Physical and Functional Interaction between p53 Mutants and Different Isoforms of p73
2000; Elsevier BV; Volume: 275; Issue: 38 Linguagem: Inglês
10.1074/jbc.m003360200
ISSN1083-351X
AutoresSabrina Strano, Eliana Munarriz, Mario Rossi, Barbara Cristofanelli, Yosef Shaul, Luisa Castagnoli, Arnold J. Levine, Ada Sacchi, Gianni Cesareni, Moshe Oren, Giovanni Blandino,
Tópico(s)Nanoplatforms for cancer theranostics
Resumop53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro andin vivo with p73α, β, γ, and δ. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage. p53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro andin vivo with p73α, β, γ, and δ. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage. fetal calf serum cytomegalovirus green fluorescent protein glutathione S-transferase hemagglutinin monoclonal antibody oligomerization domain. PAGE, polyacrylamide gel electrophoresis The p53 tumor suppressor gene is the most frequent target for genetic alterations in human cancers (1Hollstein M. Soussi T. Thomas G. von Brevern M. Bartsch H. Rec. Res. Cancer Res. 1997; 143: 369-389Crossref PubMed Scopus (55) Google Scholar). The wild type p53 protein can elicit a variety of biological effects, ranging from growth arrest to apoptosis and differentiation (2Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6698) Google Scholar, 3Hansen R. Oren M. Curr. Opin. Genet. Dev. 1997; 7: 46-51Crossref PubMed Scopus (206) Google Scholar, 4Almog N. Rotter V. Biochim. Biophys. Acta. 1998; 1378: R43-R54PubMed Google Scholar). These effects are mainly exerted by wild-type p53 through the activation of a growing plethora of p53-responsive target genes (5Oren M. J. Biol. Chem. 1999; 274: 36031-36034Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). In human cancers, the most prevalent type of p53 mutations consists of missense mutations, often within the highly conserved DNA binding core domain of the protein (2Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6698) Google Scholar,3Hansen R. Oren M. Curr. Opin. Genet. Dev. 1997; 7: 46-51Crossref PubMed Scopus (206) Google Scholar). One certain outcome of those mutations may be the elimination of cellular wild-type p53 activity. However, at variance with other tumor suppressor genes, cells with p53 mutations typically maintain expression of full-length protein. This may suggest that at least certain mutant forms of p53 can contribute actively to cancer progression through gain of function activity (6Michalovitz D. Haley O. Oren M. J. Cell. Biochem. 1991; 45: 22-29Crossref PubMed Scopus (99) Google Scholar, 7Dittmer D. Pati S. Zambetti G. Chu S. Tereseky K. Moore M. Finlay C. Levine A.J. Nat. Genet. 1993; 4: 42-46Crossref PubMed Scopus (791) Google Scholar, 8Gualberto A. Aldape K. Kozakiewicz K. Tlsty T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5166-5171Crossref PubMed Scopus (204) Google Scholar, 9Li R. Sutphin D.P. Schwartz D. Matas D. Almog N. Wolkowicz R. Goldfinger N. Pei H. Prokocimer M. Rotter V. Oncogene. 1998; 16: 3269-3278Crossref PubMed Scopus (124) Google Scholar, 10Fraizer M.W. He X. Wang J. Gu Z. Cleveland J.L. Zambetti G.P. Mol. Cell. Biol. 1998; 18: 3735-3743Crossref PubMed Scopus (174) Google Scholar, 11Blandino G. Levine A.J. Oren M. Oncogene. 1999; 18: 477-485Crossref PubMed Scopus (371) Google Scholar, 12Prives C. Hall P. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1225) Google Scholar).A new gene, p73, sharing considerable sequence homology as well as structural homology with p53, has recently been identified (13Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1533) Google Scholar, 14Jost C. Marin M. Kaelin W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (897) Google Scholar). Similarly to p53, the p73 protein can be roughly divided into three main domains: (a) the N-terminal transactivation domain, which shares 29% homology with the N-terminal part of p53; (b) the sequence-specific DNA binding domain, which shares 63% of homology with the corresponding p53 domain; and (c) the tetramerization domain, which shares 42% homology with the oligomerization domain of p53 (15Oren M. Cell. 1997; 90: 829-832Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 16Kaelin Jr., W.G. J. Natl. Cancer Inst. 1999; 91: 594-598Crossref PubMed Scopus (137) Google Scholar). Furthermore, ectopic expression of p73 can transactivate p53 responsive target genes, and also induces apoptosis in different cell lines (13Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1533) Google Scholar, 14Jost C. Marin M. Kaelin W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (897) Google Scholar). As