Regulation of the p53 Tumor Suppressor Protein
1999; Elsevier BV; Volume: 274; Issue: 51 Linguagem: Inglês
10.1074/jbc.274.51.36031
ISSN1083-351X
Autores Tópico(s)Ubiquitin and proteasome pathways
Resumoc-Jun N-terminal kinase Mutations in the p53 tumor suppressor gene occur in about 50% of all human tumors, making it the most frequent target for genetic alterations in cancer (for recent reviews on p53 see Refs. 1Hansen R. Oren M. Curr. Opin. Genet. Dev. 1997; 7: 46-51Crossref PubMed Scopus (206) Google Scholar, 2Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6697) Google Scholar, 3Agarwal M.L. Taylor W.R. Chernov M.V. Chernova O.B. Stark G.R. J. Biol. Chem. 1998; 273: 1-4Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 4Almog N. Rotter V. Biochim. Biophys. Acta. 1998; 1378: R43-R54PubMed Google Scholar, 5Prives C. Hall P.A. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1225) Google Scholar). Such mutations probably facilitate carcinogenesis primarily through abrogating the tumor suppressor activities of the wild type p53 protein, although at least some forms of tumor-associated mutant p53 proteins may also contribute overt oncogenic activities (gain of function). Excessive wild type p53 activity gives rise to a variety of cellular outcomes, most notably cell cycle arrest and apoptosis. These cellular effects of wild type p53 can reduce cancer incidence through elimination of cancer-prone cells from the replicative pool. However, such effects might become very undesirable if occurring in a normal, unperturbed cell. p53 activity must therefore be kept under tight control, being unleashed only when a cell accumulates lesions that may otherwise drive it into a cancerous state. The signals and mechanisms that regulate p53 activity, maintaining it at low levels under normal conditions and turning it on in cancer-prone cells, are the subject of this review.p53-activating SignalsUnder normal conditions, p53 is most probably latent. Consequently, it does not interfere with cell cycle progression and cell survival. Moreover, p53 knock-out mice appear in most cases to undergo proper development and maturation (6Donehower L.A. Harvey M. Slagle B.L. Mcarthur M.J. Montgomery C.A. Butel J.S. Bradley A. Nature. 1992; 356: 215-221Crossref PubMed Scopus (4009) Google Scholar), suggesting that p53 is not essential for the normal performance of cells within the body. However, a variety of conditions can lead to rapid induction of p53 activity (Fig. 1). The common denominator of these conditions is that they represent various types of stress, which are likely to favor the emergence of cancer-bound cells. Such conditions include direct DNA damage (7Kastan M.B. Onyekwere O. Sidransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar, 8Maltzman W. Czyzyk L. Mol. Cell. Biol. 1984; 4: 1689-1694Crossref PubMed Scopus (813) Google Scholar, 9Huang L.C. Clarkin K.C. Wahl G.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4827-4832Crossref PubMed Scopus (321) Google Scholar) as well as damage to components involved in the proper handling and segregation of the cellular genetic material (e.g. the mitotic spindle (10Cross S.M. Sanchez C.A. Morgan C.A. Schimke M.K. Ramel S. Idzerda R.L. Raskind W.H. Reid B.J. Science. 1995; 267: 1353-1356Crossref PubMed Scopus (675) Google Scholar)), ribonucleotide depletion (11Linke S.P. Clarkin K.C. Di L.A. Tsou A. Wahl G.M. Genes Dev. 1996; 10: 934-947Crossref PubMed Scopus (479) Google Scholar), hypoxia (12Graeber T.G. Osmanian C. Jacks T. Housman D.E. Koch C.J. Lowe S.W. Giaccia A.J. Nature. 1996; 379: 88-91Crossref PubMed Scopus (2160) Google Scholar), heat shock (13Ohnishi T. Wang X.J. Ohnishi K. Matsumoto H. Takahashi A. J. Biol. Chem. 1996; 271: 14510-14513Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), and exposure to nitric oxide (NO) (14Forrester K. Ambs S. Lupold S.E. Kapust R.B. Spillare E.A. Weinberg W.C. Felleybosco E. Wang X.W. Geller D.A. Tzeng E. Billiar T.R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2442-2447Crossref PubMed Scopus (388) Google Scholar). Accumulation of genomic aberrations is a key carcinogenic mechanism; the rapid induction of p53 activity in response to genomic damage thus serves to ensure that cells carrying such damage are effectively taken care of. Furthermore, p53 may also contribute, directly or indirectly, to particular DNA repair processes (15Smith M.L. Chen I.T. Zhan Q.M. Oconnor P.M. Fornace A.J. Oncogene. 