described method (2224) and analyzed using the CCP4 Molecular Graphics Program (CCP4MG) v. 2.10.6 (43). Structures were superposed using a built-in function within the software.

Immunofluorescence microscopy

MDA157, HME, or TMD231 cells were grown on glass coverslips and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, washed with PBS, and permeabilized in 1% Triton X-100 in PBS for 15 min. Coverslips were blocked in 5% bovine serum albumin for 1 hour in PBS/Tween before they were stained with p73 (D3G10, rabbit monoclonal; Cell Signaling Technology) and p53 (DO-1, mouse monoclonal; Santa Cruz Biotechnology) antibodies. Coverslips were then incubated with anti-rabbit Alexa Fluor 647 and anti-mouse Alexa Fluor 488 secondary antibodies before they were fixed to slides with ProLong Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (Life Technologies). Slides were visualized on a Leica SP8 MP microscope. Colocalization analysis was performed using Imaris v9.0.2, and colocalized images were created using the “RG2B_Colocolization” plugin for ImageJ. Scale bar, 25 μm (MDA157 and TMD231) or 50 μm (HME).

Colony-forming assay

Empty vector– or shp53-transduced hTert BJ cells were plated in triplicate and treated with anisomycin for 9 hours. DMSO was used as a vehicle control. Cells were stained with equal parts methanol and methylene blue for 15 min. The plate was then washed three times with deionized water to remove any excess stain and left to dry at room temperature.

GFP fluorescence assay

BJ cells expressing GFP TP53 shRNA (shp53) (44, 45) or empty (control) pLVTHM vector (a gift from the D. Trono laboratory, EPFL) were plated in quadruplicate and treated with DMSO or anisomycin (10 μM) for 48 hours. Fluorescence emitted by GFP in each well was measured at 509 nm using a SpectraMax M5 spectrophotometer (Molecular Devices).

Statistical analysis

Data were analyzed by two-tailed unpaired t tests. P values <0.05 were considered significant.


Fig. S1. Phosphorylation of Thr81 in cell lines with wild-type p53.

Fig. S2. U87 cell death.

Fig. S3. JNK activation or inhibition in HME, MDA157, and TMD231 cell lines.

Table S1. Manders’ coefficients for microscopy colocalization analysis.

Data file S1. Wild-type p53 structure [Protein Data Bank (PDB)].

Data file S2. p53 R248W structure (PDB).

Data file S3. p53 T81E structure (PDB).

Data file S4. p53 S46D structure (PDB).

Data file S5. p53 R248W/T81E structure (PDB).


Acknowledgments: We thank members of the Mayo laboratory for critical reading of the manuscript, D. Trono for the expression vector, and P. Wubbolding and P.M. Hauck for production and purification of recombinant proteins. Funding: This work was supported in part through funds provided by the Jeff Gordon Children’s Foundation, the Riley Children’s Foundation, and NIH CA172256 to L.D.M. Author contributions: Experiments were performed and analyzed by E.R.W. and C.P.M. (contributed equally) and K.E.B. and A.M.K. (contributed equally). L.D.M. conducted some experiments and wrote the article. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.
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