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[Cancer Research 61, 7030-7033, October 1, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Substitutions That Compromise the Ionizing Radiation-induced Association of p53 with 14-3-3 Proteins Also Compromise the Ability of p53 to Induce Cell Cycle Arrest1

Elena S. Stavridi2, Nabil H. Chehab2, Asra Malikzay and Thanos D. Halazonetis3

The Wistar Institute [E. S. S., N. H. C., A. M., T. D. H.], Program in Biochemistry [N. H. C.], and Department of Pathology and Laboratory Medicine [T. D. H.], University of Pennsylvania, Philadelphia, Pennsylvania 19104-4268


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Ionizing radiation (IR) induces an increase in the levels and activity of the p53 tumor suppressor protein. The increased activity is attributed to IR-induced posttranslational modifications, some of which regulate the interaction of p53 with other proteins. One of these modifications is dephosphorylation of Ser376, which leads to association of p53 with 14-3-3 proteins. To establish the significance of this interaction, we examined the function of mutant p53 proteins that do not interact with 14-3-3 proteins in vivo. These p53 mutants retained sequence-specific DNA binding activity. However, their ability to activate transcription of the endogenous p21/waf1/cip1 gene and to induce G1 arrest was compromised, suggesting that the dephosphorylation of Ser376 and the association of p53 with 14-3-3 proteins contribute to the activation of p53 in response to IR.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The p53 tumor suppressor protein is a critical component of the DNA damage checkpoint machinery. p53 transactivates genes that induce cell cycle arrest and apoptosis and genes involved in DNA repair (1 , 2) . The p53-regulated genes that induce cell cycle arrest include p21/cip1/waf1 and 14-3-3 {sigma} (3 , 4) . p21/cip1/waf1 encodes an inhibitor of cyclin-dependent kinases. 14-3-3 {sigma} is one of the 14-3-3 protein isoforms; it induces cell cycle arrest in G2 by sequestering in the cytoplasm proteins required for entry into mitosis (5 , 6) .

The mechanisms by which DNA damage activates p53 have been the subject of intense study. It appears that most DNA-damaging agents, including IR, lead to posttranslational modifications of p53 that regulate the interaction of p53 with other proteins or otherwise regulate p53 function. One of these modifications is phosphorylation of p53 on Ser20 (7, 8, 9) . This modification leads to increased p53 protein levels by inducing dissociation of p53 from Mdm2 (7 , 10 , 11) , a protein that targets p53 for degradation through the ubiquitin pathway (12, 13, 14) . Other modifications induced in response to DNA damage include phosphorylation of Ser6, Ser9, Ser15, Ser33, Ser37, Ser46, Ser392, dephosphorylation of Ser376, and acetylation of Lys320, Lys373, and Lys382 (1 , 15) .

One of the modifications, the functional significance of which is unclear, is dephosphorylation of Ser376 of p53. This modification creates a binding site for 14-3-3 proteins and leads to an association of p53 with 14-3-3 (16) . In vitro, 14-3-3 proteins enhance the sequence-specific DNA binding activity of p53, but in vivo their effect on p53 function is not known. We also do not know which 14-3-3 isoforms bind to p53 in vivo. If 14-3-3 {sigma} binds to p53 and enhances its activity, then there is potential for a positive-feedback loop driving p53 activation, because p53 transactivates the gene encoding 14-3-3 {sigma} (4) . Here, we address the functional significance of the interaction of p53 with 14-3-3 proteins and explore which of the 14-3-3 isoforms interact with p53 in irradiated cells.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Interaction of p53 with 14-3-3 in Vitro.
GST4 /14-3-3 fusion proteins expressed in Escherichia coli were incubated with glutathione Sepharose 4B beads (Pharmacia, Piscataway, NJ) in 1x IP buffer [25 mM HEPES (pH 7.4), 100 mM NaCl, 5 mM MgCl2, 100 mM EDTA, 200 ng/ml BSA, and 0.1% Tween 20). The beads were then incubated with 35S-labeled in vitro translated p53 (17) for 1 h at 4°C. p53 that remained bound to the beads after washing was visualized by autoradiography.