revealed by the comparison of the p73 and p53 sequences, the DNA binding domains share the highest homology. However, although this domain in p53 is the major site of the mutations, no mutations could be found so far in p73 despite extensive efforts (17Mai M. Yokomizo A. Qian C. Yang P. Tindall D.J. Smith D.I. Liu W. Cancer Res. 1998; 58: 2347-2349PubMed Google Scholar, 18Nomoto S. Haruki N. Kondo M. Konishi H. Takahashi T. Cancer Res. 1998; 58: 1380-1385PubMed Google Scholar, 19Takahashi H. Ichimiya S. Nimura Y. Watanabe M. Furusato M. Wakui S. Yatani R. Aizawa S. Nakagawara A. Cancer Res. 1998; 58: 2076-2077PubMed Google Scholar, 20Yokomizo A. Mai M. Tindall D. Cheng L. Bostwick D.G. Naito S. Smith D.I. Liu W. Oncogene. 1999; 18: 1629-1633Crossref PubMed Scopus (125) Google Scholar, 21Nimura Y. Mihara M. Ichimiya S. Sakiyama S. Seki S. Ohira M. Nomura N. Fujimori M. Adachi W. Amano J. He M. Ping Y.M. Nakagawara A. Int. J. Cancer. 1998; 78: 437-440Crossref PubMed Scopus (82) Google Scholar, 22Kroiss M. Bosserhoff A.K. Vogt T. Buettner R. Bogenrieder T. Landthaler M. Stolz W. Melanoma Res. 1998; 8: 504-509Crossref PubMed Scopus (35) Google Scholar, 23Tsao H. Jhang X. Maieewski P. Haluska F. Cancer Res. 1999; 59: 172-174PubMed Google Scholar). Unlike p53, p73 is not inactivated by viral oncoproteins, such as T-antigen, E6, and E1Bp55, well known inactivators of p53 (24Linzer D.I. Levine A.J. Cell. 1979; 17: 43-52Abstract Full Text PDF PubMed Scopus (1233) Google Scholar, 25Lane D.P. Crawford L.V. Nature. 1979; 278: 261-263Crossref PubMed Scopus (1744) Google Scholar, 26Marin M.C. Jost C.A. De Caprio J.A. Caput D. Kaelin Jr., W.G. Mol. Cell. Biol. 1998; 18: 6316-6324Crossref PubMed Scopus (174) Google Scholar, 27Dobbelstein M. Roth J. J. Gen. Virol. 1998; 79: 3079-3083Crossref PubMed Scopus (55) Google Scholar, 28Prabhu N. Somasundaram K. Satyamoorthy K. Herlyn M. el-Deiry W.S. Int. J. Oncol. 1998; 13: 5-9PubMed Google Scholar). It was originally reported that p73, unlike p53, is not induced upon UV irradiation, highlighting an additional difference from p53 (14Jost C. Marin M. Kaelin W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (897) Google Scholar). More recently, however, it was found that p73 is induced and tyrosine-phosphorylated by exposure of cells to DNA-damaging agents, such cisplatin and γ-radiation, suggesting a differential behavior of p73 in response to different types of DNA damage (29Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (504) Google Scholar, 30Gong J.G. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (830) Google Scholar, 31Yuan Z.M. Shioya H. Ishiko T. Sun X.G. Gu J.J. Huang Y.Y. Lu H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (539) Google Scholar). Of note, different p73 variants exist in the cell, giving rise to a family of proteins that adds a new level of complexity to the understanding of the p73 signaling in cancer cells (13Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1533) Google Scholar, 32De Laurenzi V. Costanzo A. Barcaroli D. Terrinoni A. Falco M. Annichiarico-Petruzzelli M. Levrero M. Melino G. J. Exp. Med. 1998; 188: 1763-1768Crossref PubMed Scopus (361) Google Scholar, 33De Laurenzi V. Catani M.V. Costanzo A. Terrinoni A. Corazzari M. Levrero M. Knight R.A. Melino G. Cell Death Differ. 1999; 6: 389-390Crossref PubMed Scopus (131) Google Scholar). Interestingly, it was recently reported that human tumor-derived p53 mutants can associate with p73α and interfere with its transcriptional activity and ability to induce apoptosis when co-expressed in transient transfection assays (34Di Como C.J. Gaiddon C. Prives C. Mol. Cell. Biol. 1999; 19: 1438-1449Crossref PubMed Scopus (380) Google Scholar).Here we investigate in vitro and in vivo the interaction between human tumor-derived p53 mutants and the various p73 spliced isoforms. We report that (a) the association between mutant p53 and p73 occurs under physiological conditions; (b) two different human tumor-derived p53 mutants (His175 and Gly281) associate with p73 α, β, γ, and δ when co-transfected transiently in H1299 cells; (c) the DNA binding domain of mutant p53 is sufficient for the association with p73 isoforms; (d) a region of p73 that includes the sequence-specific DNA binding and the oligomerization domains is sufficient for the association with mutant p53; (e) p73-tyrosine phosphorylation is dispensable for the association with mutant p53; and (f) human tumor-derived p53 mutants interfere with the transcriptional activity of p73α, β, γ, and δ.DISCUSSIONWe report here that human tumor-derived p53 mutants can engage in a physical