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• W1976050753 abstract "The function and stability of the tumor suppressor p53 are tightly controlled by the negative regulator mouse double minute 2 (Mdm2), which binds to p53, blocking DNA binding and targeting p53 for proteosome-mediated degradation. Following DNA damage or cellular stress, p53 is phosphorylated within the Mdm2 binding domain on threonine 18 and serine 20. To analyze the roles of these phosphorylation events, residues 18 and 20 were mutated to alanines. Transient transfection into p53-null cells demonstrated that the T18A protein can be expressed stably, but the S20A protein is very unstable, precluding further analysis. When expressed stably at low basal levels in p53-null human fibroblasts or fibrosarcoma cells, the T18A mutant accumulated 5–10-fold less well than wild-type p53 following exposure to UV. Analysis of p53-dependent transcription following UV revealed that the phosphorylation of threonine 18 is required for transactivation of the p21, Hdm2 (the human ortholog of Mdm2), and GADD45 genes. The phosphorylation of serine 33, another early event following DNA damage, is not required for p53 accumulation or p53-dependent transactivation following UV. The function and stability of the tumor suppressor p53 are tightly controlled by the negative regulator mouse double minute 2 (Mdm2), which binds to p53, blocking DNA binding and targeting p53 for proteosome-mediated degradation. Following DNA damage or cellular stress, p53 is phosphorylated within the Mdm2 binding domain on threonine 18 and serine 20. To analyze the roles of these phosphorylation events, residues 18 and 20 were mutated to alanines. Transient transfection into p53-null cells demonstrated that the T18A protein can be expressed stably, but the S20A protein is very unstable, precluding further analysis. When expressed stably at low basal levels in p53-null human fibroblasts or fibrosarcoma cells, the T18A mutant accumulated 5–10-fold less well than wild-type p53 following exposure to UV. Analysis of p53-dependent transcription following UV revealed that the phosphorylation of threonine 18 is required for transactivation of the p21, Hdm2 (the human ortholog of Mdm2), and GADD45 genes. The phosphorylation of serine 33, another early event following DNA damage, is not required for p53 accumulation or p53-dependent transactivation following UV. mouse double minute 2 casein kinase I adriamycin ionizing radiation The p53 tumor suppressor is a transcription factor that plays a critical role in maintaining genomic stability by mediating cellular growth arrest, DNA repair, and apoptosis after DNA damage. Loss of p53 function increases the potential for cell proliferation in the presence of damaged DNA (1Ljungman M. Neoplasia. 2000; 2: 208-225Crossref PubMed Scopus (186) Google Scholar, 2Prives C. Hall P.A. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1233) 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 (649) Google Scholar). It is extremely important that the activity of this mediator of cellular life and death be tightly regulated. Control of p53 function is enforced, in large part, by its negative regulator mouse double minute 2 (Mdm21 or Hdm2 in human cells). p53 and Mdm2 comprise a feedback loop. Mdm2 binds to the N-terminal domain of p53 (amino acids 17–27), blocking its transactivation function. Because Mdm2 is an E3 ubiquitin ligase, it also modifies p53 and targets it for proteosome-mediated degradation (4Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3745) Google Scholar, 5Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1604) Google Scholar, 6Kubbutat M.H. Vousden K.H. Mol. Cell. Biol. 1997; 17: 460-468Crossref PubMed Scopus (277) Google Scholar). p53, in turn, is a transcription factor for the Mdm2 gene (7Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. Cell. 1992; 69: 1237-1245Abstract Full Text PDF PubMed Scopus (2798) Google Scholar). Following DNA damage, the p53-Mdm2 interaction is lost, and the p53 protein accumulates and binds to DNA (8Fuchs S.Y. Adler V. Buschmann T. Wu X. Ronai Z. Oncogene. 