Title: Phosphorylation of p53 Serine 15 Increases Interaction with CBP
Abstract: p53 exerts its cell cycle regulatory effects through its ability to function as a sequence-specific DNA binding transcription factor. CREB-binding protein (CBP)/p300, through its interaction with the N terminus of p53, acts as a coactivator for p53 and increases the sequence-specific DNA-binding activity of p53 by acetylating its C terminus. The same N-terminal domain of p53 has recently been shown to be phosphorylated at Ser15 in response to γ-irradiation. Remarkably, we now demonstrate that phosphorylation of p53 at Ser15 increases its ability to recruit CBP/p300. The increase in CBP/p300 binding was followed by an increase in the overall level of acetylation of the C terminus of p53. These results provide a mechanism for the activation of p53-regulated genes following DNA damage, through a signaling pathway linking p53 N-terminal kinase and C-terminal acetyltransferase activities. p53 exerts its cell cycle regulatory effects through its ability to function as a sequence-specific DNA binding transcription factor. CREB-binding protein (CBP)/p300, through its interaction with the N terminus of p53, acts as a coactivator for p53 and increases the sequence-specific DNA-binding activity of p53 by acetylating its C terminus. The same N-terminal domain of p53 has recently been shown to be phosphorylated at Ser15 in response to γ-irradiation. Remarkably, we now demonstrate that phosphorylation of p53 at Ser15 increases its ability to recruit CBP/p300. The increase in CBP/p300 binding was followed by an increase in the overall level of acetylation of the C terminus of p53. These results provide a mechanism for the activation of p53-regulated genes following DNA damage, through a signaling pathway linking p53 N-terminal kinase and C-terminal acetyltransferase activities. CREB-binding protein glutathione S-transferase DNA-dependent protein kinase polyacrylamide gel electrophoresis. p53, which mediates cell cycle arrest and apoptosis in response to DNA damage, is inactivated in approximately 60% of all human cancers (1Bates S. Vousden K.H. Curr. Opin. Genet. Dev. 1996; 6: 12-18Crossref PubMed Scopus (338) Google Scholar, 2Kastan M.B. Canman C.E. Leonard C.J. Cancer Metastasis Rev. 1995; 14: 3-15Crossref PubMed Scopus (440) Google Scholar, 3Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2282) Google Scholar, 4Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6695) Google Scholar). Regulation of the cell cycle and apoptosis is achieved through the function of p53 as a transcription factor, regulating transcription of genes including p21/waf1, GADD45, mdm2, cyclin G, and bax. The structure of p53 can be divided into three main domains: an N-terminal activation domain, a central, sequence-specific DNA-binding domain, and a C-terminal domain, which mediates tetramerization and regulates sequence-specific and nonspecific DNA binding (1Bates S. Vousden K.H. Curr. Opin. Genet. Dev. 1996; 6: 12-18Crossref PubMed Scopus (338) Google Scholar, 2Kastan M.B. Canman C.E. Leonard C.J. Cancer Metastasis Rev. 1995; 14: 3-15Crossref PubMed Scopus (440) Google Scholar, 3Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2282) Google Scholar, 4Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6695) Google Scholar). The N-terminal region interacts with a number of proteins, including MDM2 (5Momand 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), TBP (6Truant R. Xiao H. Ingles C.J. Greenblatt J. J. Biol. Chem. 1993; 268: 2284-2287Abstract Full Text PDF PubMed Google Scholar), dTAFII40 and dTAFII60 (7Thut C.J. Chen J.L. Klemm R. Tjian R. Science. 1995; 267: 100-104Crossref PubMed Scopus (406) Google Scholar), hTAFII31 (8Lu H. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5154-5158Crossref PubMed Scopus (280) Google Scholar), CBP1 (9Gu W. Shi X.L. Roeder R.G. Nature. 