association with p73α, β, γ, and δ. Furthermore, this association occurs under physiological conditions. In agreement with the previously reported association between mutant p53 and p73α (34Di Como C.J. Gaiddon C. Prives C. Mol. Cell. Biol. 1999; 19: 1438-1449Crossref PubMed Scopus (380) Google Scholar), our findings contribute to the definition of a network involving mutant p53 and the various p73 isoforms in cancer cells. These data do not exclude the possibility that other human tumor-derived p53 mutants, distinct from those used in our experiments, may be unable to associate with p73 (44Shaulian E. Zauberman D. Ginsberg D. Oren M. Mol. Cell. Biol. 1992; 12: 5581-5592Crossref PubMed Scopus (322) Google Scholar). Several reports have clearly suggested that some human tumor-derived p53 mutants can exert gain of function activity (7Dittmer D. Pati S. Zambetti G. Chu S. Tereseky K. Moore M. Finlay C. Levine A.J. Nat. Genet. 1993; 4: 42-46Crossref PubMed Scopus (791) Google Scholar, 9Li R. Sutphin D.P. Schwartz D. Matas D. Almog N. Wolkowicz R. Goldfinger N. Pei H. Prokocimer M. Rotter V. Oncogene. 1998; 16: 3269-3278Crossref PubMed Scopus (124) Google Scholar,10Fraizer M.W. He X. Wang J. Gu Z. Cleveland J.L. Zambetti G.P. Mol. Cell. Biol. 1998; 18: 3735-3743Crossref PubMed Scopus (174) Google Scholar, 12Prives C. Hall P. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1225) Google Scholar). This activity is dependent on the type of the p53 mutation, as well as on the cell context in which the biological gain of function is measured. We and others have previously reported that conformational mutants such as p53His175, but not DNA contact mutants, can increase the resistance to etoposide or contribute to genomic instability by abrogating the mitotic spindle checkpoint and consequently leading to polyploidy of human cells (8Gualberto A. Aldape K. Kozakiewicz K. Tlsty T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5166-5171Crossref PubMed Scopus (204) Google Scholar, 11Blandino G. Levine A.J. Oren M. Oncogene. 1999; 18: 477-485Crossref PubMed Scopus (371) Google Scholar). We are currently investigating whether association with p73 is a specific property of gain of function p53 mutants. Of note, the finding that the core domain of mutant p53 is sufficient for the association with p73 highlights the potential role of this domain as a module for protein-protein interaction. Further investigation employing yeast two-hybrid screening or an immunoaffinity approach is needed to verify whether the core domain of mutant p53 plays an important role in the gain of function activity. Moreover, in view of the substantial similarity of the core domains between p73 and p63, another member of the p53 family, it is of interest to find out whether p63 can also participate in that network (46Yang A. Kaghad M. Wang Y. Gillet E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1830) Google Scholar, 47Yang A. Schweitzer R. Sun D. Kaghad M. Walker N. Bronson R.T. Tabin C. Sharpe A. Caput D. Crum C. Mckeon F. Nature. 1999; 398: 714-718Crossref PubMed Scopus (1891) Google Scholar, 48Mills A.A. Zheng B. Wang X.-J. Vogel H. Roop D.R. Bradley A. Nature. 1999; 398: 708-713Crossref PubMed Scopus (1688) Google Scholar, 49Kaelin Jr., W.G. Oncogene. 1999; 18: 7701-7705Crossref PubMed Scopus (158) Google Scholar). It is conceivable that other proteins interacting either with mutant p53 or with p73 might also interfere with the biological outcome of the entire network (29Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (504) Google Scholar, 30Gong J.G. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (830) Google Scholar, 31Yuan Z.M. Shioya H. Ishiko T. Sun X.G. Gu J.J. Huang Y.Y. Lu H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (539) Google Scholar, 50Higashino F. Pipas M.J. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15683-15687Crossref PubMed Scopus (50) Google Scholar, 51Zeng X. Chen L. Jost C. Maya R. Keller D. Wang X. Kaelin Jr., W.G. Oren M. Chen J. Lu H. Mol. Cell. Biol. 1998; 19: 3257-3266Crossref Scopus (303) Google Scholar, 52Balint E. Bates S. Vousden K.H. Oncogene. 