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• W2000450562 abstract "Previously, we found that the protein kinase C (PKC) inhibitor H7 stimulates p53 to accumulate in a form incapable of inducing transcription from p53-dependent promoters. We concluded that H7 inhibits constitutive C-terminal phosphorylation of p53, which regulates its turnover in unstressed cells. We now show that p53 and its inhibitor MDM2 (HDM2 in human cells) are together in the nuclei of H7-treated cells and can be co-immunoprecipitated. Despite this association of p53 with the ubiquitin ligase MDM2, ubiquitinated p53 was not detected in H7-treated cells. Furthermore, co-treatment with H7 and the proteosome inhibitor LLnL prevented the accumulation of ubiquitinated p53 that was observed in cells treated solely with LLnL. In addition, treatment of cells with the PKC activator phorbol ester stimulated the ubiquitination of p53 and reduced its ability to accumulate after stress. H7 did not induce the phosphorylation of human p53 on Ser-15 (Ser-18 in mouse protein), a modification that occurs in response to DNA damage and leads to the release of MDM2 and to transactivation by p53. We conclude that phosphorylation of the C-terminal domain of p53 by PKC increases its ubiquitination and degradation in unstressed cells. Previously, we found that the protein kinase C (PKC) inhibitor H7 stimulates p53 to accumulate in a form incapable of inducing transcription from p53-dependent promoters. We concluded that H7 inhibits constitutive C-terminal phosphorylation of p53, which regulates its turnover in unstressed cells. We now show that p53 and its inhibitor MDM2 (HDM2 in human cells) are together in the nuclei of H7-treated cells and can be co-immunoprecipitated. Despite this association of p53 with the ubiquitin ligase MDM2, ubiquitinated p53 was not detected in H7-treated cells. Furthermore, co-treatment with H7 and the proteosome inhibitor LLnL prevented the accumulation of ubiquitinated p53 that was observed in cells treated solely with LLnL. In addition, treatment of cells with the PKC activator phorbol ester stimulated the ubiquitination of p53 and reduced its ability to accumulate after stress. H7 did not induce the phosphorylation of human p53 on Ser-15 (Ser-18 in mouse protein), a modification that occurs in response to DNA damage and leads to the release of MDM2 and to transactivation by p53. We conclude that phosphorylation of the C-terminal domain of p53 by PKC increases its ubiquitination and degradation in unstressed cells. Ataxia telangiectasia mutated protein kinase C phosphate-buffered saline CREB-binding protein p300/CBP-associated factor alternative reading frame The tumor suppressor p53 is inactivated in most human tumors (1Levine A.J. Momand J. Finlay C.A. Nature. 1991; 351: 453-456Crossref PubMed Scopus (3665) Google Scholar,2Nigro J.M. Baker S.J. Preisinger A.C. Jessup J.M. Hostetter R. Cleary K. Bigner S.H. Davidson N. Baylin S. Devilee P. Nature. 1989; 342: 705-708Crossref PubMed Scopus (2573) Google Scholar). The protective role of p53 in normal cells depends on its ability to function as a transcription factor, activated in response to DNA damage and other stresses such as hypoxia (3Alarcon R. Koumenis C. Geyer R.K. Maki C.G. Giaccia A.J. Cancer Res. 1999; 59: 6046-6051PubMed Google Scholar), thermal stress (4Nitta M. Okamura H. Aizawa S. Yamaizumi M. Oncogene. 1997; 15: 561-568Crossref PubMed Scopus (79) Google Scholar, 5Ohnishi T. Wang X. Ohnishi K. Takahashi A. Oncogene. 1998; 16: 1507-1511Crossref PubMed Scopus (50) Google Scholar), or nucleotide starvation (6Chernova O.B. Chernov M.V. Ishizaka Y. Agarwal M.L. Stark G.R. Mol. Cell. Biol. 1998; 18: 536-545Crossref PubMed Scopus (57) Google Scholar, 7Linke S.P. Clarkin K.C. Di Leonardo A. Tsou A. Wahl G.M. Genes Dev. 1996; 10: 934-947Crossref PubMed Scopus (483) Google Scholar). Activated p53 moves to the nucleus, where it binds to specific DNA sequences and regulates the expression of many genes involved in the cell cycle, apoptosis, and DNA repair (8Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar,9Oren M. J. Biol. Chem. 1999; 274: 36031-36034Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). The