1998; 17: 2543-2547Crossref PubMed Scopus (210) Google Scholar). The properties of human tumors and Mdm2-null mice exemplify the importance of this regulatory loop. Mdm2-null mice do not survive because of overexpression of p53, but Mdm2-null, p53-null mice do survive (9Jones S.N. Roe A.E. Donehower L.A. Bradley A. Nature. 1995; 378: 206-208Crossref PubMed Scopus (1070) Google Scholar). Mdm2 was first identified as a protein overexpressed in a mouse tumor cell line carrying an amplification of the gene in double minute chromosomes (10Fakharzadeh S.S. Trusko S.P. George D.L. EMBO J. 1991; 10: 1565-1569Crossref PubMed Scopus (628) Google Scholar). Hdm2 has been found to be amplified in human tumors (11Oliner J.D. Kinzler K.W. Meltzer P.S. George D.L. Vogelstein B. Nature. 1992; 358: 80-83Crossref PubMed Scopus (1806) Google Scholar). In these cases, overexpression of Mdm2 or Hdm2 depletes cellular p53. In unstressed cells, p53 is present at a low basal level in a complex with Mdm2 (8Fuchs S.Y. Adler V. Buschmann T. Wu X. Ronai Z. Oncogene. 1998; 17: 2543-2547Crossref PubMed Scopus (210) Google Scholar). Following DNA damage, the human p53 N-terminal region is phosphorylated on serines 6, 9, 15, 20, 33, and 37 and threonine 18. The association with Mdm2 is lost (8Fuchs S.Y. Adler V. Buschmann T. Wu X. Ronai Z. Oncogene. 1998; 17: 2543-2547Crossref PubMed Scopus (210) Google Scholar, 12Higashimoto Y. Saito S. Tong X.H. Hong A. Sakaguchi K. Appella E. Anderson C.W. J. Biol. Chem. 2000; 275: 23199-23203Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 13Sakaguchi K. Saito S. Higashimoto Y. Roy S. Anderson C.W. Appella E. J. Biol. Chem. 2000; 275: 9278-9283Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 14Shieh S.Y. Taya Y. Prives C. EMBO J. 1999; 18: 1815-1823Crossref PubMed Scopus (266) Google Scholar, 15Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (597) Google Scholar, 16Shieh S.-Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1760) Google Scholar, 17Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (712) Google Scholar). These post-translational modifications also enable p53 to bind to DNA and transactivate its target genes (reviewed in Ref. 1Ljungman M. Neoplasia. 2000; 2: 208-225Crossref PubMed Scopus (186) Google Scholar). Mdm2 binds to p53 within a region spanning amino acids 17–27 (18Kussie P.H. Gorina S. Marechal V. Elenbaas B. Moreau J. Levine A.J. Pavletich N.P. Science. 1996; 274: 948-953Crossref PubMed Scopus (1808) Google Scholar). Threonine 18 and serine 20 are the only p53 phosphorylation sites within the p53-Mdm2 interaction domain; therefore, they are the most likely to regulate the p53-Mdm2 interaction. Consistent with this idea, p53-derived peptides phosphorylated on threonine 18 or serine 20, but not on serines 15, 33, or 37, showed reduced affinity for Mdm2 (13Sakaguchi K. Saito S. Higashimoto Y. Roy S. Anderson C.W. Appella E. J. Biol. Chem. 2000; 275: 9278-9283Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 19Bottger V. Bottger A. Garcia-Echeverria C. Ramos Y.F. Van der Eb A.J. Jochemsen A.G. Lane D.P. Oncogene. 1999; 18: 189-199Crossref PubMed Scopus (151) Google Scholar, 20Dumaz N. Meek D.W. EMBO J. 1999; 18: 7002-7010Crossref PubMed Scopus (390) Google Scholar, 21Unger T. Juven-Gershon T. Moallem E. Berger M. Vogt S.R. Lozano G. Oren M. Haupt Y. EMBO J. 1999; 18: 1805-1814Crossref PubMed Scopus (315) Google Scholar). Threonine 18 is phosphorylated in vitro by casein kinase I (CKI) (13Sakaguchi K. Saito S. Higashimoto Y. Roy S. Anderson C.W. Appella E. J. Biol. Chem. 2000; 275: 9278-9283Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 22Dumaz N. Milne D.M. Meek D.W. FEBS Lett. 1999; 463: 312-316Crossref PubMed Scopus (109) Google Scholar). The role of CKI in the DNA damage response is not known. However, CKI recognition sites are phosphorylated more efficiently when a negative charge is present three residues away in the direction of the N terminus, creating a recognition site for this ubiquitously expressed kinase (reviewed in Ref. 23Gross S.D. Anderson R.A. Cell. Signal. 