1999; 18: 3923-3929Crossref PubMed Scopus (194) Google Scholar, 53Zeng X. Li X. Miller A. Yuan Z. Yuan W. Kwok R.P.S. Goodman R. Lu H. Mol. Cell. Biol. 2000; 20: 1299-1310Crossref PubMed Scopus (82) Google Scholar, 54Scharnhorst V. Dekker P. van der Eb A.J. Jochensen A.G. J. Biol. Chem. 2000; 275: 10202-10211Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar).The studies performed to date suggest that p73 is rarely mutated in human cancers and that ectopic expression of p73 induces apoptosis in cancer cells. Thus, agents that increase p73 expression may provide new potential anticancer treatments. Conversely, proteins inactivating or interfering with p73 might have an impact upon cellular properties, clinical responses to therapy, and prognosis of a tumor. Our data, showing that human tumor-derived p53 mutants can interact with p73 not only physically but also functionally, might implicate mutant p53 as a likely candidate for such type of proteins. Along this line, an intriguing question can be raised: how does mutant p53 inactivate p73? In accordance with our novel finding that both the core and the oligomerization domains of p73 are involved in the association with mutant p53, we might depict two possible scenarios. On the one hand, mutant p53 binding the oligomerization domain of p73 can interfere with the formation of p73 homo-oligomers. On the other hand, mutant p53 binding the core domain of p73 can interfere with its binding to DNA.The recent observation that p73-deficient mice do not exhibit an increase in spontaneous tumors suggests that the association between mutant p53 and p73 might have an impact mainly upon tumor chemoresistance rather than on tumor development (55Yang A. Walker N. Bronson R. Kaghad M. Oosterwegel M. Bonnin J. Vagner C. Bonnet H. Dikkes P. Sharpe A. McKeon F. Caput D. Nature. 2000; 404: 99-103Crossref PubMed Scopus (878) Google Scholar). If this prediction is proven correct, one would expect that cells from p73-deficient mice will be less prone to killing by anticancer drugs than their wild type counterparts.The establishment of a precise role for the association between mutant p53 and p73 might be very useful for anticancer treatment. To this end, further evidence needs to be collected. The p53 tumor suppressor gene is the most frequent target for genetic alterations in human cancers (1Hollstein M. Soussi T. Thomas G. von Brevern M. Bartsch H. Rec. Res. Cancer Res. 1997; 143: 369-389Crossref PubMed Scopus (55) Google Scholar). The wild type p53 protein can elicit a variety of biological effects, ranging from growth arrest to apoptosis and differentiation (2Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6698) Google Scholar, 3Hansen R. Oren M. Curr. Opin. Genet. Dev. 1997; 7: 46-51Crossref PubMed Scopus (206) Google Scholar, 4Almog N. Rotter V. Biochim. Biophys. Acta. 1998; 1378: R43-R54PubMed Google Scholar). These effects are mainly exerted by wild-type p53 through the activation of a growing plethora of p53-responsive target genes (5Oren M. J. Biol. Chem. 1999; 274: 36031-36034Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). In human cancers, the most prevalent type of p53 mutations consists of missense mutations, often within the highly conserved DNA binding core domain of the protein (2Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6698) Google Scholar,3Hansen R. Oren M. Curr. Opin. Genet. Dev. 1997; 7: 46-51Crossref PubMed Scopus (206) Google Scholar). One certain outcome of those mutations may be the elimination of cellular wild-type p53 activity. However, at variance with other tumor suppressor genes, cells with p53 mutations typically maintain expression of full-length protein. This may suggest that at least certain mutant forms of p53 can contribute actively to cancer progression through gain of function activity (6Michalovitz D. Haley O. Oren M. J. Cell. Biochem. 1991; 45: 22-29Crossref PubMed Scopus (99) Google Scholar, 7Dittmer D. Pati S. Zambetti G. Chu S. Tereseky K. Moore M. Finlay C. Levine A.J. Nat. Genet. 1993; 4: 42-46Crossref PubMed Scopus (791) Google Scholar, 8Gualberto A. Aldape K. Kozakiewicz K. Tlsty T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5166-5171Crossref PubMed Scopus (204) Google Scholar, 9Li R. Sutphin D.P. Schwartz D. Matas D. Almog N. Wolkowicz R. Goldfinger N. Pei H. Prokocimer M. Rotter V. Oncogene. 1998; 16: 3269-3278Crossref PubMed Scopus (124) Google Scholar, 10Fraizer M.W. He X. Wang J. Gu Z. Cleveland J.L. Zambetti G.P. Mol. Cell. Biol. 1998; 18: 3735-3743Crossref PubMed Scopus (174) Google Scholar, 11Blandino G. Levine A.J. Oren M. Oncogene. 1999; 18: 477-485Crossref PubMed Scopus (371) Google Scholar, 12Prives C. Hall P. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1225) Google Scholar). A new gene, p73, sharing considerable sequence homology as well as structural homology with p53, has recently been identified (13Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1533) Google Scholar, 14Jost C. Marin M. Kaelin W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (897) Google Scholar). Similarly to p53, the p73 protein can be roughly divided into three main domains: (a) the N-terminal transactivation domain, which shares 29% homology with the N-terminal part of p53; (b) the sequence-specific DNA binding domain, which shares 63% of homology with the corresponding p53 domain; and (c) the tetramerization domain, which shares 42% homology with the oligomerization domain of p53 (15Oren M. Cell. 1997; 90: 829-832Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 16Kaelin Jr., W.G. J. Natl. Cancer Inst. 1999; 91: 594-598Crossref PubMed Scopus (137) Google Scholar). Furthermore, ectopic expression of p73 can transactivate p53 responsive target genes, and also induces apoptosis in different cell lines (13Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1533) Google Scholar, 14Jost C. Marin M. Kaelin W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (897) Google Scholar). As revealed by the comparison of the p73 and p53 sequences, the DNA binding domains share the highest homology. However, although this domain in p53 is the major site of the mutations, no mutations could be found so far in p73 despite extensive efforts (17Mai M. Yokomizo A. Qian C. Yang P. Tindall D.J. Smith D.I. Liu W. Cancer Res. 1998; 58: 2347-2349PubMed Google Scholar, 18Nomoto S. Haruki N. Kondo M. Konishi H. Takahashi T. Cancer Res. 1998; 58: 1380-1385PubMed Google Scholar, 19Takahashi H. Ichimiya S. Nimura Y. Watanabe M. Furusato M. Wakui S. Yatani R. Aizawa S. Nakagawara A. Cancer Res. 1998; 58: 2076-2077PubMed Google Scholar, 20Yokomizo A. Mai M. Tindall D. Cheng L. Bostwick D.G. Naito S. Smith D.I. Liu W. Oncogene. 1999; 18: 1629-1633Crossref PubMed Scopus (125) Google Scholar, 21Nimura Y. Mihara M. Ichimiya S. Sakiyama S. Seki S. Ohira M. Nomura N. Fujimori M. Adachi W. Amano J. He M. Ping Y.M. Nakagawara A. Int. J. Cancer. 1998; 78: 437-440Crossref PubMed Scopus (82) Google Scholar, 22Kroiss M. Bosserhoff A.K. Vogt T. Buettner R. Bogenrieder T. Landthaler M. Stolz W. Melanoma Res. 1998; 8: 504-509Crossref PubMed Scopus (35) Google Scholar, 23Tsao H. Jhang X. Maieewski P. Haluska F. Cancer Res. 1999; 59: 172-174PubMed Google Scholar). Unlike p53, p73 is not inactivated by viral oncoproteins, such as T-antigen, E6, and E1Bp55, well known inactivators of p53 (24Linzer D.I. Levine A.J. Cell. 1979; 17: 43-52Abstract Full Text PDF PubMed Scopus (1233) Google Scholar, 25Lane D.P. Crawford L.V. Nature. 1979; 278: 261-263Crossref PubMed Scopus (1744) Google Scholar, 26Marin M.C. Jost C.A. De Caprio J.A. Caput D. Kaelin Jr., W.G. Mol. Cell. Biol. 1998; 18: 6316-6324Crossref PubMed Scopus (174) Google Scholar, 27Dobbelstein M. Roth J. J. Gen. Virol. 1998; 79: 3079-3083Crossref PubMed Scopus (55) Google Scholar, 28Prabhu N. Somasundaram K. Satyamoorthy K. Herlyn M. el-Deiry W.S. Int. J. Oncol. 1998; 13: 5-9PubMed Google Scholar). It was originally reported that p73, unlike p53, is not induced upon UV irradiation, highlighting an additional difference from p53 (14Jost C. Marin M. Kaelin W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (897) Google Scholar). More recently, however, it was found that p73 is induced and tyrosine-phosphorylated by exposure of cells to DNA-damaging agents, such cisplatin and γ-radiation, suggesting a differential behavior of p73 in response to different types of DNA damage (29Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (504) Google Scholar, 30Gong J.G. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (830) Google Scholar, 31Yuan Z.M. Shioya H. Ishiko T. Sun X.G. Gu J.J. Huang Y.Y. Lu H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (539) Google Scholar). Of note, different p73 variants exist in the cell, giving rise to a family of proteins that adds a new level of complexity to the understanding of the p73 signaling in cancer cells (13Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1533) Google Scholar, 32De Laurenzi V. Costanzo A. Barcaroli D. Terrinoni A. Falco M. Annichiarico-Petruzzelli M. Levrero M. Melino G. J. Exp. Med. 1998; 188: 1763-1768Crossref PubMed Scopus (361) Google Scholar, 33De Laurenzi V. Catani M.V. Costanzo A. Terrinoni A. Corazzari M. Levrero M. Knight R.A. Melino G. Cell Death Differ. 