1997; 387: 819-823Crossref PubMed Scopus (520) Google Scholar, 10Scolnick D.M. Chehab N.H. Stavridi E.S. Lien M.C. Caruso L. Moran E. Berger S.L. Halazonetis T.D. Cancer Res. 1997; 57: 3693-3696PubMed Google Scholar), the p62 subunit of TFIIH (11Xiao H. Pearson A. Coulombe B. Truant R. Zhang S. Regier J.L. Triezenberg S.J. Reinberg D. Flores O. Ingles C.J. Greenblatt J. Mol. Cell. Biol. 1994; 14: 7013-7024Crossref PubMed Scopus (327) Google Scholar), and the adenovirus E1B 55-kDa protein (12Teodoro J.G. Branton P.E. J. Virol. 1997; 71: 3620-3627Crossref PubMed Google Scholar). It also contains a number of sites that are phosphorylated in vitroby casein kinase I (13Knippschild U. Milne D.M. Campbell L.E. DeMaggio A.J. Christenson E. Hoekstra M.F. Meek D.W. Oncogene. 1997; 15: 1727-1736Crossref PubMed Scopus (143) Google Scholar, 14Meek D.W. Campbell L.E. Jardine L.J. Knippschild U. McKendrick L. Milne D.M. Biochem. Soc. Trans. 1997; 25: 416-419Crossref PubMed Scopus (24) Google Scholar), DNA-dependent protein kinase (DNA-PK) (15Lees-Miller S.P. Sakaguchi K. Ullrich S.J. Appella E. Anderson C.W. Mol. Cell. Biol. 1992; 12: 5041-5049Crossref PubMed Scopus (463) Google Scholar), and c-Jun N-terminal kinase (16Milne D.M. Campbell L.E. Campbell D.G. Meek D.W. J. Biol. Chem. 1995; 270: 5511-5518Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Recently, the cyclin-dependent kinase-activating kinase, which is a cyclin-dependent kinase 7/cyclinH/p36MAT1complex, was also shown to phosphorylate human p53 (17Ko L.J. Shieh S.Y. Chen X. Jayaraman L. Tamai K. Taya Y. Prives C. Pan Z.Q. Mol. Cell. Biol. 1997; 17: 7220-7229Crossref PubMed Scopus (149) Google Scholar). Importantly, Ser15 has been shown to be phosphorylated in vivo in response to ionizing radiation (18Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (709) Google Scholar, 19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar) and is hyperphosphorylated in human T-cell lymphotrophic virus I-transformed cells (20Pise-Masison C.A. Radonovich M. Sakaguchi K. Appella E. Brady J.N. J. Virol. 1998; 72: 6348-6355Crossref PubMed Google Scholar). Phosphorylation of Ser15 has been shown to inhibit binding of TFIID in vitro (20Pise-Masison C.A. Radonovich M. Sakaguchi K. Appella E. Brady J.N. J. Virol. 1998; 72: 6348-6355Crossref PubMed Google Scholar), and Ser15/Ser37 phosphorylation has been shown to correlate inversely with MDM2 binding (19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar).The coactivator proteins CBP and p300 mediate transcriptional activation through a number of transcription activators, including CREB, NF-κB, c-Myb, and nuclear hormone receptors (21Kwok R.P. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1279) Google Scholar, 22Chrivia J.C. Kwok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1758) Google Scholar, 23Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (709) Google Scholar, 24Dai P. Akimaru H. Tanaka Y. Hou D.X. Yasukawa T. Kanei-Ishii C. Takahashi T. Ishii S. Genes Dev. 1996; 10: 528-540Crossref PubMed Scopus (303) Google Scholar, 25Chakravarti D. LaMorte V.J. Nelson M.C. Nakajima T. Schulman I.G. Juguilon H. Montminy M. Evans R.M. Nature. 1996; 383: 99-103Crossref PubMed Scopus (842) Google Scholar). Recently, p300 and CBP have also been shown to associate with p53 in vitro and in vivo and to cooperate with p53 in transactivation of a cotransfected reporter plasmid or the endogenousp21 gene. The interaction of the N terminus of p53 with the C-terminal region of CBP is thought to be important for this cooperative activity (9Gu W. Shi X.L. Roeder R.G. Nature. 1997; 387: 819-823Crossref PubMed Scopus (520) Google Scholar, 10Scolnick D.M. Chehab N.H. Stavridi E.S. Lien M.C. Caruso L. Moran E. Berger S.L. Halazonetis T.D. Cancer Res. 1997; 57: 3693-3696PubMed Google Scholar, 26Avantaggiati M.L. Ogryzko V. Gardner K. Giordano A. Levine A.S. Kelly K. Cell. 1997; 89: 1175-1184Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 27Lill N.L. Grossman S.R. Ginsberg D. DeCaprio J. Livingston D.M. Nature. 