p53 response has three distinct aspects: stabilization of the protein leads to its accumulation, activation of its ability to bind to DNA allows it to localize to specific promoters, and activation of its transactivation domain allows it to interact with the basal transcriptional machinery. All three aspects are regulated by post-translational modification on multiple sites, especially phosphorylation (10Jayaraman L. Prives C. Cell Mol. Life Sci. 1999; 55: 76-87Crossref PubMed Scopus (124) Google Scholar). Stress-induced N-terminal phosphorylations increase the stability of p53 and its ability to interact with the transcriptional machinery, whereas phosphorylation and acetylation of the C-terminal domain facilitates DNA binding. In unstressed cells, the level of wild-type p53 is low because of its short half-life (5–20 min). After induction, the lifetime increases severalfold (11Maltzman W. Czyzyk L. Mol. Cell. Biol. 1984; 4: 1689-1694Crossref PubMed Scopus (818) Google Scholar, 12Price B.D. Calderwood S.K. Oncogene. 1993; 8: 3055-3062PubMed Google Scholar). The proteosome-mediated degradation of p53 is regulated by ubiquitination. The oncoprotein MDM2 (HDM2 in human cells) is a ubiquitin ligase that modifies p53 in its C-terminal domain after it binds to the N-terminal domain (13Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1604) Google Scholar). p53 in turn regulates the transcription of mdm2 (14Barak Y. Juven T. Haffner R. Oren M. EMBO J. 1993; 12: 461-468Crossref PubMed Scopus (1181) Google Scholar), thus forming a feedback loop (15Wu X. Bayle J.H. Olson D. Levine A.J. Genes Dev. 1993; 7: 1126-1132Crossref PubMed Scopus (1645) Google Scholar) that keeps the level of p53 low. The importance of this regulation is highlighted by the embryonic lethality of a homozygous deletion ofmdm2 and the rescue of this lethality by the additional deletion of both copies of the p53 gene (16Montes de Oca L.R. Wagner D.S. Lozano G. Nature. 1995; 378: 203-206Crossref PubMed Scopus (1211) Google Scholar). MDM2 also regulates the activity of p53. When bound to the N-terminal transactivation domain, MDM2 inhibits the ability of p53 to interact with transcription factors such as the TATA-binding protein (17Thut C.J. Goodrich J.A. Tjian R. Genes Dev. 1997; 11: 1974-1986Crossref PubMed Scopus (232) Google Scholar). Stresses induce multiple phosphorylations of the N-terminal domain of p53 (18Appella E. Anderson C.W. Pathol. Biol.(Paris). 2000; 48: 227-245PubMed Google Scholar, 19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1760) Google Scholar, 20Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (711) Google Scholar). Phosphorylation of Thr-18 and Ser-20 of human protein, located in the HDM2 binding site (21Bottger A. Bottger V. Garcia-Echeverria C. Chene P. Hochkeppel H.K. Sampson W. Ang K. Howard S.F. Picksley S.M. Lane D.P. J. Mol. Biol. 1997; 269: 744-756Crossref PubMed Scopus (226) Google Scholar, 22Chehab 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), negatively regulates interaction of the two proteins (22Chehab 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, 23Dumaz N. Milne D.M. Meek D.W. FEBS Lett. 1999; 463: 312-316Crossref PubMed Scopus (109) Google Scholar, 24Shieh S.Y. Taya Y. Prives C. EMBO J. 1999; 18: 1815-1823Crossref PubMed Scopus (266) Google Scholar, 25Unger 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). Ser-20 is phosphorylated by the serine-threonine kinase Chk2 in an ATM1-dependent manner (26Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288Crossref PubMed Google Scholar, 27Hirao 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 (1051) Google Scholar). Less is known about the phosphorylation of Thr-18. The S20A mutation of p53 greatly enhances its binding to MDM2 and decreases its stability dramatically. The phosphorylation of Ser-15, strongly associated with the response to DNA damage (28Burma S. Kurimasa A. Xie G. Taya Y. Araki R. Abe M. Crissman H.A. Ouyang H. Li G.C. Chen D.J. J. Biol. Chem. 1999; 274: 17139-17143Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 29Fiscella 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, 30Kapoor M. Hamm R. Yan W. Taya Y. Lozano G. Oncogene. 