Interaction of p53 with 14-3-3 in Vivo.
U2OS osteosarcoma cells were either mock irradiated or exposed to 9 Gy of IR or 50 J/m2 UV light. Whole cell extracts were prepared 2 h after exposure to IR or 16 h after exposure to UV light by lysis in 1x extraction buffer [50 mM Tris (pH 8), 120 mM NaCl, 0.5% NP40, 1 mM DTT, 0.4 µg/ml Pefabloc SC, 2 µg/ml pepstatin, 0.2 µM wortmannin, 0.1 µM staurosporine, 15 mM NaF, and 1 mM sodium vanadate]. 14-3-3 was precipitated using isoform-specific antibodies or antibody K19, which recognizes all 14-3-3 isoforms (Santa Cruz Biotechnology, Santa Cruz, CA). Coprecipitated p53 was detected by immunoblotting with antibody DO7 (Calbiochem, San Diego, CA). The interaction of HA-tagged p53IND proteins with endogenous 14-3-3 was performed using U2OS cells transiently transfected with 2.5 µg of plasmids encoding p53 and 27.5 µg of pBC12/PLseap carrier plasmid (7) . Antibody Y11 (Santa Cruz Biotechnology) was used to recognizes HA-tagged p53IND that coprecipitated with 14-3-3.

DNA Binding Assay.
U2OS cells were transfected with 2.5 µg of plasmid encoding p53 and 27.5 µg of pBC12/PLseap carrier plasmid (7) . Twenty-four h later, the cells were exposed to 9 Gy of IR or were mock irradiated, and 1 h later, the cells were lysed using 1x extraction buffer. Oligonucleotides BCV4A and TT3 (18) with biotin tags at their 5-prime ends were coupled to streptavidin-agarose beads and incubated with the cell lysates for 1 h at 4°C in 1x extraction buffer containing a single-stranded oligonucleotide as nonspecific competitor DNA (18) . HA-tagged p53 bound to the beads was detected by immunoblotting with antibody Y11.

Transcription Activation Assays.
Saos2 cells were transfected by calcium phosphate precipitation with 0.1 µg of plasmid expressing p53 and 29 µg of pBC12/PLseap carrier plasmid (7) . Twenty-four h later, the cells were exposed to 9 Gy of IR, and 24 h later, the cells were lysed by scraping in 0.5 ml of 2x RIPA buffer [40 mM Tris (pH 7.4), 2 mM EDTA, 300 mM NaCl, 20 mM KCl, 2% NP40, 0.2% Triton-X, and 0.2% SDS]. p21/cip1/waf1 protein levels were monitored by immunoblotting using a specific monoclonal antibody (Calbiochem, San Diego, CA). Alternatively, cells were transfected with 1 µg of plasmid expressing p53 and 29 µg of the p53-specific reporter plasmid pEp21-TK-SEAP. Alkaline phosphatase activity was determined 48 h later (19) .

Cell Cycle Arrest.
U2OS osteosarcoma cells were transfected by calcium phosphate precipitation with 2.5 µg of a plasmid expressing p53IND, 5 µg of a plasmid expressing a dominant-negative p53 mutant (p53Trp248), 1 µg of a plasmid expressing green fluorescent protein (as a marker), and 24 µg of pBC12/PLseap carrier plasmid (7 , 10) . Twenty-four h later, the cells were exposed to 5 Gy of IR or were mock irradiated. The cells were harvested 12 h later, resuspended in 200 µl of 0.4% paraformaldehyde in PBS, and incubated for 12 min at 37°C and subsequently for 10 min on ice. The fixed cells were overlaid with 1800 µl of cold (-20°C) methanol with gentle vortexing. After a 10-min incubation on ice, the cells were washed in 1x PBS-TF (PBS with 0.1% Tween 20 and 2% fetal bovine serum) and incubated in 1 ml of PBS-TF containing 20 µl of RNase (Life Technologies, Inc., Grand Island, NY) and 10 µl of propidium iodide (Boehringer Mannheim, Indianapolis, IN) for 1 h at 37°C. Flow cytometry analysis was performed on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
14-3-3 Isoform Specificity.
The question of whether all or specific 14-3-3 isoforms bind to p53 was addressed both in vitro and in vivo. The in vitro studies examined the seven known human genes encoding 14-3-3 isoforms. We generated plasmid vectors that allowed each 14-3-3 gene product to be expressed as a GST fusion protein; the GST/14-3-3 fusion proteins were immobilized on glutathione beads and examined for their ability to capture full-length wild-type p53. Because the interaction between p53 and 14-3-3 proteins requires Ser378 of p53 to be phosphorylated, we used a p53 protein bearing three amino acid substitutions, including substitution of Ser378 with Ala as a negative control. 35S-labeled wild-type p53 translated in vitro was captured specifically by the GST/14-3-3 {gamma} isoform, whereas the mutant p53 protein was not captured by any of the GST/14-3-3 fusion proteins (Fig. 1A)Citation .