1998; 10: 699-711Crossref PubMed Scopus (269) Google Scholar). For example, serine 15, which is phosphorylated by stress-activated kinases such as DNA-PKII, ATM, and ATR, is three residues away from threonine 18. The phosphorylation of serine 15 may make threonine 18 more susceptible to phosphorylation by CKI (24Tibbetts R.S. Brumbaugh K.M. Williams J.M. Sarkaria J.N. Cliby W.A. Shieh S.Y. Taya Y. Prives C. Abraham R.T. Genes Dev. 1999; 13: 152-157Crossref PubMed Scopus (867) Google Scholar, 25Banin 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 (1710) Google Scholar, 26Canman 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 (1712) Google Scholar, 27Khanna K.K. Lavin M.F. Oncogene. 1993; 8: 3307-3312PubMed Google Scholar, 28Lees-Miller S.P. Sakaguchi K. Ullrich S.J. Appella E. Anderson C.W. Mol. Cell. Biol. 1992; 12: 5041-5049Crossref PubMed Scopus (465) Google Scholar, 29Wang Y. Eckhart W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4231-4235Crossref PubMed Scopus (73) Google Scholar). In support of this idea, efficient phosphorylation of threonine 18 by CKI was observed only after serine 15 had been phosphorylated (13Sakaguchi K. Saito S. Higashimoto Y. Roy S. Anderson C.W. Appella E. J. Biol. Chem. 2000; 275: 9278-9283Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 22Dumaz N. Milne D.M. Meek D.W. FEBS Lett. 1999; 463: 312-316Crossref PubMed Scopus (109) Google Scholar). Serine 20 is phosphorylated in vitro by Chk1 and Chk2, DNA damage-responsive kinases that lie downstream of ATM, which is activated by double-strand breaks (30Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288Crossref PubMed Google Scholar, 31Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (694) Google Scholar, 32Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300Crossref PubMed Google Scholar, 33Chaturvedi P. Eng W.K. Zhu Y. Mattern M.R. Mishra R. Hurle M.R. Zhang X. Annan R.S. Lu Q. Faucette L.F. Scott G.F. Li X. Carr S.A. Johnson R.K. Winkler J.D. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar). Mutation of serine 20 reduces the stability of p53 after DNA damage (34Chehab N.H. Malikzay A. Stavridi E.S. Halazonetis T.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13777-13782Crossref PubMed Scopus (461) Google Scholar). Furthermore,Chk2 is a tumor suppressor gene because germline mutation ofChk2 results in Li-Fraumeni Syndrome (LFS), a highly penetrant cancer predisposition syndrome usually caused by germline mutations of p53 (35Bell D.W. Varley J.M. Szydlo T.E. Kang D.H. Wahrer D.C. Shannon K.E. Lubratovich M. Verselis S.J. Isselbacher K.J. Fraumeni J.F. Birch J.M. Li F.P. Garber J.E. Haber D.A. Science. 1999; 286: 2528-2531Crossref PubMed Scopus (764) Google Scholar, 36Malkin 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 (3075) Google Scholar). Mutations in Chk2 have been identified in human tumors, and p53 is not stabilized in cells from Chk2-null mice following DNA damage (37Wu X. Webster S.R. Chen J. J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 38Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1050) Google Scholar). Taken together, this evidence suggests a critical role for Chk2 in signaling to p53, probably through the phosphorylation of serine 20. Use of phosphospecific antibodies has demonstrated that both threonine 18 and serine 20 are phosphorylated after DNA damage, suggesting a role for these sites in regulating p53-Mdm2 association and therefore, in p53 protein stability (14Shieh S.Y. Taya Y. Prives C. EMBO J. 1999; 18: 1815-1823Crossref PubMed Scopus (266) Google Scholar, 24Tibbetts R.S. Brumbaugh K.M. Williams J.M. Sarkaria J.N. Cliby W.A. Shieh S.Y. Taya Y. Prives C. Abraham R.T. Genes Dev. 1999; 13: 152-157Crossref PubMed Scopus (867) Google Scholar, 34Chehab N.H. Malikzay A. Stavridi E.S. Halazonetis T.