2000; 19: 358-364Crossref PubMed Scopus (79) Google Scholar), also affects the p53-MDM2 interaction (31Dumaz N. Meek D.W. EMBO J. 1999; 18: 7002-7010Crossref PubMed Scopus (390) Google Scholar).In vitro experiments have revealed that the phosphatidylinositol 3-kinase family members ATM, ATM-Rad3-related (ATR), and DNA-dependent protein kinase can phosphorylate Ser-15 (28Burma S. Kurimasa A. Xie G. Taya Y. Araki R. Abe M. Crissman H.A. Ouyang H. Li G.C. Chen D.J. J. Biol. Chem. 1999; 274: 17139-17143Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 32Banin 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 (1711) Google Scholar, 33Canman 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, 34Lakin N.D. Hann B.C. Jackson S.P. Oncogene. 1999; 18: 3989-3995Crossref PubMed Scopus (111) Google Scholar), reducing 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 (1760) Google Scholar). Phosphorylation of Ser-15 stimulates p53-dependent transactivation and increases the interaction of p53 with the histone acetyltransferases CBP/p300 and PCAF, co-activators of transcription (31Dumaz N. Meek D.W. EMBO J. 1999; 18: 7002-7010Crossref PubMed Scopus (390) Google Scholar, 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 (363) Google Scholar). Oncogenes modulate the p53-MDM2 interaction independently of post-translational modification. ARF, a product of the p16INK4a locus, binds to MDM2, preventing the degradation of p53. Adenovirus E1a, MYC, and RAS promote the accumulation of p53 by inducing the expression of ARF (36de 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 (548) Google Scholar, 37Eischen C.M. Weber J.D. Roussel M.F. Sherr C.J. Cleveland J.L. Genes Dev. 1999; 13: 2658-2669Crossref PubMed Scopus (712) Google Scholar, 38Palmero I. Pantoja C. Serrano M. Nature. 1998; 395: 125-126Crossref PubMed Scopus (546) Google Scholar). When ARF is overexpressed, ARF/MDM2 complexes accumulate in the nucleoli, preventing p53 from moving from the nucleus to the cytosol, where it can be degraded (39Weber J.D. Kuo M.L. Bothner B. Di Giammarino E.L. Kriwacki R.W. Roussel M.F. Sherr C.J. Mol. Cell. Biol. 2000; 20: 2517-2528Crossref PubMed Scopus (244) Google Scholar). The C-terminal regulatory domain of p53 regulates its ability to bind to DNA, and deletion of this domain activates DNA binding constitutively (40Hupp T.R. Sparks A. Lane D.P. Cell. 1995; 83: 237-245Abstract Full Text PDF PubMed Scopus (448) Google Scholar). Phosphorylation of C-terminal serine residues or binding of the antibody PAb421 to a C-terminal epitope activates the DNA binding activity of p53 (41Hupp T.R. Lane D.P. Cold Spring Harb. Symp. Quant. Biol. 1994; 59: 195-206Crossref PubMed Scopus (106) Google Scholar), as does the acetylation of C-terminal lysine residues by CBP/p300 and PCAF (42Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar). The C-terminal domain is also involved in regulating the degradation of p53, because deletion of as few as 16 C-terminal amino acids significantly decreases MDM2-dependent turnover (43Kubbutat M.H. Ludwig R.L. Ashcroft M. Vousden K.H. Mol. Cell. Biol. 1998; 18: 5690-5698Crossref PubMed Scopus (172) Google Scholar). We demonstrated previously that the constitutive phosphorylation of the C-terminal domain of p53, probably by protein kinase C (PKC), is likely to help to regulate the stability of p53 in unstressed cells. Treatment of mouse or human fibroblasts with the PKC inhibitors H7 or bisindolylmaleimide I increased the half-life of p53, thus causing it to accumulate. In vivo labeling with [32P]orthophosphate of cells and analysis of p53-specific phosphopeptides revealed that the phosphorylation of a peptide from the C-terminal domain that contains a consensus PKC site was reduced upon treatment with H7 (44Chernov M.V. Ramana C.V. Adler V.V. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2284-2289Crossref PubMed Scopus (74) Google Scholar). We have now investigated the mechanism by which p53 is stabilized in response to H7 and demonstrate that preventing the constitutive C-terminal phosphorylation of p53 by PKC prevents ubiquitination without affecting the formation of p53-MDM2 complexes. Stimulation of PKC by phorbol ester induced the ubiquitination of p53 in response to UV and inhibited its accumulation. Mouse (12)1/CA fibroblasts carrying a p53-responsive β-galactosidase reporter construct have been described (45Chernov M.V. Stark G.R. Oncogene. 