1995; 10: 1053-1059PubMed Google Scholar, 16Offer H. Wolkowicz R. Matas D. Blumenstein S. Livneh Z. Rotter V. FEBS Lett. 1999; 450: 197-204Crossref PubMed Scopus (124) Google Scholar). The pivotal role of p53 in maintaining genomic integrity has earned it the nickname “guardian of the genome” (17Lane D.P. Nature. 1992; 358: 15-16Crossref PubMed Scopus (4436) Google Scholar). In addition, p53 activity is triggered by a variety of oncogenic proteins, including Myc, Ras, adenovirus E1A, and β-catenin (18Hermeking H. Eick D. Science. 1994; 265: 2091-2093Crossref PubMed Scopus (699) Google Scholar, 19Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3887) Google Scholar, 20Debbas M. White E. Gene Dev. 1993; 7: 546-554Crossref PubMed Scopus (831) Google Scholar, 21Damalas A. Ben-Ze'ev A. Simcha I. Shtutman M. Leal J.F. Zhurinsky J. Geiger B. Oren M. EMBO J. 1999; 18: 3054-3063Crossref PubMed Scopus (208) Google Scholar), providing a direct link between oncogenic processes and the tumor suppressor action of p53 (see below).Regulation of p53 Gene ExpressionAs described later, induction of the p53 response upon stress occurs largely through alterations in the p53 protein. Changes in the rate of transcription of the p53 gene play a minor role, if any, in such induction. Consequently, the transcriptional regulation of the p53 gene has received very little attention during recent years. This need not imply that the regulation of p53gene expression is totally irrelevant. In fact, it was observed long ago that p53 mRNA levels rise substantially upon serum stimulation (22Reich N.C. Levine A.J. Nature. 1984; 308: 199-201Crossref PubMed Scopus (376) Google Scholar). This rise may be because of the presence of binding sites for serum-induced factors in the p53 promoter (23Ginsberg D. Oren M. Yaniv M. Piette J. Oncogene. 1990; 5: 1285-1290PubMed Google Scholar) as well as to the ability of the p53 gene to bind the c-Myc protein and to be transcriptionally stimulated by excess c-Myc (24Reisman D. Elkind N.B. Roy B. Beamon J. Rotter V. Cell Growth Differ. 1993; 4: 57-65PubMed Google Scholar). The induction of an anti-proliferative gene, p53, by serum and growth factors may at first glance seem paradoxical. However, it does make good sense. Cells undergoing DNA replication and extensive proliferation are at higher risk of acquiring DNA damage and giving rise to multiple cancer-prone progeny than quiescent cells. Induction of higher p53 mRNA levels under such conditions places the cells in a state of anticipation; as long as there is no DNA damage or other stress, p53 remains latent and does not interfere with normal cellular transactions. However, if conditions emerge that call for a p53 response, the presence of higher levels of p53 mRNA ensures that such a response will be rapid and effective.Activation of p53 by Post-transcriptional MechanismsExposure of cells to p53-activating signals can lead within a relatively short time to a marked elevation in p53 protein. To some extent, this can be achieved by increased translation of the p53 mRNA, probably involving relief of a translational repression mechanism operating through the 3′-untranslated region of this mRNA (25Fu L.N. Minden M.D. Benchimol S. EMBO J. 1996; 15: 4392-4401Crossref PubMed Scopus (131) Google Scholar). There also exists evidence that p53 itself can inhibit p53 synthesis through binding to its own mRNA (26Mosner J. Mummenbrauer T. Bauer C. Sczakiel G. Grosse F. Deppert W. EMBO J. 1995; 14: 4442-4449Crossref PubMed Scopus (266) Google Scholar, 27Fontoura B.M.A. Atienza C.A. Sorokina E.A. Morimoto T. Carroll R.B. Mol. Cell. Biol. 1997; 17: 3146-3154Crossref PubMed Scopus (59) Google Scholar). Yet, it is generally accepted that the accumulation of active p53 in response to stress occurs mainly through post-translational