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13777-13782Crossref PubMed Scopus (461) Google Scholar, 39Sakaguchi 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 (1024) Google Scholar). Phosphorylation of serine 20 after exposure of cells to ionizing radiation (IR) or UV correlates with the timing of p53 protein accumulation, which occurs 1–2 h after IR and 4 h after UV (13Sakaguchi K. Saito S. Higashimoto Y. Roy S. Anderson C.W. Appella E. J. Biol. Chem. 2000; 275: 9278-9283Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 14Shieh S.Y. Taya Y. Prives C. EMBO J. 1999; 18: 1815-1823Crossref PubMed Scopus (266) Google Scholar). The timing of threonine 18 phosphorylation has not been studied as carefully, although one report does show phosphorylation of this site 2 h after IR (13Sakaguchi K. Saito S. Higashimoto Y. Roy S. Anderson C.W. Appella E. J. Biol. Chem. 2000; 275: 9278-9283Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Thus, the phosphorylations of threonine 18 and serine 20 are relatively early events following DNA damage and are likely to play a role in the dissociation of p53 from Mdm2. Serine 33 is also phosphorylated very soon after DNA is damaged, within 1 h following irradiation with UV or IR (14Shieh S.Y. Taya Y. Prives C. EMBO J. 1999; 18: 1815-1823Crossref PubMed Scopus (266) Google Scholar, 15Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (597) Google Scholar, 39Sakaguchi 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 (1024) Google Scholar). Serine 33 is phosphorylated by Cdk-activating kinase (CAK), a component of the RNA polymerase II holoenzyme (40Ko 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). This interaction could provide a signal to p53 from RNA polymerase that has been stalled by polymerase poisons or bulky DNA lesions. We have previously restored wild-type and phosphorylation-site mutant p53 cDNAs to p53-null fibroblasts by using a tetracycline-regulated system (41Bean L.J.H. Stark G.R. Oncogene. 2001; 20: 1076-1084Crossref PubMed Scopus (56) Google Scholar). This system allowed us to express p53 at low basal levels in cells with a normal p53 response and to examine the properties of these mutant proteins. In the current study, we examine the effect of mutating additional residues that are phosphorylated soon after DNA-damaging events on p53 accumulation and p53-dependent transactivation following DNA damage. pTO and pTA.hygro were previously reported (42Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4267) Google Scholar). pTO.neo and pTO.p53 wt.neo were previously described (41Bean L.J.H. Stark G.R. Oncogene. 2001; 20: 1076-1084Crossref PubMed Scopus (56) Google Scholar). pTO.p53.S20A.neo and pTO.p53.S33A.neo were generated by PCR-SOEing (41Bean L.J.H. Stark G.R. Oncogene. 2001; 20: 1076-1084Crossref PubMed Scopus (56) Google Scholar) using the S20A mutagenic forward primer, 5′-GGAAACATTTGCAGACCTATGG-3′; reverse mutagenic primer, 5′-CCATAGGTCTGCAAATGTTTCC-3′; S33A forward mutagenic primer, 5′-CAACGTTCTGGCCCCCTTGCC-3′; and reverse mutagenic primer, 5′-GGCAAGGGGGCCAGAACGTTG-3′. The T18A mutation was created by amplifying pTO.p53.neo, using the long forward primer T18A.l-F (5′-GTCACTGCCATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAGCATTTTC-3′) with the reverse primer p53UTR-R (5′-GGCTGGGGCGCGGAGCTGG-3′). The PCR product was gel-purified, digested with NcoI, and ligated into pBS.p53 and then pTO.neo as previously described (41Bean L.J.H. Stark G.R. Oncogene. 2001; 20: 1076-1084Crossref PubMed Scopus (56) Google Scholar). The resulting construct, pTO.p53.T18A-P.neo and all previously described pTO.p53 constructs contain a proline residue at amino acid 72. pTO.p53.T18A-R.neo was constructed by removing the p53 NcoI N-terminal fragment from p53-18ApCB6+ (a gift from Margaret Ashcroft, National Institutes of Heath, Bethesda, MD) and ligating it into pBS.p53 and pTO.neo. This construct contains an arginine residue at amino acid 72. Cells were grown in Dulbecco's minimal essential medium (Invitrogen), supplemented with antibiotics and 10% fetal bovine serum (Invitrogen) in a humidified atmosphere containing 10% CO2. MDAH041, a spontaneously immortalized Li-Fraumeni skin fibroblast cell line, was described previously (43Bischoff F.Z. Strong L.C. Yim S.O. Pratt D.R. Siciliano M.J. Giovanella B.C. Tainsky M.A. Oncogene. 