1997; 14: 2503-2510Crossref PubMed Scopus (43) Google Scholar). RKO cells were obtained from Bert Vogelstein (Johns Hopkins Oncology Center, Baltimore). All cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum. H7 and phorbol ester were obtained from Sigma. Total RNA was extracted by using the TRIzol reagent (Life Technologies, Inc.) according to the manufacturer's protocol. Protein extractions and Western analyses were performed as described by Chernov and Stark (45Chernov M.V. Stark G.R. Oncogene. 1997; 14: 2503-2510Crossref PubMed Scopus (43) Google Scholar) with slight modifications. Briefly, the cells were washed with ice-cold phosphate-buffered saline (PBS), scraped into 1 ml of ice-cold PBS, and collected in Eppendorf tubes. After centrifugation at 2000 ×g for 5 min at 4 °C, the cells were resuspended in up to 100 µl of NET (50 mm Tris-HCl, pH 7.4, 150 mmNaCl, 5 mm EDTA, and 1% Nonidet P-40) buffer with 0.5 mm phenylmethanesulfonyl fluoride, 2 mmbenzamidine, 1 µg/ml each aprotinin, leupeptin, and pepstatin, and 1 mm sodium vanadate and incubated for 10 min on ice. After vortexing for 20 s, the debris was precipitated by centrifugation at 16,000 × g for 15 min at 4 °C. Mouse p53 was detected with the monoclonal antibodies PAb421 and PAb240 (gifts of A. Levine, Rockefeller University, New York, NY). Human p53 was detected by using the DO-1 antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology Inc.). MDM2 and HDM2 were detected by using antibody 2A10 (a gift of M. E. Perry, University of Wisconsin, Madison, WI). Human or mouse p53 phosphorylated on Ser-15 was detected by using the phospho-p53 (Ser-15) antibody (New England Biolabs Inc.). Horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (Bio-Rad) and goat anti-rabbit antibodies (Rockland Immunochemicals Inc.) were detected with the Renaissance chemoluminescence reagent (PerkinElmer Life Sciences). Cells grown on coverslips were fixed with 2% formaldehyde in PBS and then with methanol at room temperature for 12 min each, washed five times with PBS, and blocked for 1 h in Hanks' solution with 5% fetal calf serum and 20 mm Hepes pH 7.8. The coverslips were incubated overnight at 4 °C with primary anti-MDM2 antibody IF2 (CalBiochem). After three washes in PBS-Tween 20, the cells were treated for 1 h at room temperature with secondary fluorescein-conjugated anti-mouse immunoglobulin (Sigma). The cells were washed three times in PBS-Tween 20 with 1 µg/ml 4′,6-diamidino-2-phenylindole (DAPI) in the second wash, and the coverslips were mounted by using 20 µl of Vectashield (Vector Laboratories Inc.). p53-HDM2 complexes were immunoprecipitated with anti-HDM2 SMP14 (BD Biosciences/PharMingen) according to Picksley et al. (46Picksley S.M. Vojtesek B. Sparks A. Lane D.P. Oncogene. 1994; 9: 2523-2529PubMed Google Scholar), except that 0.5% Nonidet P-40 was used. Briefly, the cells were lysed for 20 min on ice, and the lysates were cleared by centrifugation for 15 min at 16,000 × g. Antibody SMP14 (2 µg) was added to 10 mg of total protein, and the mixture was rocked at 4 °C for 1.5 h. Antibody complexes were captured on GammaBind G-Sepharose (Amersham Pharmacia Biotech) by rocking the mixture at 4 °C for 1 h. The Sepharose-bound complex was washed four times in cold lysis buffer, and the proteins were denatured in 2× SDS-polyacrylamide gel electrophoresis loading buffer (100 mm Tris-HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, 20% glycerol, and 1.3 m β-mercaptoethanol) and separated by SDS-polyacrylamide gel electrophoresis (10% acrylamide). After the transfer, the membranes were cut to separate p53 from HDM2; p53 was detected by using DO-1-horseradish peroxidase (Santa Cruz Biotechnology Inc.), and HDM2 was detected with 2A10. For analysis of ubiquitination, p53 was immunoprecipitated by using anti-p53 polyclonal antibody C-19 (Santa Cruz Biotechnology Inc.) according to the same protocol. We examined the ability of the p53 