mechanisms. Pivotal is the increase in the protein half-life of p53. p53 is usually a very labile protein, turning over with a half-life sometimes as short as a few minutes (28Rogel A. Popliker M. Webb C.A. Oren M. Mol. Cell. Biol. 1985; 5: 2851-2855Crossref PubMed Scopus (329) Google Scholar). In response to DNA damage and other types of stress, p53 is markedly stabilized (7Kastan M.B. Onyekwere O. Sidransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar, 8Maltzman W. Czyzyk L. Mol. Cell. Biol. 1984; 4: 1689-1694Crossref PubMed Scopus (813) Google Scholar). A rapid increase in p53 concentration without a need for de novo transcription is particularly advantageous in cells with severely damaged genomes. In addition, there is most probably a qualitative conversion of p53 from latent to active form. The best documented change concerns the sequence-specific DNA binding activity of p53. p53 operates as a gene-specific transcriptional activator, which relies on its ability to bind defined sequence elements within target genes (1Hansen R. Oren M. Curr. Opin. Genet. Dev. 1997; 7: 46-51Crossref PubMed Scopus (206) Google Scholar, 2Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6697) Google Scholar, 3Agarwal M.L. Taylor W.R. Chernov M.V. Chernova O.B. Stark G.R. J. Biol. Chem. 1998; 273: 1-4Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 4Almog N. Rotter V. Biochim. Biophys. Acta. 1998; 1378: R43-R54PubMed Google Scholar, 5Prives C. Hall P.A. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1225) Google Scholar). The sequence-specific DNA binding activity of p53 is subject to constitutive negative regulation, primarily through its inhibitory C-terminal domain (29Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Cell. 1992; 71: 875-886Abstract Full Text PDF PubMed Scopus (859) Google Scholar, 30Bayle J.H. Elenbaas B. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5729-5733Crossref PubMed Scopus (115) Google Scholar, 31Wolkowicz R. Peled A. Elkind N.B. Rotter V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6842-6846Crossref PubMed Scopus (35) Google Scholar). Relief of this inhibition upon exposure to stress results in increased DNA binding (32Hupp T.R. Lane D.P. J. Biol. Chem. 1995; 270: 18165-18174Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 33Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2152) Google Scholar, 34Waterman M.J. Stavridi E.S. Waterman J.L. Halazonetis T.D. Nat. Genet. 1998; 19: 175-178Crossref PubMed Scopus (402) Google Scholar) and consequently increased biochemical and biological activity. The transcriptional activity of p53 may also be induced by changes in other regions,e.g. modifications within its N-terminal transactivation domain, enabling a more efficient recruitment of components of the transcription machinery (35Lambert P.F. Kashanchi F. Radonovich M.F. Shiekhattar R. Brady J.N. J. Biol. Chem. 1998; 273: 33048-33053Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Finally, p53 activation may also involve a change in subcellular localization; whereas latent p53 may often be cytoplasmic, at least during part of the cell cycle (36Shaulsky G. Ben-Zeev A. Rotter V. Oncogene. 1990; 5: 1707-1711PubMed Google Scholar), exposure to stress results in its accumulation in the nucleus, where it is expected to exert its biochemical activities.The p53-Mdm2 LoopA key player in the regulation of p53 is the Mdm2 protein. Mdm2 is the product of an oncogene, whose excess activity facilitates several types of human cancer (for reviews see Refs. 37Lozano G. Montes de Oca Luna R. Biochim. Biophys. Acta. 1998; 1377: M55-M59PubMed Google Scholar, 38Freedman D.A. Wu L. Levine A.J. Cell. Mol. Life Sci. 1999; 55: 96-107Crossref PubMed Scopus (480) Google Scholar, 39Juven-Gershon T. Oren M. Mol. Med. 1999; 5: 71-83Crossref PubMed Google Scholar). Mdm2 exhibits a unique relationship with p53. On the one hand, the Mdm2 protein binds to p53 and inactivates it (40Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. Cell. 1992; 69: 1237-1245Abstract Full Text PDF PubMed Scopus (2776) Google Scholar, 41Chen J.D. Wu X.W. Lin J.Y. Levine A.J. Mol. Cell. Biol. 1996; 16: 2445-2452Crossref PubMed Scopus (332) Google Scholar, 42Haupt Y. Barak Y. Oren M. EMBO J. 1996; 15: 1596-1606Crossref PubMed Scopus (204) Google Scholar). The binding occurs right within the p53 transactivation domain, interfering with recruitment of basal transcription machinery components (43Thut C.J. Chen J.L. Klemm R. Tjian R. Science. 