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  		Fig. 1. Interaction of p53 with specific 14-3-3 isoforms. A, interaction of GST/14-3-3 isoforms with 35S-labeled in vitro translated wild-type p53 or p53 with alanines at positions 376–8. The latter protein serves as a negative control, because it does not bind to 14-3-3. B, coimmunoprecipitation of p53 with specific 14-3-3 isoforms. Lysates prepared from nonirradiated cells (NR) or cells exposed to IR or UV light were immunoprecipitated (IP) with 14-3-3 isoform-specific antibodies or antibody K19 and coprecipitated p53 was detected by immunoblotting (IB). As a control, buffer only (Cells -) was used instead of the cell lysates. C, specificity of 14-3-3 isoform-specific antibodies. GST/14-3-3 isoforms were immunoblotted with the isoform-specific antibodies or with antibody K19, which recognizes all 14-3-3 isoforms.

 
In vivo, the isoform specificity of the interaction between p53 and 14-3-3 was examined by coimmunoprecipitation analysis of the endogenous wild-type p53 and 14-3-3 proteins using 14-3-3 isoform-specific antibodies and lysates prepared from U2OS osteosarcoma cells that were either not irradiated or exposed to IR or UV light. p53 and 14-3-3 interact in cells exposed to IR but not in nonirradiated cells or in cells exposed to UV light (10) . Thus, for each 14-3-3 isoform we had a negative control. The {gamma}, {epsilon}, and {tau} isoforms exhibited an IR-specific interaction with wild-type p53, whereas no interaction could be detected with the {sigma} and {zeta} isoforms (Fig. 1B)Citation . The K19 antibody, which recognizes all 14-3-3 isoforms, served as a positive control (Fig. 1B)Citation , and the isoform-specificity of the antibodies, which were obtained commercially, was verified by immunoblotting using recombinant GST/14-3-3 fusion proteins (Fig. 1C)Citation .

Functional Significance of the p53/14-3-3 Interaction.
To examine the functional significance of the p53/14-3-3 interaction, we assembled a panel of COOH-terminal p53 mutants that interacted with 14-3-3 proteins to varying degrees and examined their function in tissue culture cells. The panel consisted of p53 mutants with single substitutions of Ser376 to Ala (A376), Thr377 to Ala (A377) or Ser378 to Ala (A378) and a p53 mutant with all of these three amino acid substitutions (A376–8). These mutants were selected because the interaction between p53 and 14-3-3 in vitro and in vivo is regulated by the phosphorylation states of Ser376 and Ser378 (16) .

The functional properties of the COOH-terminal p53 mutants were first examined in U2OS osteosarcoma cells, which have been used extensively to study p53 activation in response to DNA damage (7 , 10 , 16) . Because U2OS cells express wild-type p53, the COOH-terminal p53 mutants were modified in two ways (Fig. 2A)Citation : (a) an NH2-terminal HA tag was inserted to distinguish them from endogenous p53; and (b) seven amino acid substitutions were introduced in the tetramerization domain. The modified domain, hereafter referred to as IND (independent), allows the COOH-terminal p53 mutants to form tetramers but prevents hetero-oligomerization with endogenous p53 (20) . Transient transfection of U2OS cells with 0.1 µg of plasmid DNA encoding HA-tagged p53IND with a wild-type (p53INDwt) or mutant (p53INDA376, A377, A378, and A376–8) COOH terminus led to low levels of p53 protein expression, which increased significantly in response to IR or UV light (data not shown; Ref. 7 ). The DNA damage-induced p53 stabilization was a handicap for this study, which focuses on regulation of p53 functional activity. However, transfecting the cells with 2.5 µg of plasmid DNA led to higher levels of p53 protein, which did not increase further in response to DNA damage (Fig. 2B)Citation , allowing us to study the effects of DNA damage on p53 activity independently of its effects on p53 protein levels.



