Biogenesis of p53 Involves Cotranslational Dimerization of Monomers and Posttranslational Dimerization of Dimers

Precisely how mutant p53 exerts a dominant negative effect over wild type p53 has been an enigma. To understand how wild type and mutant p53 form hetero-oligomers, we studied p53 biogenesis in vitro. We show here that p53 dimers are formed cotranslationally (on the polysome), whereas tetramers are formed posttranslationally (by the dimerization of dimers in solution). Coexpression of wild type and mutant p53 therefore results in 50% of the p53 generated being heterotetramers comprised of a single species: wild type dimer/mutant dimer. Using hot spot mutants of p53 and a variety of natural target sites, we show that all wild type/mutant heterotetramers manifest impaired DNA binding activity. This impairment is not due to the mutant dimeric subunit inhibiting association of the complex with DNA but rather due to the lack of significant contribution (positive cooperativity) from the mutant partner. For all heterotetramers, bias in binding is particularly pronounced against those sequences in genes responsible for apoptosis rather than cell growth arrest. These results explain the molecular basis of p53 dominant negative effect and suggest a functional role in the regulation of p53 tetramerization. Precisely how mutant p53 exerts a dominant negative effect over wild type p53 has been an enigma. To understand how wild type and mutant p53 form hetero-oligomers, we studied p53 biogenesis in vitro. We show here that p53 dimers are formed cotranslationally (on the polysome), whereas tetramers are formed posttranslationally (by the dimerization of dimers in solution). Coexpression of wild type and mutant p53 therefore results in 50% of the p53 generated being heterotetramers comprised of a single species: wild type dimer/mutant dimer. Using hot spot mutants of p53 and a variety of natural target sites, we show that all wild type/mutant heterotetramers manifest impaired DNA binding activity. This impairment is not due to the mutant dimeric subunit inhibiting association of the complex with DNA but rather due to the lack of significant contribution (positive cooperativity) from the mutant partner. For all heterotetramers, bias in binding is particularly pronounced against those sequences in genes responsible for apoptosis rather than cell growth arrest. These results explain the molecular basis of p53 dominant negative effect and suggest a functional role in the regulation of p53 tetramerization. electrophoretic mobility shift assay The importance of p53 as a tumor suppressor has been well documented. Over half of all human cancers are mutated in the gene encoding p53, and many viruses can induce transformation of the host cell through p53 inactivation (Ref. 1.Hollstein M. Shomer B. Greenblatt M. Soussi T. Hovig E. Montesano R. Harris C.C. Nucleic Acids Res. 1996; 24: 141-146Crossref PubMed Scopus (456) Google Scholar; reviewed in Refs. 2.Levine A.J. Virology. 1990; 177: 419-426Crossref PubMed Scopus (150) Google Scholar and 3.Liu Y. Kulesz-Martin M. Carcinogenesis. 2001; 22: 851-860Crossref PubMed Scopus (130) Google Scholar). Furthermore, inheriting a germline p53 mutation characteristic of Li-Fraumeni syndrome confers a strong predisposition to cancer because 50% of those afflicted acquire cancer by age 30 (4.Malkin D. Li F.P. Strong L.C. Fraumeni Jr., J.F. Nelson C.E. Kim D.H. Kassel J. Gryka M.A. Bischoff F.Z. Tainsky M.A. Friend S.H. Science. 1990; 250: 1233-1238Crossref PubMed Scopus (3087) Google Scholar, 5.Malkin D. Cancer Genet. Cytogenet. 1993; 66: 83-92Abstract Full Text PDF PubMed Scopus (127) Google Scholar). The role of p53 in tumorigenesis was also demonstrated in an animal tumor model, in which p53−/− mice were found to be more prone to cancer development (primarily lymphomas) compared with their wild type (p53+/+) and heterozygous (p53+/−) littermates (6.Donehower L.A. Harvey M. Slagle B.L. McArthur M.J. Montgomery Jr., C.A. Butel J.S. Bradley A. Nature. 1992; 356: 215-221Crossref PubMed Scopus (4054) Google Scholar, 7.Harvey M. McArthur M.J. Montgomery Jr., C.A. Butel J.S. Bradley A. Donehower L.A. Nat. Genet. 1993; 5: 225-229Crossref PubMed Scopus (492) Google Scholar). Loss of p53 or its function is therefore clearly linked to tumor formation. As “guardian of the genome,” p53 is activated by a number of genotoxic and stress signals such as ionizing radiation, ultraviolet light, ribonucleotide depletion, hypoxia, oxidative stress, heat shock, and exposure to nitric oxide (reviewed in Ref. 8.Ljungmann M. Neoplasia. 2000; 2: 208-225Crossref PubMed Scopus (186) Google Scholar). Critical to the tumor-suppressing function of activated p53 is its ability to bind sequence-specific DNA sites and induce the transcription of genes involved in cell cycle arrest, DNA repair, and apoptosis. This is illustrated by the fact that most p53 mutations occur in its DNA-binding domain and affect its sequence-specific DNA binding ability (1.Hollstein M. Shomer B. Greenblatt M. Soussi T. Hovig E. Montesano R. Harris C.C. Nucleic Acids Res. 1996; 24: 141-146Crossref PubMed Scopus (456) Google Scholar). Such mutations (called “hot spot” mutations) can be divided into two categories; class I mutations affect residues that make direct contact with DNA, whereas class II mutations occur at residues crucial for maintaining the conformation of the DNA-binding domain (9.Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2158) Google Scholar). The consensus p53 binding site consists of two or more copies of the 10-bp half-site 5′-PuPuPuC(A/T)(A/T)GPyPyPy-3′ separated by up to 13 bp (10.el-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1752) Google Scholar). Minor variations to this consensus sequence are found in all p53 target genes. Each half-site consists of two inverted repeat 5-bp quarter sites. p53 binds to this consensus DNA sequence as a pair of clamps, with the two monomers within each dimer binding to two consecutive quarter sites within a half-site (11.McLure K.G. Lee P.W. EMBO J. 1998; 17: 3342-3350Crossref PubMed Scopus (205) Google Scholar). The two dimers within the tetramer therefore bind to the two half-sites in juxtaposition to each other, resulting in overall enhanced stability of the p53-DNA complex. The lack of such cooperative binding in a single dimer-half-site interaction accounts for the drastically reduced stability of the dimer-half-site complex relative to that of the tetramer-full site complex. This is also reflected by the observation that in contrast to full sites, half-sites do not confer transcriptional responsiveness to p53. 