1999; 6: 389-390Crossref PubMed Scopus (131) Google Scholar). Interestingly, it was recently reported that human tumor-derived p53 mutants can associate with p73α and interfere with its transcriptional activity and ability to induce apoptosis when co-expressed in transient transfection assays (34Di Como C.J. Gaiddon C. Prives C. Mol. Cell. Biol. 1999; 19: 1438-1449Crossref PubMed Scopus (380) Google Scholar). Here we investigate in vitro and in vivo the interaction between human tumor-derived p53 mutants and the various p73 spliced isoforms. We report that (a) the association between mutant p53 and p73 occurs under physiological conditions; (b) two different human tumor-derived p53 mutants (His175 and Gly281) associate with p73 α, β, γ, and δ when co-transfected transiently in H1299 cells; (c) the DNA binding domain of mutant p53 is sufficient for the association with p73 isoforms; (d) a region of p73 that includes the sequence-specific DNA binding and the oligomerization domains is sufficient for the association with mutant p53; (e) p73-tyrosine phosphorylation is dispensable for the association with mutant p53; and (f) human tumor-derived p53 mutants interfere with the transcriptional activity of p73α, β, γ, and δ. DISCUSSIONWe report here that human tumor-derived p53 mutants can engage in a physical association with p73α, β, γ, and δ. Furthermore, this association occurs under physiological conditions. In agreement with the previously reported association between mutant p53 and p73α (34Di Como C.J. Gaiddon C. Prives C. Mol. Cell. Biol. 1999; 19: 1438-1449Crossref PubMed Scopus (380) Google Scholar), our findings contribute to the definition of a network involving mutant p53 and the various p73 isoforms in cancer cells. These data do not exclude the possibility that other human tumor-derived p53 mutants, distinct from those used in our experiments, may be unable to associate with p73 (44Shaulian E. Zauberman D. Ginsberg D. Oren M. Mol. Cell. Biol. 1992; 12: 5581-5592Crossref PubMed Scopus (322) Google Scholar). Several reports have clearly suggested that some human tumor-derived p53 mutants can exert gain of function activity (7Dittmer D. Pati S. Zambetti G. Chu S. Tereseky K. Moore M. Finlay C. Levine A.J. Nat. Genet. 1993; 4: 42-46Crossref PubMed Scopus (791) Google Scholar, 9Li R. Sutphin D.P. Schwartz D. Matas D. Almog N. Wolkowicz R. Goldfinger N. Pei H. Prokocimer M. Rotter V. Oncogene. 1998; 16: 3269-3278Crossref PubMed Scopus (124) Google Scholar,10Fraizer M.W. He X. Wang J. Gu Z. Cleveland J.L. Zambetti G.P. Mol. Cell. Biol. 1998; 18: 3735-3743Crossref PubMed Scopus (174) Google Scholar, 12Prives C. Hall P. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1225) Google Scholar). This activity is dependent on the type of the p53 mutation, as well as on the cell context in which the biological gain of function is measured. We and others have previously reported that conformational mutants such as p53His175, but not DNA contact mutants, can increase the resistance to etoposide or contribute to genomic instability by abrogating the mitotic spindle checkpoint and consequently leading to polyploidy of human cells (8Gualberto A. Aldape K. Kozakiewicz K. Tlsty T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5166-5171Crossref PubMed Scopus (204) Google Scholar, 11Blandino G. Levine A.J. Oren M. Oncogene. 1999; 18: 477-485Crossref PubMed Scopus (371) Google Scholar). We are currently investigating whether association with p73 is a specific property of gain of function p53 mutants. Of note, the finding that the core domain of mutant p53 is sufficient for the association with p73 highlights the potential role of this domain as a module for protein-protein interaction. Further investigation employing yeast two-hybrid screening or an immunoaffinity approach is needed to verify whether the core domain of mutant p53 plays an important role in the gain of function activity. Moreover, in view of the substantial similarity of the core domains between p73 and p63, another member of the p53 family, it is of interest to find out whether p63 can also participate in that network (46Yang A. Kaghad M. Wang Y. Gillet E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1830) Google Scholar, 47Yang A. Schweitzer R. Sun D. Kaghad M. Walker N. Bronson R.T. Tabin C. Sharpe A. Caput D. Crum C. Mckeon F. Nature. 1999; 398: 714-718Crossref PubMed Scopus (1891) Google Scholar, 48Mills A.A. Zheng B. Wang X.-J. Vogel H. Roop D.R. Bradley A. Nature. 1999; 398: 708-713Crossref PubMed Scopus (1688) Google Scholar, 49Kaelin Jr., W.G. Oncogene. 1999; 18: 7701-7705Crossref PubMed Scopus (158) Google Scholar). It is conceivable that other proteins interacting either with mutant p53 or with p73 might also interfere with the biological outcome of the entire network (29Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (504) Google Scholar, 30Gong J.G. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (830) Google Scholar, 31Yuan Z.M. Shioya H. Ishiko T. Sun X.G. Gu J.J. Huang Y.Y. Lu H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (539) Google Scholar, 50Higashino F. Pipas M.J. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15683-15687Crossref PubMed Scopus (50) Google Scholar, 51Zeng X. Chen L. Jost C. Maya R. Keller D. Wang X. Kaelin Jr., W.G. Oren M. Chen J. Lu H. Mol. Cell. Biol. 1998; 19: 3257-3266Crossref Scopus (303) Google Scholar, 52Balint E. Bates S. Vousden K.H. Oncogene. 1999; 18: 3923-3929Crossref PubMed Scopus (194) Google Scholar, 53Zeng X. Li X. Miller A. Yuan Z. Yuan W. Kwok R.P.S. Goodman R. Lu H. Mol. Cell. Biol. 2000; 20: 1299-1310Crossref PubMed Scopus (82) Google Scholar, 54Scharnhorst V. Dekker P. van der Eb A.J. Jochensen A.G. J. Biol. Chem. 2000; 275: 10202-10211Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar).The studies performed to date suggest that p73 is rarely mutated in human cancers and that ectopic expression of p73 induces apoptosis in cancer cells. Thus, agents that increase p73 expression may provide new potential anticancer treatments. Conversely, proteins inactivating or interfering with p73 might have an impact upon cellular properties, clinical responses to therapy, and prognosis of a tumor. Our data, showing that human tumor-derived p53 mutants can interact with p73 not only physically but also functionally, might implicate mutant p53 as a likely candidate for such type of proteins. Along this line, an intriguing question can be raised: how does mutant p53 inactivate p73? In accordance with our novel finding that both the core and the oligomerization domains of p73 are involved in the association with mutant p53, we might depict two possible scenarios. On the one hand, mutant p53 binding the oligomerization domain of p73 can interfere with the formation of p73 homo-oligomers. On the other hand, mutant p53 binding the core domain of p73 can interfere with its binding to DNA.The recent observation that p73-deficient mice do not exhibit an increase in spontaneous tumors suggests that the association between mutant p53 and p73 might have an impact mainly upon tumor chemoresistance rather than on tumor development (55Yang A. Walker N. Bronson R. Kaghad M. Oosterwegel M. Bonnin J. Vagner C. Bonnet H. Dikkes P. Sharpe A. McKeon F. Caput D. Nature. 2000; 404: 99-103Crossref PubMed Scopus (878) Google Scholar). If this prediction is proven correct, one would expect that cells from p73-deficient mice will be less prone to killing by anticancer drugs than their wild type counterparts.The establishment of a precise role for the association between mutant p53 and p73 might be very useful for anticancer treatment. To this end, further evidence needs to be collected. We report here that human tumor-derived p53 mutants can engage in a physical association with p73α, β, γ, and δ. Furthermore, this association occurs under physiological conditions. In agreement with the previously reported association between mutant p53 and p73α (34Di Como C.J. Gaiddon C. Prives C. Mol. Cell. Biol. 1999; 19: 1438-1449Crossref PubMed Scopus (380) Google Scholar), our findings contribute to the definition of a network involving mutant p53 and the various p73 isoforms in cancer cells. These data do not exclude the possibility that other human tumor-derived p53 mutants, distinct from those used in our experiments, may be unable to associate with p73 (44Shaulian E. Zauberman D. Ginsberg D. Oren M. Mol. Cell. Biol. 1992; 12: 5581-5592Crossref PubMed Scopus (322) Google Scholar). Several reports have clearly suggested that some human tumor-derived p53 mutants can exert gain of function activity (7Dittmer D. Pati S. Zambetti G. Chu S. Tereseky K. Moore M. Finlay C. Levine A.J. Nat. Genet. 1993; 4: 42-46Crossref PubMed Scopus (791) Google Scholar, 9Li R. Sutphin D.P. Schwartz D. Matas D. Almog N. Wolkowicz R. Goldfinger N. Pei H. Prokocimer M. Rotter V. Oncogene. 1998; 16: 3269-3278Crossref PubMed Scopus (124) Google Scholar,10Fraizer M.W. He X. Wang J. Gu Z. Cleveland J.L. Zambetti G.P. Mol. Cell. Biol. 1998; 18: 3735-3743Crossref PubMed Scopus (174) Google Scholar, 12Prives C. Hall P. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1225) Google Scholar). This activity is dependent on the type of the p53 mutation, as well as on the cell context in which the biological gain of function is measured. We and others have previously reported that conformational mutants such as p53His175, but not DNA contact mutants, can increase the resistance to etoposide or contribute to genomic instability by abrogating the mitotic spindle checkpoint and consequently leading to polyploidy of human cells (8Gualberto A. Aldape K. Kozakiewicz K. Tlsty T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5166-5171Crossref PubMed Scopus (204) Google Scholar, 11Blandino G. Levine A.J. Oren M. Oncogene. 