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  		Fig. 2. Interaction of p53 COOH-terminal mutants with 14-3-3 proteins in U2OS cells. A, diagrammatic representations of wild-type p53 (p53wt) and p53IND. HA, hemagglutinin tag; Tx, transactivation domain; DNA-B, sequence-specific DNA binding domain; 4, native tetramerization domain; IND, modified independent tetramerization domain; R, COOH-terminal regulatory region, where the alanine substitutions targeting residues 376–378 map. B, protein levels of p53INDwt in mock-irradiated (0 Gy) and irradiated (5 Gy) U2OS cells. IB, immunoblot. C, interaction between p53INDwt and endogenous 14-3-3 in mock-irradiated and irradiated U2OS cells. IP Ab, immunoprecipitation antibody; IB, immunoblot. D, protein levels of p53INDwt and COOH-terminal p53IND mutants in irradiated (5 Gy) U2OS cells. Note that the COOH-terminal mutants with Ala at position 376, unlike p53INDwt and the rest of the mutants, do not migrate as doublets. The basis for this difference in electrophoretic migration is not understood. IB, immunoblot. E, interaction between p53INDwt and COOH-terminal p53IND mutants with endogenous 14-3-3 in irradiated U2OS cells. IB, immunoblot.

 
The ectopically expressed p53INDwt protein behaved similarly to endogenous wild-type p53 in that it interacted with 14-3-3 proteins only in cells exposed to IR (Fig. 2C)Citation . The COOH-terminal substitutions had no effect on p53 protein expression (Fig. 2D)Citation , but as expected, some of the substitutions interfered with the ability of p53 to interact with 14-3-3 proteins (Fig. 2E)Citation . Specifically, the interaction of p53 with 14-3-3 was abrogated by substitution of Ser376, unaffected by substitution of Thr377 and weakened by substitution of Ser378. Thus, these mutants, which differ in their ability to associate with 14-3-3 proteins in vivo, were used to study the functional significance of the p53/14-3-3 interaction.

Sequence-specific DNA binding was examined by transfecting the panel of p53 COOH-terminal mutants in U2OS cells, preparing cell lysates, and analyzing the DNA binding activities of the ectopically expressed p53 proteins in these lysates. The lysates were prepared 1 h after exposure of the cells to IR or from mock-irradiated cells. The HA-tagged p53IND proteins were examined for their ability to bind to beads coated with oligonucleotides containing the specific p53 DNA binding site or a nonspecific DNA site. p53INDwt bound to beads coated with the specific oligonucleotide but not to beads coated with the nonspecific oligonucleotide, establishing the sequence specificity of the assay (Fig. 3A)Citation . Exposure of the cells to IR did not affect the sequence-specific DNA binding activity. Furthermore, the p53IND proteins with mutant 14-3-3 binding sites bound the specific DNA as efficiently as p53INDwt (Fig. 3B)Citation . Thus, the association of p53 with 14-3-3 proteins did not affect the sequence-specific DNA binding activity of p53 in this assay.



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  		Fig. 3. Sequence-specific DNA binding of wild-type and mutant p53 proteins expressed in U2OS cells by transient transfection. A, cell lysates containing HA-tagged p53INDwt prepared from nonirradiated (0 Gy) or irradiated (5 Gy) cells were incubated with beads coated with oligonucleotides containing the specific (Sp) p53 binding site or a nonspecific (NS) site. Bound p53 was detected by immunoblotting. B, cell lysates containing HA-tagged p53IND proteins with wild-type or mutant COOH termini were incubated with beads coated with oligonucleotides containing the specific (Sp) p53 binding site. Bound p53 was detected by immunoblotting.