1991; 6: 183-186PubMed Google Scholar) as were MDAH041 cells stably transfected with pTA (44Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (801) Google Scholar). The Mwt (previously described in Ref. 41Bean L.J.H. Stark G.R. Oncogene. 2001; 20: 1076-1084Crossref PubMed Scopus (56) Google Scholar as TRwt cells), M18, and M33 cell lines were generated by calcium phosphate transfection of pTO.p53.neo constructs into MDAH041.pTA (hygro) cells in the presence of tetracycline (1 μg/ml). M18-R clones have an arginine residue at amino acid 72. All others have a proline residue at amino acid 72. Selection of stable clones was done with G418 (400 μg/ml active). Clones screened by Western transfer for p53 expression were prepared by first removing tetracycline for 24 h, then treating the cells with adriamycin (ADR) (200 ng/ml) for an additional 24 h to increase the levels of p53. Clones Mwt-3, M18-2R, M18-3R, M33-2, and M33-3 had more p53 protein than Mwt-2; therefore, they were used in the presence of tetracycline (0.01 μg/ml) to normalize the basal p53 levels to that of Mwt-2 cells. The 3-7 cells were derived from HT1080 cells through a mutagenesis strategy described in Agarwal et al.(45Agarwal M.L. Ramana C.V. Hamilton M. Taylor W.R. DePrimo S.E. Bean L.J.H. Agarwal A. Agarwal M.K. Wolfman A. Stark G.R. Oncogene. 2001; 20: 2527-2536Crossref PubMed Scopus (45) Google Scholar) and express 99% less p53 mRNA and p53 protein than parental HT1080 cells. The 3-7.pTA cells were generated by calcium phosphate-mediated transfection of 3-7 cells with pTA.hygro (described in Ref. 42Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4267) Google Scholar). Stable clones were selected in 250 μg/ml hygromycin. Because the pTA protein is required for pTO promoter function, positive transfectants were identified by their capability to express luciferase following calcium phosphate-mediated transient transfection of pTO.luciferase. Luciferase expression was assayed with the Promega assay system. SV2-βgal was co-transfected as a control. A single 3-7.pTA clone, selected for an intermediate level of pTA expression, was chosen as the parental 3-7.pTA line. Hwt, H18, and H33 clones were generated by transfection of 3-7.pTA cells with pTO.p53.neo, pTO.p53.T18A-P.neo, or pTO.p53.S33A.neo in the presence of hygromycin and 1 μg/ml tetracycline. Stable clones were selected in 600 μg/ml (active) G418 and screened for p53 expression by Western analysis. Four separate Hwt, H18, or H33 clones expressing similar low p53 basal levels were identified. Clones were maintained individually. For each experiment, Hwt, H18, or H33 cells were counted using a hemocytometer, pooled in equal numbers, and plated for 24 h prior to treatment. Western transfers onto polyvinylidene difluoride (PVDF) membranes (Millipore) were performed with whole cell extracts after separation by SDS-polyacrylamide gel electrophoresis (10% acrylamide). Protein concentrations of lysates were determined by the Bradford method (Bio-Rad), and equal quantities of protein were loaded for each sample. To detect p53, the cells were lysed in 20 mm Tris hydrochloride, pH 7.4; 1% Nonidet P-40; 150 mm NaCl; 5 mm EDTA; 1 mmphenylmethane sulfonyl fluoride; 10 μg/ml aprotinin; 25 μg/ml leupeptin; and 1 μg/ml pepstatin A. The membranes were probed with the DO-1 monoclonal antibody to p53 (Santa Cruz Biotechnology), which was detected with a goat anti-mouse antibody conjugated to horseradish peroxidase (Bio-Rad) using enhanced chemiluminescence (Dupont Pharmaceuticals). To quantify the levels of p53, the membranes were analyzed by using enhanced chemifluorescence (Dupont Pharmaceuticals) and read using a StormImager (Molecular Dynamics). The results were analyzed with ImageQuant software. Serine 15 phosphorylation was detected using phospho-p53 (Ser15) antibody (Perkin Elmer Life Sciences). Total RNA was extracted with the Trizol reagent (Invitrogen), separated by electrophoresis in a denaturing agarose