1997; 387: 823-827Crossref PubMed Scopus (593) Google Scholar). Both p300 and CBP have histone acetyltransferase activity (28Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1523) Google Scholar, 29Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2366) Google Scholar). Significantly, the acetyltransferase activity of p300 has been shown to recognize the C-terminal region of p53 as a substrate, and the acetylation of this regulatory region by p300 increases the sequence-specific binding of p53 in vitro (30Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2152) Google Scholar).We present evidence that phosphorylation of p53 in its N-terminal domain increases the association of p53 and CBP/p300 in vitro and that there is a corresponding increase in the acetylation of p53. Ser15 appears to be critical for the interaction of p53 and CBP/p300. These results provide a possible mechanism linking phosphorylation and acetylation signals in the amplification of the p53 response to ionizing radiation.DISCUSSIONp53 is a critical protein in the response of a cell to DNA damage. It is involved both in the cell cycle arrest that allows DNA repair to take place and in the induction of apoptosis should the damage be too severe to allow recovery. The transcriptional activation activity of the N-terminal domain of p53 is important for both these functions (3Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2282) Google Scholar,4Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6695) Google Scholar, 33Zhu J. Zhou W. Jiang J. Chen X. J. Biol. Chem. 1998; 273: 13030-13036Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Moreover, it is becoming increasingly apparent that phosphorylation of specific amino acids in the N terminus regulates the activity of the protein. For example, phosphorylation of Ser15 and Ser37 by DNA-PK in vitrodecreases the binding of MDM2 (19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar) and phosphorylation of Ser15 alone impairs TFIID binding (20Pise-Masison C.A. Radonovich M. Sakaguchi K. Appella E. Brady J.N. J. Virol. 1998; 72: 6348-6355Crossref PubMed Google Scholar). Importantly, γ-radiation-induced Ser15 phosphorylation correlates with a decrease in MDM2 binding by p53 in cell extracts (19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar), allowing the escape of p53 from the inhibitory effects of MDM2 (34Chen J. Lin J. Levine A.J. Mol. Med. 1995; 1: 142-152Crossref PubMed Google Scholar, 35Chen J. Wu X. Lin J. Levine A.J. Mol. Cell. Biol. 1996; 16: 2445-2452Crossref PubMed Scopus (331) Google Scholar, 36Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2810) Google Scholar). At the same time, the increase in the ability of p53 to recruit CBP/p300 following phosphorylation on these residues, as shown in this report, provides a positive mechanism for increasing its transcriptional activity. It will be of interest to determine whether the increase in p53-CBP/p300 interaction represents an increase in binding affinity or binding stoichiometry.CBP/p300 associates with p53 in vivo and in vitro(9Gu W. Shi X.L. Roeder R.G. Nature. 1997; 387: 819-823Crossref PubMed Scopus (520) Google Scholar, 10Scolnick D.M. Chehab N.H. Stavridi E.S. Lien M.C. Caruso L. Moran E. Berger S.L. Halazonetis T.D. Cancer Res. 1997; 57: 3693-3696PubMed Google Scholar, 26Avantaggiati M.L. Ogryzko V. Gardner K. Giordano A. Levine A.S. Kelly K. Cell. 1997; 89: 1175-1184Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 27Lill N.L. Grossman S.R. Ginsberg D. DeCaprio J. Livingston D.M. Nature. 1997; 387: 823-827Crossref PubMed Scopus (593) Google Scholar) and, in transfection experiments, cooperates with p53 in activation of a cotransfected reporter plasmid (9Gu W. Shi X.L. Roeder R.G. Nature. 1997; 387: 819-823Crossref PubMed Scopus (520) Google Scholar, 26Avantaggiati M.L. Ogryzko V. Gardner K. Giordano A. Levine A.S. Kelly K. Cell. 1997; 89: 1175-1184Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 27Lill N.L. Grossman S.R. Ginsberg D. DeCaprio J. Livingston D.M. Nature. 