1K. G. McLure, D. D. Sweet, and P. W. K. Lee, unpublished data. Although there is little doubt that tumors often arise through deletion or mutation of both p53 alleles (the two-hit model) (12.Knudsen Jr., A.G. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 820-823Crossref PubMed Scopus (5622) Google Scholar), there is increasing evidence that a point mutation or deletion in a single allele could result in increased susceptibility to cancer (13.Nigro J.M. Baker S.J. Preisinger A.C. Jessup J.M. Hostetter R. Cleary K. Bigner S.H. Davidson N. Baylin S. Devilee P. Glover T. Collins F.S. Weston A. Modali R. Harris C.C. Vogelstein B. 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We here that dimerization of p53 the whereas in of wild type and mutant p53 results in a single of p53 type dimer/mutant 50% of p53. the of hot spot mutants of p53 of which is of strong binding to and a variety of natural p53 target sites, we show that all heterotetramers bind to sites compared with the wild type p53 Such impaired binding is not due to of wild type p53 but rather is due to the lack of binding by the mutant dimeric subunit (positive We this model, which a number of to as the basis of the p53 dominant negative The dominant negative effect is more pronounced with sequences linked to than those responsible for cell growth arrest, the that the lack of cell a more role than of cell growth in cancer The were type human and human both from T. EMBO J. PubMed Scopus Google Scholar). and were by with the and p53 mutants were also a from T. was by DNA with and with the For of were to of of of of of and of For p53 was a was to the with were at for of were in and with of the p53 or an of of The was to the and for an 30 The were in and in The were for to their The were in and with and to at was with DNA binding of of of DNA of of of of and of DNA was to the p53-DNA an of was in the The were at for to and in a (11.McLure K.G. Lee P.W. EMBO J. 1998; 17: 3342-3350Crossref PubMed Scopus (205) Google Scholar). The were and to at was a and or with The sequences of and were (11.McLure K.G. Lee P.W. EMBO J. 1998; 17: 3342-3350Crossref PubMed Scopus (205) Google Scholar), and the are in I that were to the DNA with natural p53 target sequence of target W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1752) Google K.G. Lee P.W. 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This that dimers within a tetramer of p53 can with and that are in This tetramers over dimers because the of p53 and is (11.McLure K.G. Lee P.W. EMBO J. 1998; 17: 3342-3350Crossref PubMed Scopus (205) Google Scholar). of dimer tetramers is with the of p53 being a the dimerization of was that in wild type p53 the mutant p53 can induce a mutant conformation in the wild type J. 1991; Full Text PDF PubMed Scopus Google Scholar), resulting in the dominant negative that p53 tetramers are formed by dimerization of dimers that in Li-Fraumeni a wild type and a mutant p53 a single of heterotetramers be we that the two dimers within a p53 tetramer are K.G. Lee P.W. EMBO J. PubMed Scopus Google Scholar), be of to the binding of heterotetramers to the consensus sequence To this we with of the most p53 mutants in human cancers (1.Hollstein M. Shomer B. Greenblatt M. Soussi T. Hovig E. Montesano R. Harris C.C. 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PubMed Scopus Google with wild type p53 tetramer Although the clearly that all the wild type/mutant heterotetramers manifest reduced DNA binding compared with the wild type the of this and both the mutant and the target the mutants to the dominant negative effect over the wild type For its in the has little or affect the binding of the to the or whereas the heterotetramers bind to sequences with reduced This could explain p53 mutant is of A.M. 1993; Google Scholar, M. A. M. J. Res. 1994; Google Scholar, A.M. 1994; Google Scholar). the exerts a dominant negative effect the wild type here this effect is not as pronounced as those by the is that being the most p53 mutation in human is the dominant negative by most results that mutants and are the most dominant negative mutants of the which could in explain Li-Fraumeni with mutations at of J.M. M. G. J. A.M. J.M. PubMed Scopus Google mutation in allele is to wild type p53 from the observation to the target For all the mutants the sequence that cell growth is by the to the dominant negative of sequences that apoptosis such as and all manifest for the we found the of the to be (by than those of or not This bias was also wild type p53 not Such binding that p53 mutants were more dominant negative for of apoptosis than for growth in human cancer cell S. 2000; PubMed Scopus Google Scholar). also mutants are not dominant negative in for growth T. M. Kassel J. Friend S.H. Cancer Res. 1994; Google Scholar, J. J.M. J.D. D. J. 1996; PubMed Scopus Google Scholar). by p53 biogenesis in we for the that p53 dimerization whereas interaction in a wild type and mutant p53 type of heterotetramers is wild type dimer. These heterotetramers manifest reduced binding for all p53 target sequences not because the mutant dimer a mutant conformation the wild type but because has for DNA and therefore to the overall stability of the p53-DNA complex. was particularly for sequences that apoptosis rather than cell growth arrest, that in cell an role in cancer development in Li-Fraumeni We for the and p53 mutant and for with

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