gel, transferred to Hybond-N+ nylon membranes (Amersham Biosciences) by capillary action, and probed with32P-labeled probes for HDM, p21, GADD45, or GAPDH. Quantitation was performed by using a PhosphorImager (Molecular Dynamics), and the results were analyzed with ImageQuant software. All treatments were performed 24 h after removal of tetracycline. The UV dose in all cases was measured by using a Traceable Ultra Violet Light Meter (Control Company). The optimal dose for each cell type was the dose that gave the highest level of p53 accumulation without down-regulating p53-responsive genes. MHAD041-derived clones were treated with 25 J/m2, and HT1080-derived clones were treated with 20 J/m2. UV irradiation was performed after removal of most of the culture medium. The optimal adriamycin dose was determined by a dose response curve. Maximal p53 accumulation was achieved at 200 ng/ml medium in MDAH041-derived cells and 300 ng/ml medium in HT1080-derived cells. An estimate of the mean for each individual clone was calculated as best linear unbiased estimates, where individual clones were considered to be random effects and each mean was regressed toward the overall mean for each clone type (46Robinson G. Stat. Sci. 1991; 6: 15-51Crossref Scopus (1078) Google Scholar). The overall mean for each type of clone was estimated as a least squares mean, with individual clones considered to be random effects. Calculated p-values among clone types were adjusted for multiple comparisons using the Tukey-Kramer method to maintain an overall 0.05 significance level for each hypothesis (47Kramer C. Biometrics. 1956; 12: 310Crossref Google Scholar). Standard errors for each clone type and individual clone were calculated as the square root of the variance of the mean, which was a function of the within- and between-clone variability. Analyses were done using Mixed Procedure (SAS Statistical Software). To examine the importance of threonine 18 or serine 20 in regulating p53 stability and function, pTO.p53wt, pTO.p53.T18A-P, and pTO.p53.S20A were transfected transiently into p53-null MDAH041 cells. Western analysis revealed that, although the wild-type and T18A mutant proteins were expressed at high levels, the S20A mutant protein was nearly undetectable (Fig. 1). Northern analysis revealed high levels of all three mRNAs, indicating that it is the S20A protein and not the S20A mRNA that is unstable (Fig. 1). Consistent with the data from transient transfections, clones stably expressing the S20A protein could not be generated. These results indicate that serine 20 is required for basal stability of the p53 protein. We have used a tetracycline-regulated system to express wild-type and phosphorylation site mutant p53 proteins at low basal levels in MDAH041 fibroblasts (41Bean L.J.H. Stark G.R. Oncogene. 2001; 20: 1076-1084Crossref PubMed Scopus (56) Google Scholar). Expression of relatively high levels of wild-type p53 in these cells results in the transactivation of downstream genes and in growth arrest (44Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (801) Google Scholar). Six independent 041-derived clones stably expressing T18A were isolated. Clones M18-1P and M18-2P have a proline residue at position 72, whereas M18-1R through M18-4R have an arginine residue at this position. All other wild-type and mutant clones have a proline at amino acid 72. This naturally occurring polymorphism at position 72 does not affect the accumulation of p53 after DNA damage (see below). The accumulation of the T18A mutant protein in response to UV was impaired compared with that of the wild-type clone Mwt3 (Fig.2A). To quantify the difference between the accumulation of these two proteins, the levels of p53 were measured by chemifluorescence 18 h after irradiation with UV (Fig. 2B). For each clone, the mean percentage increase and the standard error were determined as described in “Materials and Methods.” The average percentage increase for all T18A clones was determined and compared with the wild-type average. Analysis of variance for pairwise comparisons of each type of clone indicated that the accumulation of the protein in M18 clones (average 87%) was significantly lower than in wild-type clones (average 460%;p < 0.001). Therefore, threonine 18 is required for the p53 protein to accumulate efficiently following irradiation with UV in MDAH041 cells. A second p53-null cell line was also utilized. Following mutagenesis (45Agarwal M.L. Ramana C.V. Hamilton M. Taylor W.R. DePrimo S.E. Bean L.J.H. Agarwal A. Agarwal M.K. Wolfman A. Stark G.R. Oncogene. 