1997; 387: 823-827Crossref PubMed Scopus (593) Google Scholar) or the endogenous p21/waf1 gene (10Scolnick D.M. Chehab N.H. Stavridi E.S. Lien M.C. Caruso L. Moran E. Berger S.L. Halazonetis T.D. Cancer Res. 1997; 57: 3693-3696PubMed Google Scholar). Further, p300 is specifically required for p53 transactivation of the mdm2 promoter (37Thomas A. White E. Genes Dev. 1998; 12: 1975-1985Crossref PubMed Scopus (70) Google Scholar). Increased binding of CBP/p300 by p53 could activate transcription by two means: first, by increasing recruitment of the coactivator into the transcriptional complex, and second, by increasing the specific DNA binding activity of p53 following acetylation of the C terminus (30Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2152) Google Scholar). It will be of interest, therefore, to determine the effect of CBP/p300 activation domain and acetyltransferase mutants on p53-CBP/p300 activation of independent genes.Phosphorylation on Ser15 occurs following DNA damage induced by γ-radiation or chemicals, and mutation of this residue to alanine interferes with the cell cycle-arresting properties of p53 (38Fiscella M. Ullrich S.J. Zambrano N. Shields M.T. Lin D. Lees-Miller S.P. Anderson C.W. Mercer W.E. Appella E. Oncogene. 1993; 8: 1519-1528PubMed Google Scholar). γ-Irradiation induces double strand breaks in DNA; the ends of DNA so generated can activate DNA-PK through its Ku DNA-binding subunits, and so DNA-PK might be thought an attractive candidate for transmitting the DNA damage signal, via p53, to the cell cycle arrest pathway (39Carter T. Vancurova I. Sun I. Lou W. DeLeon S. Mol. Cell. Biol. 1990; 10: 6460-6471Crossref PubMed Scopus (245) Google Scholar, 40Gottlieb T.M. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1019) Google Scholar, 41Lees-Miller S.P. Chen Y.R. Anderson C.W. Mol. Cell. Biol. 1990; 10: 6472-6481Crossref PubMed Scopus (357) Google Scholar). The observation that murine scid cells, which are deficient in the catalytic subunit of DNA-PK, show normal apoptotic responses or arrest in G1 and G2following γ-irradiation (42Gurley K.E. Kemp C.J. Carcinogenesis. 1996; 17: 2537-2542Crossref PubMed Scopus (41) Google Scholar, 43Rathmell W.K. Kaufmann W.K. Hurt J.C. Byrd L.L. Chu G. Cancer Res. 1997; 57: 68-74PubMed Google Scholar) appeared inconsistent with this hypothesis, but it has been reported that these cells constitutively phosphorylate p53 on Ser15 (19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar), and low but detectable levels of DNA-PK activity have been detected in scid MEF cells (44Woo R. McLure K.G. Lees-Miller S.P. Rancourt D.E. Lee P.W.K. Nature. 1998; 394: 700-704Crossref PubMed Scopus (293) Google Scholar). Interestingly, Woo et al. (44Woo R. McLure K.G. Lees-Miller S.P. Rancourt D.E. Lee P.W.K. Nature. 1998; 394: 700-704Crossref PubMed Scopus (293) Google Scholar) have recently reported that DNA-PK is necessary for the induction of p53-specific DNA binding in response to DNA damage. However, it is not yet established whether DNA-PK acts on p53 directly or indirectly in vivo, and it is important to note that Woo et al. (44Woo R. McLure K.G. Lees-Miller S.P. Rancourt D.E. Lee P.W.K. Nature. 1998; 394: 700-704Crossref PubMed Scopus (293) Google Scholar) found that in addition to DNA-PK, a radiation-inducible factor in nuclear extracts was required for the induction of the specific DNA binding activity of p53; possibly, other, constitutive factors in these extracts are also required. These nuclear extracts would also contain CBP/p300, but these proteins are not known to have intrinsic radiation-inducible activities, and other factors are likely to be involved. The ATM kinase (which, like DNA-PK, is a member of the phosphatidylinositol 3-kinase (PI 3-kinase) family and which appears to be involved in the normal response of the cell to ionizing radiation (reviewed in Ref. 45Rotman G. Shiloh Y. Cancer Surv. 1997; 29: 285-304PubMed Google Scholar)) can also phosphorylate p53 at Ser15 (46Banin S. Moyal L. Shieh S.Y. 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, 47Canman C.E. Lim D.-S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1692) Google Scholar) and could therefore also be responsible for increased CBP/p300 recruitment to p53. ATM kinase activity is induced following exposure of cells to ionizing radiation or radiomimetic drugs (46Banin S. Moyal L. Shieh S.Y. 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, 47Canman C.E. Lim D.