1995; 267: 100-104Crossref PubMed Scopus (406) Google Scholar, 44Lu H. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5154-5158Crossref PubMed Scopus (280) Google Scholar). Moreover, Mdm2 can actively repress transcription when tethered to p53 (45Thut C.J. Goodrich J.A. Tjian R. Gene Dev. 1997; 11: 1974-1986Crossref PubMed Scopus (230) Google Scholar). Importantly, Mdm2 binding can also lead to complete elimination of p53 through proteolytic degradation. On the other hand, p53 binds specifically to the mdm2 gene and stimulates its transcription (46Barak Y. Juven T. Haffner R. Oren M. EMBO J. 1993; 12: 461-468Crossref PubMed Scopus (1167) Google Scholar, 47Wu X.W. Bayle J.H. Olson D. Levine A.J. Genes Dev. 1993; 7: 1126-1132Crossref PubMed Scopus (1621) Google Scholar). This duality defines a negative feedback loop (Fig. 2), which probably serves to keep p53 in tight check and to terminate the p53 signal once the triggering stress has been effectively dealt with. In some situations, mdm2transcription is induced later than that of other p53 target genes (48Perry M.E. Piette J. Zawadzki J.A. Harvey D. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11623-11627Crossref PubMed Scopus (239) Google Scholar, 49Wu L. Levine A.J. Mol. Med. 1997; 3: 441-451Crossref PubMed Google Scholar); this may set a time window within which p53 is allowed to exert freely its biochemical and biological effects. The critical importance of the p53-Mdm2 loop is best illustrated by the analysis ofmdm2 knock-out mice. Inactivation of the mdm2gene results in early embryonal lethality, but this is completely prevented by simultaneous inactivation of p53 (50Jones S.N. Roe A.E. Donehower L.A. Bradley A. Nature. 1995; 378: 206-208Crossref PubMed Scopus (1057) Google Scholar, 51Montes de Oca Luna R. Wagner D.S. Lozano G. Nature. 1995; 378: 203-206Crossref PubMed Scopus (1196) Google Scholar). Conceivably, in the absence of functional Mdm2 protein, p53 becomes strongly deregulated to the extent that its excess activity leads to embryonic death. The other side of the coin is revealed in certain human cancers; excessive Mdm2 expression, achieved through mdm2 gene amplification (52Oliner J.D. Kinzler K.W. Meltzer P.S. George D.L. Vogelstein B. Nature. 1992; 358: 80-83Crossref PubMed Scopus (1790) Google Scholar) or other mechanisms (53Landers J.E. Haines D.S. Strauss J.F. George D.L. Oncogene. 1994; 9: 2745-2750PubMed Google Scholar), can lead to constitutive inhibition of p53 and thereby promote cancer without a need to alter the p53 gene itself. It should be kept in mind, however, that excess Mdm2 can also promote cancer independently of p53 (54Lundgren K. Montes de Oca Luna R. McNeill Y.B. Emerick E.P. Spencer B. Barfield C.R. Lozano G. Rosenberg M.P. Finlay C.A. Genes Dev. 1997; 11: 714-725Crossref PubMed Scopus (212) Google Scholar,55Sun P. Dong P. Dai K. Hannon G.J. Beach D. Science. 1998; 282: 2270-2272Crossref PubMed Scopus (184) Google Scholar).FIG. 2The p53-Mdm2 autoregulatory loop. The p53 protein binds to the mdm2 gene and activates its transcription. The resultant Mdm2 protein binds to p53 and blocks its activity.View Large Image Figure ViewerDownload (PPT)Regulation of p53 Protein DegradationMuch of the activation of p53 is achieved through p53 protein stabilization. This realization has markedly accelerated research on p53 degradation. It is now well established that the rapid demise of p53 is achieved largely through the ubiquitin-proteasome pathway (56Maki C.G. Huibregtse J.M. Howley P.M. Cancer Res. 1996; 56: 2649-2654PubMed Google Scholar), although a role for other proteolytic enzymes such as calpain has also been implied (57Kubbutat M.H.G. Vousden K.H. Mol. Cell. Biol. 1997; 17: 460-468Crossref PubMed Scopus (275) Google Scholar). Mdm2 plays a pivotal role here as well. Elevated Mdm2 levels result in rapid p53 degradation, which is dependent on the ability of the two proteins to engage in direct binding (58Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3658) Google Scholar, 59Kubbutat M.H.G. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2812) Google Scholar). Furthermore, interference with p53-Mdm2 binding by monoclonal antibodies or competitor peptides results in a dramatic stabilization and accumulation of p53 in non-stressed cells (60Bottger A. Bottger V. Sparks A. Liu W.L. Howard S.F. Lane D.P. Curr. Biol. 1997; 7: 860-869Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). This strongly argues that the low basal levels of p53 in such cells are due primarily to continuous Mdm2-promoted degradation.How does Mdm2 promote p53 degradation? When the proteolytic activity of the proteasome is blocked by specific inhibitors, excess Mdm2 augments the accumulation of ubiquitinated forms of p53 (58Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3658) Google Scholar, 59Kubbutat M.H.G. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2812) Google Scholar), suggesting that Mdm2 facilitates p53 ubiquitination. Strong support for this conclusion was provided by showing that Mdm2 can directly functionin vitro as a p53-specific E3 ubiquitin-protein ligase, which covalently attaches ubiquitin groups to p53 (61Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1587) Google Scholar, 62Honda R. Yasuda H. EMBO J. 1999; 18: 22-27Crossref PubMed Scopus (612) Google Scholar) (Fig. 3 A). It remains to be determined whether Mdm2 operates alone in vivo or is part of a larger E3 complex.FIG. 3Regulation of p53-mediated p53 degradation. The binding of Mdm2 to p53 promotes the ubiquitination of p53 and its subsequent degradation by the proteasome (A). DNA damage induces covalent modifications of p53 and Mdm2, particularly phosphorylation (indicated by (P)). Phosphorylation within the Mdm2-p53 binding interface can block binding and thereby protect p53 from degradation (B). Phosphorylation of Mdm2 within domain(s) required for its biochemical activity will also block p53 degradation, even if Mdm2-p53 binding is maintained (C). Deregulated oncoproteins induce the synthesis of ARF, which binds to Mdm2 and prevents its action (D). Ub, ubiquitin monomer. N andC denote the amino and carboxyl terminus, respectively, of each protein.View Large Image Figure ViewerDownload (PPT)Although Mdm2 emerges as the key regulator of p53 stability, other mechanisms for p53 ubiquitination and degradation also exist. Of particular interest is the possible role of the c-Jun N-terminal kinase (JNK)1; in vitroand in vivo studies suggest that the binding of JNK to p53 results in ubiquitination and proteolytic removal of p53 (63Fuchs S.Y. Adler V. Buschmann T. Yin Z. Wu X. Jones S.N. Ronai Z. Genes Dev. 1998; 12: 2658-2663Crossref PubMed Scopus (280) Google Scholar). The “division of labor” between Mdm2 and JNK is presently unclear; however, there are strong indications that it changes during the cell cycle (63Fuchs S.Y. Adler V. Buschmann T. Yin Z. Wu X. Jones S.N. Ronai Z. Genes Dev. 1998; 12: 2658-2663Crossref PubMed Scopus (280) Google Scholar).Covalent Modifications of p53Rapid post-translational activation of signaling proteins is often achieved through covalent modifications, particularly protein phosphorylation. It was thus conceivable that the rapid stabilization and activation of the p53 protein upon stress also involves stress-induced covalent modifications of p53. Indeed, there is mounting evidence in support of this conjecture. p53 becomes phosphorylated on multiple sites in vivo in response to various types of stress, and many stress-activated kinases can phosphorylate p53in vitro (reviewed in Refs. 5Prives C. Hall P.A. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1225) Google Scholar and 64Fuchs S.Y. Fried V.A. Ronai Z. Oncogene. 1998; 17: 1483-1490Crossref PubMed Scopus (136) Google Scholar, 65Meek D.W. Cell. Signal. 