2001; 20: 2527-2536Crossref PubMed Scopus (45) Google Scholar), the 3-7 clone was isolated from HT1080 fibrosarcoma cells, which express high levels of wild-type p53. p53 mRNA and protein expression are reduced by 99% in 3-7 cells (data not shown). The levels of exogenous p53 must be carefully regulated in HT1080-derived cells because, no accumulation was observed after DNA damage in clones that express a high level of p53. Four 3-7-derived clones expressing wild-type (Hwt clones) and four expressing the T18A mutant (H18 clones) were isolated and compared with a pool of Hwt cells. Accumulation of the T18A mutant protein was also compared with wild-type protein in 3-7 cells. Western analysis showed that pooled H18 clones accumulated less p53 protein than pooled Hwt clones (Fig. 2D). The percentage increase in wild-type or mutant p53 was quantified over the 24-h time course and is represented graphically. As in MDAH041 cells, threonine 18 is required for p53 to accumulate efficiently after UV damage. Because serine 15 is required for p53 to accumulate after UV damage, its phosphorylation was analyzed in M18 and H18 clones after UV irradiation using a phosphospecific antibody (Fig. 2, C andD). Phosphorylation of serine 15 was observed in both M18 and H18 clones, albeit at a much lower level than in wild-type controls. This deficiency corresponds to the lower levels of mutant protein after UV damage. These results could not be quantified by StormImager analysis because of the relatively weak phosphospecific antibody signal. Four MDAH041-derived clones stably expressing low basal levels of the S33A mutant protein (M33 clones) were isolated and compared with Mwt clones for p53 accumulation following DNA damage. Four 3-7-derived clones expressing the S33A mutant proteins (H33 clones) were also isolated, pooled, and compared with pooled Hwt clones. p53 in Mwt clones and M33 clones accumulated similarly for 24 h following UV (Fig.3A). The phosphorylation of serine 15 was normal in M33 clones compared with Mwt clones. p53 accumulation was quantified by chemifluorescence 18 h after irradiation with UV in four independent clones. Analysis of variance for pairwise comparisons between clone types revealed no significant difference between wild-type and mutant clones (Fig. 3B). Similarly, there was no difference in accumulation or serine 15 phosphorylation between wild-type and S33A mutant proteins in pooled 3-7 clones following irradiation with UV (Fig. 3C). Because serine 33 is phosphorylated very soon after irradiation with UV (14Shieh S.Y. Taya Y. Prives C. EMBO J. 1999; 18: 1815-1823Crossref PubMed Scopus (266) Google Scholar,39Sakaguchi 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 (1024) Google Scholar), the accumulation of p53 in Mwt and M33 clones 1, 2, and 3 h and Hwt and H33 pools 30, 60, and 90 min after exposure to UV was examined. The accumulation and serine 15 phosphorylation of the wild-type and S33A proteins were similar in both cell systems (Fig. 3D). Mwt, M18, and M33 clones were treated with 200 ng/ml adriamycin, and the accumulation of p53 was compared with Mwt clones over a 24-h time course. The accumulation of p53 in all three was similar (data not shown). The phosphorylation of serine 15 was observed in wild-type, M18, and M33" @default.
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• W1976050753 title "Regulation of the Accumulation and Function of p53 by Phosphorylation of Two Residues within the Domain That Binds to Mdm2" @default.
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