 
We subsequently examined the ability of the p53 COOH-terminal mutants to activate expression of the endogenous p21/cip1/waf1 gene. For these studies, expression of the endogenous wild-type p53 protein in U2OS cells complicated the analysis, because only a subset of the transfected cells actually expressed the p53 COOH-terminal mutants, and both the endogenous and ectopically expressed p53 could contribute to p21/cip1/waf1 expression. We therefore decided to pursue analysis of the transcriptional activity of the p53 COOH-terminal mutants in Saos2 osteosarcoma cells, which do not express endogenous p53. The mutants interacted with endogenous 14-3-3 in irradiated Saos2 cells as described above for U2OS cells (Fig. 4A)Citation , and their ability to activate transcription of the endogenous p21/cip1/waf1 gene correlated with their ability to interact with 14-3-3 (Fig. 4B)Citation . In contrast, all p53 COOH-terminal mutants activated transcription from a reporter plasmid (Fig. 4C)Citation , suggesting that the effect of 14-3-3 on p53 transcriptional activity is not evident in transient reporter assays.



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  		Fig. 4. Transcriptional activities of wild-type p53 and p53 COOH-terminal mutants in Saos2 cells. A, interaction of p53 COOH-terminal mutants with endogenous 14-3-3 proteins in irradiated Saos2 cells. IB, immunoblot. B, transactivation of the endogenous p21/cip1/waf1 gene, as indicated by determining the levels of p21/waf1/cip1 protein in cells transiently transfected with the indicated p53 proteins. IB, immunoblot. C, transcriptional activities of the same p53 mutants using a reporter plasmid that contains the p53 binding site present in the p21/waf1/cip1 gene. The tumor-derived p53 mutant p53W248 serves as a negative control.

 
The ability of the p53 COOH-terminal mutants to induce cell cycle arrest was studied in U2OS osteosarcoma cells. The research design involved coexpressing three proteins in transiently transfected cells: green fluorescent protein to mark the efficiently transfected cells; a dominant-negative, tumor-derived p53 mutant to inactivate endogenous wild-type p53; and one of the p53IND proteins described above. Parental (nontransfected) U2OS cells arrested predominantly in G2 after exposure to IR, whereas cells expressing p53INDwt showed a significant fraction of cells arresting in G1 (Fig. 5)Citation . The COOH-terminal mutants that did not interact with 14-3-3 proteins were unable to induce G1 arrest; p53INDA378, which interacted weakly with 14-3-3 proteins, induced partial G1 arrest; and p53INDA377, which interacted strongly with 14-3-3 proteins, induced G1 arrest as efficiently as p53INDwt (Fig. 5)Citation .



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  		Fig. 5. Cell cycle arrest activities of wild-type p53 and p53 COOH-terminal mutants in U2OS cells. The cells were either not transfected or transfected with plasmids encoding the indicated proteins. All transfected cells express a tumor-derived p53 mutant (p53W248), and some transfected cells additionally express a p53IND protein, as indicated. The cells were either mock-irradiated (0 Gy) or exposed to 5 Gy IR.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The first question that we addressed in this study is that of isoform specificity in the interaction between p53 and 14-3-3. There are seven distinct genes encoding 14-3-3 isoforms in mammals, and these are highly related to each other at the amino acid sequence level. Yet, despite the high similarity, the isoforms differ significantly in their affinity for specific ligands (21) . Indeed, we observed that in vitro the {gamma} isoform bound p53 with significantly higher affinity than the other isoforms. In vivo, we observed an interaction of p53 with 14-3-3 {gamma} but also with isoforms {epsilon} and {tau}. This could reflect different posttranslational modifications of p53 or 14-3-3 in vitro and in vivo, differences in the relative abundance of the 14-3-3 isoforms in vivo, and/or heterodimerization of different 14-3-3 isoforms in vivo. Interestingly, we did not observe a significant interaction between p53 and 14-3-3 {sigma}. If these two proteins interacted, then we would have the potential for a positive feedback loop leading to p53 activation, because p53 induces expression of the 14-3-3 {sigma} gene (4) and binding of 14-3-3 to p53 enhances its functional activity.