1998; 10: 159-166Crossref PubMed Scopus (177) Google Scholar, 66Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1171) Google Scholar, 67Jayaraman L. Prives C. Cell. Mol. Life Sci. 1999; 55: 76-87Crossref PubMed Scopus (122) Google Scholar). A potential outcome of such phosphorylation might be the stabilization of p53 through inhibition of p53 ubiquitination and degradation. The pivotal role of Mdm2 in these processes suggests several likely scenarios. For instance, because degradation requires the binding of Mdm2 to p53 (58Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3658) Google Scholar,59Kubbutat M.H.G. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2812) Google Scholar), phosphorylation of residues positioned within the binding interface of either protein may interfere with binding and lead to p53 stabilization (Fig. 3 B). In the case of p53, several candidate sites within its Mdm2-binding domain have been identified which are modified in response to DNA damage and whose phosphorylation reduces the affinity of p53 for Mdm2 (68Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1728) Google Scholar, 69Shieh S.Y. Taya Y. Prives C. EMBO J. 1999; 18: 1815-1823Crossref PubMed Scopus (266) Google Scholar, 70Unger T. Juven-Gershon T. Moallem E. Berger M. Vogt Sionov R. Lozano G. Oren M. Haupt Y. EMBO J. 1999; 18: 1805-1814Crossref PubMed Scopus (315) Google Scholar). Of particular interest are serines 15 and 20 and threonine 18 of human p53, all located within or very close to the Mdm2-binding domain of p53. Serine 15 has been studied particularly closely, as it is the site of p53 phosphorylation by the ATM kinase (71Banin S. Moyal L. Shieh S. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1695) Google Scholar, 72Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1693) Google Scholar), whose activity is required for p53 stabilization in response to ionizing radiation and some other types of DNA damage (73Kastan M.B. Zhan Q.M. Eldeiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2923) Google Scholar, 74Khanna K.K. Beamish H. Yan J. Hobson K. Williams R. Dunn I. Lavin M.F. Oncogene. 1995; 11: 609-618PubMed Google Scholar). It should be noted that although the idea that such phosphorylation events are responsible for p53 stabilization is very attractive, the in vivo relevance of this idea has been challenged recently (75Ashcroft M. Kubbutat M.H. Vousden K.H. Mol. Cell. Biol. 1999; 19: 1751-1758Crossref PubMed Scopus (376) Google Scholar, 76Blattner C. Tobiasch E. Litfen M. Rahmsdorf H.J. Herrlich P. Oncogene. 1999; 18: 1723-1732Crossref PubMed Scopus (132) Google Scholar). Hence, the effect of p53 phosphorylation on stability may depend on the intracellular context and particularly on the availability of alternative mechanisms for p53 degradation.Stabilization of p53 might be achieved by modifying not only p53 but also Mdm2. In a simple scenario, Mdm2 may become phosphorylated in a manner that disrupts its interaction with p53 (Fig. 3 B). In fact, a candidate phosphorylation site within Mdm2 has been described (77Mayo L.D. Turchi J.J. Berberich S.J. Cancer Res. 1997; 57: 5013-5016PubMed Google Scholar). Alternatively, phosphorylated Mdm2 may retain p53 binding but become impaired with regard to its E3 ubiquitin ligase activity (Fig. 3 C). This may particularly apply to the C-terminal part of Mdm2, known to be required for p53 ubiquitination (61Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1587) Google Scholar, 78Kubbutat M.H. Ludwig R.L. Levine A.J. Vousden K.H. Cell Growth Differ. 1999; 10: 87-92PubMed Google Scholar).Finally, both p53 and Mdm2 may also be subject to other types of modifications. Acetylation of p53, leading to increased DNA binding, has been well documented (33Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2152) Google Scholar, 79Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1013) Google Scholar). p53 glycosylation has also been reported, and it too may increase DNA binding (80Shaw P. Freeman J. Bovey R. Iggo R. Oncogene. 