1999; 18: 477-485Crossref PubMed Scopus (371) Google Scholar). We are currently investigating whether association with p73 is a specific property of gain of function p53 mutants. Of note, the finding that the core domain of mutant p53 is sufficient for the association with p73 highlights the potential role of this domain as a module for protein-protein interaction. Further investigation employing yeast two-hybrid screening or an immunoaffinity approach is needed to verify whether the core domain of mutant p53 plays an important role in the gain of function activity. Moreover, in view of the substantial similarity of the core domains between p73 and p63, another member of the p53 family, it is of interest to find out whether p63 can also participate in that network (46Yang A. Kaghad M. Wang Y. Gillet E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1830) Google Scholar, 47Yang A. Schweitzer R. Sun D. Kaghad M. Walker N. Bronson R.T. Tabin C. Sharpe A. Caput D. Crum C. Mckeon F. Nature. 1999; 398: 714-718Crossref PubMed Scopus (1891) Google Scholar, 48Mills A.A. Zheng B. Wang X.-J. Vogel H. Roop D.R. Bradley A. Nature. 1999; 398: 708-713Crossref PubMed Scopus (1688) Google Scholar, 49Kaelin Jr., W.G. Oncogene. 1999; 18: 7701-7705Crossref PubMed Scopus (158) Google Scholar). It is conceivable that other proteins interacting either with mutant p53 or with p73 might also interfere with the biological outcome of the entire network (29Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (504) Google Scholar, 30Gong J.G. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (830) Google Scholar, 31Yuan Z.M. Shioya H. Ishiko T. Sun X.G. Gu J.J. Huang Y.Y. Lu H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (539) Google Scholar, 50Higashino F. Pipas M.J. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15683-15687Crossref PubMed Scopus (50) Google Scholar, 51Zeng X. Chen L. Jost C. Maya R. Keller D. Wang X. Kaelin Jr., W.G. Oren M. Chen J. Lu H. Mol. Cell. Biol. 1998; 19: 3257-3266Crossref Scopus (303) Google Scholar, 52Balint E. Bates S. Vousden K.H. Oncogene. 1999; 18: 3923-3929Crossref PubMed Scopus (194) Google Scholar, 53Zeng X. Li X. Miller A. Yuan Z. Yuan W. Kwok R.P.S. Goodman R. Lu H. Mol. Cell. Biol. 2000; 20: 1299-1310Crossref PubMed Scopus (82) Google Scholar, 54Scharnhorst V. Dekker P. van der Eb A.J. Jochensen A.G. J. Biol. Chem. 2000; 275: 10202-10211Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The studies performed to date suggest that p73 is rarely mutated in human cancers and that ectopic expression of p73 induces apoptosis in cancer cells. Thus, agents that increase p73 expression may provide new potential anticancer treatments. Conversely, proteins inactivating or interfering with p73 might have an impact upon cellular properties, clinical responses to therapy, and prognosis of a tumor. Our data, showing that human tumor-derived p53 mutants can interact with p73 not only physically but also functionally, might implicate mutant p53 as a likely candidate for such type of proteins. Along this line, an intriguing question can be raised: how does mutant p53 inactivate p73? In accordance with our novel finding that both the core and the oligomerization domains of p73 are involved in the association with mutant p53, we might depict two possible scenarios. On the one hand, mutant p53 binding the oligomerization domain of p73 can interfere with the formation of p73 homo-oligomers. On the other hand, mutant p53 binding the core domain of p73 can interfere with its binding to DNA. The recent observation that p73-deficient mice do not exhibit an increase in spontaneous tumors suggests that the association between mutant p53 and p73 might have an impact mainly upon tumor chemoresistance rather than on tumor development (55Yang A. Walker N. Bronson R. Kaghad M. Oosterwegel M. Bonnin J. Vagner C. Bonnet H. Dikkes P. Sharpe A. McKeon F. Caput D. Nature. 2000; 404: 99-103Crossref PubMed Scopus (878) Google Scholar). If this prediction is proven correct, one would expect that cells from p73-deficient mice will be less prone to killing by anticancer drugs than their wild type counterparts. The establishment of a precise role for the association between mutant p53 and p73 might be very useful for anticancer treatment. To this end, further evidence needs to be collected. We thank G. Del Sal, G. Melino, W. G. Kaelin, Jr., and B. Vogelstein for expression plasmids and D. Lane for DO-1 antibody.
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