-S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1692) Google Scholar), and it has been shown that cells deficient for the ATM gene exhibit delayed and reduced phosphorylation of p53 Ser15 following exposure to ionizing radiation (18Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (709) Google Scholar). Although we have used DNA-PK as a reagent for the site-specific phosphorylation of these regulatory sites, it is entirely possible that a different kinase is responsible for the phosphorylation seen in vivo. In fact, it is quite possible that redundant phosphorylation pathways operate. Alternatively, different kinases could be involved according to the cell type and DNA-damaging agent.The question arises of why phosphorylation is utilized for regulation of the p53-CBP/p300 interaction. The answer may lie in the increasingly apparent complexity of the p53 N terminus and its interactions. Not only is the phosphorylation state known to vary with respect to stimulus (13Knippschild U. Milne D.M. Campbell L.E. DeMaggio A.J. Christenson E. Hoekstra M.F. Meek D.W. Oncogene. 1997; 15: 1727-1736Crossref PubMed Scopus (143) Google Scholar, 14Meek D.W. Campbell L.E. Jardine L.J. Knippschild U. McKendrick L. Milne D.M. Biochem. Soc. Trans. 1997; 25: 416-419Crossref PubMed Scopus (24) Google Scholar, 18Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (709) Google Scholar, 48Meek D. Semin. Cancer Biol. 1994; 5: 203-210PubMed Google Scholar, 49Kapoor M. Lozano G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2834-2837Crossref PubMed Scopus (193) Google Scholar), but recent work has identified separate activation subdomains, one existing within the first 42 amino acids and the second lying just C-terminally in amino acids 43–63 (33Zhu J. Zhou W. Jiang J. Chen X. J. Biol. Chem. 1998; 273: 13030-13036Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 50Candau R. Scolnick D.M. Darpino P. Ying C.Y. Halazonetis T.D. Berger S.L. Oncogene. 1997; 15: 807-816Crossref PubMed Scopus (124) Google Scholar). Furthermore, it appears that these separate regions mediate the activation of different subsets of p53 target genes. The second domain has been implicated in directing apoptosis (33Zhu J. Zhou W. Jiang J. Chen X. J. Biol. Chem. 1998; 273: 13030-13036Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Phosphorylation of the p53 N terminus might therefore be used to control protein interactions, regulating transcription of downstream genes and thereby providing a subtle and powerful means for determining the response of the cell to DNA damage.Given the variety of p53 phosphorylation patterns seen in response to different stimuli (13Knippschild U. Milne D.M. Campbell L.E. DeMaggio A.J. Christenson E. Hoekstra M.F. Meek D.W. Oncogene. 1997; 15: 1727-1736Crossref PubMed Scopus (143) Google Scholar, 14Meek D.W. Campbell L.E. Jardine L.J. Knippschild U. McKendrick L. Milne D.M. Biochem. Soc. Trans. 1997; 25: 416-419Crossref PubMed Scopus (24) Google Scholar, 18Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (709) Google Scholar, 48Meek D. Semin. Cancer Biol. 1994; 5: 203-210PubMed Google Scholar, 49Kapoor M. Lozano G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2834-2837Crossref PubMed Scopus (193) Google Scholar), it will be of importance to determine which p53 response pathway(s) stimulates the association of p53 with CBP/p300. Such a response might be restricted to certain types of damage. Certainly, recent reports that UV, but not ionizing radiation, induces phosphorylation of murine p53 on Ser389suggest that different DNA-damaging agents induce different phosphorylation pathways (49Kapoor M. Lozano G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2834-2837Crossref PubMed Scopus (193) Google Scholar). This observation raises the interesting possibility that the response to ionizing radiation, including double-strand break repair, activates p53 binding by acetylation, but the response to UV, including nucleotide excision repair, utilizes phosphorylation of Ser389 or its apparent human equivalent, Ser392. These various pathways would not only allow differentiation between alternative damage responses but also the temporal regulation of the activities of p53, presumably concluding with the restoration of MDM2 