1996; 12: 921-930PubMed Google Scholar). The role of covalent modifications in p53 activation by stress remains a very challenging area of research.In addition to covalent modifications, protein-protein interactions also play a key role in regulating cellular p53 levels and activity. Such interactions and their implications are the subject of numerous studies, and it is impossible to discuss them thoroughly here. The reader is referred to several recent comprehensive reviews (5Prives C. Hall P.A. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1225) Google Scholar, 67Jayaraman L. Prives C. Cell. Mol. Life Sci. 1999; 55: 76-87Crossref PubMed Scopus (122) Google Scholar,81Hupp T.R. Cell Mol. Life Sci. 1999; 55: 88-95Crossref PubMed Scopus (48) Google Scholar).The ARF ConnectionThe ability of Mdm2 to promote p53 ubiquitination can be modulated not only by covalent modifications but also by the binding of other regulatory proteins. The most vivid and perhaps most important example is provided by the ARF protein. This small protein arises through translation of an alternative reading frame derived from theINK4A tumor suppressor gene (82Kamijo T. Zindy F. Roussel M.F. Quelle D.E. Downing J.R. Ashmun R.A. Grosveld G. Sherr C.J. Cell. 1997; 91: 649-659Abstract Full Text Full Text PDF PubMed Scopus (1373) Google Scholar). Of note, ARF binds to Mdm2 and to a lesser extent also to p53, and this binding prevents Mdm2-mediated p53 proteolysis (83Kamijo T. Weber J.D. Zambetti G. Zindy F. Roussel M.F. Sherr C.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8292-8297Crossref PubMed Scopus (783) Google Scholar, 84Pomerantz J. Schreiber-Agus N. Liegeois N. Silverman A. Alland L. Chin L. Potes J. Chen K. Orlow I. Lee H.-W. Cordon-Cardo C. DePinho R.A. Cell. 1998; 92: 713-723Abstract Full Text Full Text PDF PubMed Scopus (1324) Google Scholar, 85Zhang Y. Xiong Y. Yarbrough W.G. Cell. 1998; 92: 725-734Abstract Full Text Full Text PDF PubMed Scopus (1392) Google Scholar), apparently by blocking the E3 ligase activity of Mdm2 (62Honda R. Yasuda H. EMBO J. 1999; 18: 22-27Crossref PubMed Scopus (612) Google Scholar). The interaction between Mdm2 and ARF is therefore another attractive candidate for modulation by stress signals.p53 and Oncogenic StressOf particular interest is the activation of the p53 response by oncogenic stress, such as the deregulated expression of oncoproteins like adenovirus E1A, Ras, Myc, and β-catenin (Fig. 1). Although the importance of this response to tumor suppression is very obvious (Fig. 4 A), its biochemical basis has remained unknown until the discovery of ARF and its role in p53 stabilization. Recent work has revealed that excess activity of several oncoproteins leads to massive induction of ARF (86Zindy F. Eischen C.M. Randle D.H. Kamijo T. Cleveland J.L. Sherr C.J. Roussel M.F. Genes Dev. 1998; 12: 2424-2433Crossref PubMed Scopus (1055) Google Scholar, 87de Stanchina E. McCurrach M.E. Zindy F. Shieh S.Y. Ferbeyre G. Samuelson A.V. Prives C. Roussel M.F. Sherr C.J. Lowe S.W. Genes Dev. 1998; 12: 2434-2442Crossref PubMed Scopus (544) Google Scholar, 88Palmero I. Pantoja C. Serrano M. Nature. 1998; 395: 125-126Crossref PubMed Scopus (541) Google Scholar). This induction is primarily because of enhanced transcription, at least some of which is mediated through the E2F transcription factor (89Bates S. Phillips A.C. Clark P.A. Stott F. Peters G. Ludwig R.L. Vousden K.H. Nature. 1998; 395: 124-125Crossref PubMed Scopus (810) Google Scholar). The induced ARF protein then binds to Mdm2, thus preventing p53 ubiquitination and degradation (Fig. 3 D). Obviously, the inhibitory effects of p53 are not triggered when Myc or Ras proteins are recruited as part of a properly orchestrated growth response, initiated by the binding of a growth factor to its receptor, or else such cells would not be able to execute a mitogenic response. The question that comes to mind is: how can p53 tell between such “healthy” activation of Ras, Myc, or E2F and one that occurs independently of a proper growth signal and may lead to cancer? One possible difference may lie in the more transient nature of the activation in the first case. However, it is also conceivable that when a cell is exposed to a growth factor, one arm of the response d
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