The second question that we wanted to address is whether the interaction between p53 and 14-3-3 proteins is functionally significant. Analysis of the function of p53 mutants that are defective in their ability to interact with 14-3-3 suggests that 14-3-3 enhances p53 function. However, we cannot exclude the possibility that the substitution we introduced affected not only the interaction of p53 with 14-3-3 but also some other p53-protein interaction or some p53 posttranslational modification. Thus, it is formally possible that the functional defects were not attributable to the disruption of the p53/14-3-3 interaction. Nevertheless, we think that the p53/14-3-3 interaction is functionally important, because p53/14-3-3 binding and p53 activity correlated well in our panel of p53 mutants. The mechanism by which 14-3-3 proteins could enhance p53 function remains elusive. We used an ex vivo DNA binding assay, and it appears that 14-3-3 proteins do not affect the sequence-specific DNA binding activity of p53. However, this needs to be examined more carefully using chromosome-immunoprecipitation assays. 14-3-3 proteins also did not appear to affect the intracellular localization of p53 (data not shown). Instead, the results raise the possibility that 14-3-3 enhances the transcriptional activity of p53. Interestingly, a similar role for 14-3-3 proteins has been proposed in plant cells, where 14-3-3 proteins have been shown to facilitate interaction of sequence-specific DNA binding transcription factors with the basal transcription machinery (22) . Further analysis of the p53 mutants described in this study may help elucidate the mechanisms by which p53 exerts its tumor suppressor effect.


   ACKNOWLEDGMENTS
 
We thank Bert Vogelstein for the gift of the antibody that recognizes 14-3-3 {sigma}. We also thank Alastair Aitken, Philip Leder, Jules Shafer, Giovanni Rovera, and Clayton Buck for support and helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grant DAMD17-99-1-9455 provided by the Department of Defense and Grant CA76367 from the National Cancer Institute. N. H. C. was supported by Wistar Institute NIH Training Grant CA09171. Back

2 These authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104-4268. E-mail: halazonetis{at}wistar.upenn.edu Back

4 The abbreviations used are: GST, glutathione S-transferase; IR, ionizing radiation; HA, hemagglutinin. Back

Received 6/14/01. Accepted 8/15/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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Y. Pereg, S. Lam, A. Teunisse, S. Biton, E. Meulmeester, L. Mittelman, G. Buscemi, K. Okamoto, Y. Taya, Y. Shiloh, et al.
Differential Roles of ATM- and Chk2-Mediated Phosphorylations of Hdmx in Response to DNA Damage.
Mol. Cell. Biol., September 1, 2006; 26(18): 6819 - 6831.
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D. Bridges and G. B. G. Moorhead
14-3-3 Proteins: A Number of Functions for a Numbered Protein
Sci. Signal., August 9, 2005; 2005(296): re10 - re10.
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A. Benzinger, N. Muster, H. B. Koch, J. R. Yates III, and H. Hermeking
Targeted Proteomic Analysis of 14-3-3{varsigma}, a p53 Effector Commonly Silenced in Cancer
Mol. Cell. Proteomics, June 1, 2005; 4(6): 785 - 795.
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J. M. A. Moreira, G. Ohlsson, F. E. Rank, and J. E. Celis
Down-regulation of the Tumor Suppressor Protein 14-3-3{sigma} Is a Sporadic Event in Cancer of the Breast
Mol. Cell. Proteomics, April 1, 2005; 4(4): 555 - 569.
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S. E. M. Meek, W. S. Lane, and H. Piwnica-Worms
Comprehensive Proteomic Analysis of Interphase and Mitotic 14-3-3-binding Proteins
J. Biol. Chem., July 30, 2004; 279(31): 32046 - 32054.
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D. Bridges and G. B. G. Moorhead
14-3-3 Proteins: A Number of Functions for a Numbered Protein
Sci. Signal., July 20, 2004; 2004(242): re10 - re10.
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E. S. Stavridi and T. D. Halazonetis
p53 and stress in the ER
Genes & Dev., February 1, 2004; 18(3): 241 - 244.
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H.-Y. Yang, Y.-Y. Wen, C.-H. Chen, G. Lozano, and M.-H. Lee
14-3-3{sigma} Positively Regulates p53 and Suppresses Tumor Growth
Mol. Cell. Biol., October 15, 2003; 23(20): 7096 - 7107.
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