inhibition, p53 degradation, and resumption of the cell cycle. p53, which mediates cell cycle arrest and apoptosis in response to DNA damage, is inactivated in approximately 60% of all human cancers (1Bates S. Vousden K.H. Curr. Opin. Genet. Dev. 1996; 6: 12-18Crossref PubMed Scopus (338) Google Scholar, 2Kastan M.B. Canman C.E. Leonard C.J. Cancer Metastasis Rev. 1995; 14: 3-15Crossref PubMed Scopus (440) Google Scholar, 3Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2282) Google Scholar, 4Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6695) Google Scholar). Regulation of the cell cycle and apoptosis is achieved through the function of p53 as a transcription factor, regulating transcription of genes including p21/waf1, GADD45, mdm2, cyclin G, and bax. The structure of p53 can be divided into three main domains: an N-terminal activation domain, a central, sequence-specific DNA-binding domain, and a C-terminal domain, which mediates tetramerization and regulates sequence-specific and nonspecific DNA binding (1Bates S. Vousden K.H. Curr. Opin. Genet. Dev. 1996; 6: 12-18Crossref PubMed Scopus (338) Google Scholar, 2Kastan M.B. Canman C.E. Leonard C.J. Cancer Metastasis Rev. 1995; 14: 3-15Crossref PubMed Scopus (440) Google Scholar, 3Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2282) Google Scholar, 4Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6695) Google Scholar). The N-terminal region interacts with a number of proteins, including MDM2 (5Momand 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), TBP (6Truant R. Xiao H. Ingles C.J. Greenblatt J. J. Biol. Chem. 1993; 268: 2284-2287Abstract Full Text PDF PubMed Google Scholar), dTAFII40 and dTAFII60 (7Thut C.J. Chen J.L. Klemm R. Tjian R. Science. 1995; 267: 100-104Crossref PubMed Scopus (406) Google Scholar), hTAFII31 (8Lu H. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5154-5158Crossref PubMed Scopus (280) Google Scholar), CBP1 (9Gu W. Shi X.L. Roeder R.G. Nature. 1997; 387: 819-823Crossref PubMed Scopus (520) Google Scholar, 10Scolnick D.M. Chehab N.H. Stavridi E.S. Lien M.C. Caruso L. Moran E. Berger S.L. Halazonetis T.D. Cancer Res. 1997; 57: 3693-3696PubMed Google Scholar), the p62 subunit of TFIIH (11Xiao H. Pearson A. Coulombe B. Truant R. Zhang S. Regier J.L. Triezenberg S.J. Reinberg D. Flores O. Ingles C.J. Greenblatt J. Mol. Cell. Biol. 1994; 14: 7013-7024Crossref PubMed Scopus (327) Google Scholar), and the adenovirus E1B 55-kDa protein (12Teodoro J.G. Branton P.E. J. Virol. 1997; 71: 3620-3627Crossref PubMed Google Scholar). It also contains a number of sites that are phosphorylated in vitroby casein kinase I (13Knippschild U. Milne D.M. Campbell L.E. DeMaggio A.J. Christenson E. Hoekstra M.F. Meek D.W. Oncogene. 1997; 15: 1727-1736Crossref PubMed Scopus (143) Google Scholar, 14Meek D.W. Campbell L.E. Jardine L.J. Knippschild U. McKendrick L. Milne D.M. Biochem. Soc. Trans. 1997; 25: 416-419Crossref PubMed Scopus (24) Google Scholar), DNA-dependent protein kinase (DNA-PK) (15Lees-Miller S.P. Sakaguchi K. Ullrich S.J. Appella E. Anderson C.W. Mol. Cell. Biol. 1992; 12: 5041-5049Crossref PubMed Scopus (463) Google Scholar), and c-Jun N-terminal kinase (16Milne D.M. Campbell L.E. Campbell D.G. Meek D.W. J. Biol. Chem. 1995; 270: 5511-5518Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Recently, the cyclin-dependent kinase-activating kinase, which is a cyclin-dependent kinase 7/cyclinH/p36MAT1complex, was also shown to phosphorylate human p53 (17Ko L.J. Shieh S.Y. Chen X. Jayaraman L. Tamai K. Taya Y. Prives C. Pan Z.Q. Mol. Cell. Biol. 1997; 17: 7220-7229Crossref PubMed Scopus (149) Google Scholar). Importantly, Ser15 has been shown to be phosphorylated in vivo in response to ionizing radiation (18Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (709) Google Scholar, 19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar) and is hyperphosphorylated in human T-cell lymphotrophic virus I-transformed cells (20Pise-Masison C.A. Radonovich M. Sakaguchi K. Appella E. Brady J.N. J. Virol. 1998; 72: 6348-6355Crossref PubMed Google Scholar). 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