that accumulated in mouse (12)1/CA cells treated with H7 to activate transcription, compared with the p53 that accumulated in cells treated with the proteosome inhibitor LLnL (20Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (711) Google Scholar). Both treatments caused a substantial increase in the levels of p53 (Fig.1A, lanes 3 and4), comparable with the levels found in UV-irradiated cells (Fig. 1 A, lane 2). Despite the high levels of p53, there was only a very small increase in β-galactosidase, the expression of which is regulated in these cells by a p53-dependent promoter (Fig. 1 B). However, when the cells were treated with either inhibitor for 6 h and then irradiated with UV, there was a substantial induction of β-galactosidase (Fig. 1 B), indicating that H7 does not activate pathways that respond to DNA damage and that it causes latent p53 to accumulate by an unknown mechanism. The level and activity of p53 are regulated by HDM2 (MDM2 in mouse cells). Decreased levels of HDM2 or its compartmentalization in nucleoli prevents HDM2 from binding to p53 and transporting it out of the nuclei, leading to its accumulation. We demonstrated previously that p53 accumulates in the nuclei of H7-treated cells (44Chernov M.V. Ramana C.V. Adler V.V. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2284-2289Crossref PubMed Scopus (74) Google Scholar). To determine the role of HDM2 in this process, we examined the expression levels and location of this protein. The amount of hdm2 mRNA in RKO cells treated with H7 remains the same for 1–2 h, then decreases (Fig.2A). The amount of HDM2 protein changes little up to 6 h after treatment (Fig.2 B). Similar observations were made in mouse cells (data not shown). During this time, the level of p53 protein increases significantly. RKO cells treated with H7 for 4 h showed no change in HDM2 localization or level compared with untreated cells (Fig.2 C). The dispersed pattern of staining indicates an even distribution of HDM2 in the nucleus, suggesting that it is available to interact with p53. These data suggest that compartmentalization or changes in the amount of HDM2 are unlikely to be responsible for the accumulation of p53. Phosphorylation of the N-terminal domain is the hallmark of the response of p53 to DNA damage and is important for stabilizing and activating the protein. The phosphorylation of murine Ser-18 (Ser-15 in human p53) by several different protein kinases following many types of cellular insults indicates that modification of this site is pivotal in p53-mediated stress responses (19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1760) Google Scholar, 20Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (711) Google Scholar, 28Burma S. Kurimasa A. Xie G. Taya Y. Araki R. Abe M. Crissman H.A. Ouyang H. Li G.C. Chen D.J. J. Biol. Chem. 1999; 274: 17139-17143Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 32Banin 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 (1711) Google Scholar, 33Canman 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, 34Lakin N.D. Hann B.C. Jackson S.P. Oncogene. 1999; 18: 3989-3995Crossref PubMed Scopus (111) Google Scholar). The latency of the p53 that accumulates after proteosomes are inhibited by LLnL is due to the absence of N-terminal phosphorylation, including the phosphorylation of Ser-15 (19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1760) Google Scholar). Because latent p53 accumulated in cells treated with either H7 or LLnL, we compared the phosphorylation of Ser-18/Ser-15 after either treatment in mouse (12)1/CA fibroblasts and human RKO cells. The cells were treated with UV, H7, and LLnL, individually or in combination, and the phosphorylation of Ser-18/Ser-15 was measured by using anti-P-Ser-15 antibodies. The antibodies PAb240 (mouse) and DO-1 (human) were used to determine the total amount of p53 in each sample. Treatment with either H7 or LLnL did not lead to phosphorylation of Ser-18/Ser-15 after 6 h (Fig.3A, lanes 3 and4). A little phosphorylation of Ser-15 was observed in RKO cells 12 h after treatment with H7, probably because of the stress of prolonged exposure to the inhibitor (Fig. 3 B). This late, low level phosphorylation of Ser-15 is consistent with our earlier result that a low level of p53-dependent gene expression is observed in cells treated with H7 for a long time (44Chernov M.V. Ramana C.V. Adler V.V. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2284-2289Crossref PubMed Scopus (74) Google Scholar). As expected, simultaneous treatment with H7 plus UV, or with LLnL plus UV, induced significant phosphorylation of Ser-18/Ser-15 in either mouse or human cells (Fig. 3 A, lanes 5 and 6). The amount of phosphorylated p53 in co-treated cells was equal to or higher than the levels in cells treated with UV alone (Fig. 3 A, compare lanes 3 and 4 to 5 and6). The levels of Ser-18/Ser-15 phosphorylation correlate with the amounts of p53 protein. The interaction of p53 with HDM2 is a critical event in p53 degradation. In RKO cells treated with H7, we did not detect any phosphorylation of Ser-15. In contrast, the phosphorylation of Ser-15 is frequently observed in parallel with stress-induced dissociation of the p53-HDM2 complex. Furthermore, p53 and HDM2 are both in the nuclei of H7-treated cells (Fig. 2 C). The p53-HDM2 interaction can be blocked by the phosphorylation of Thr-18 or Ser-20, which are in the HDM2 binding site of p53. To analyze the mechanism of p53 stabilization more directly, we tested for the presence of p53-HDM2 complexes (Fig.4). RKO cells were irradiated with UV or treated with H7, and HDM2 was immunoprecipitated 1, 2, 3, or 4 h after treatment. As a positive control, cells were treated with LLnL for 4 h. Proteosome inhibitors cause the accumulation of unmodified p53, which is in a complex with HDM2 (19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1760) Google Scholar). A fraction of each extract was analyzed by direct Western to monitor the levels of p53 and HDM2 (Fig. 4 A). The levels of HDM2 were comparable in H7- and UV-treated cells, remained the same for up to 3 h after treatment, and started to decrease in the case of H7 (or to increase in the case of UV) (Fig. 4 A, lanes 2–5). LLnL caused significant accumulation of both proteins (Fig. 4 A,lane 6), as expected, because both HDM2 and p53 are degraded by proteosomes (47Chang Y.C. Lee Y.S. Tejima T. Tanaka K. Omura S. Heintz N.H. Mitsui Y. Magae J. Cell Growth Differ. 1998; 9: 79-84PubMed Google Scholar). The amount of HDM2 immunoprecipitated from the cells by using the SMP14 antibody reflected the changes shown by direct Western analysis (Fig. 4 B). In contrast, the amount of p53 co-immunoprecipitated with HDM2 differed dramatically after these two treatments. In response to H7, the levels of p53 increased gradually for 4 h, despite declining amounts of HDM2, indicating that the accumulated p53 binds to HDM2. As expected, the p53 that accumulated after UV treatment was not bound to HDM2 and only a slight increase in co-precipitated p53 was observed after 4 h, in parallel with the increase in the level of HDM2. In the positive control, large amounts of p53 were complexed with HDM2 after treatment with LLnL (Fig.4 B, lane 13). Degradation by proteosomes is the major way in which p53 levels are regulated. When p53 accumulates because proteosomes are inhibited, one can observe several differently ubiquitinated forms (48Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3750) Google Scholar). In untreated cells, the lifetimes of the ubiquitinated forms of p53 are so short that they are not readily detected. We determined whether treatment with H7 affects the ubiquitination of p53. Total p53, immunoprecipitated from (12)1/CA or RKO cells using polyclonal antibodies specific for the C-terminal domain, was analyzed. No ubiquitinated p53 was detected in control cells or in cells treated with UV or H7 (Fig.5). As expected, treatment with LLnL induced the accumulation of several ubiquitinated forms with higher apparent molecular weights. Consistent with the observed phosphorylation of the N-terminal domain and the loss of MDM2 binding, treatment with UV reduced the levels of ubiquitination in LLnL treated samples. Surprisingly, despite the presence of p53-HDM2 complexes in H7-treated mouse cells, treatment with H7 completely abolished the ubiquitination of p53. A similar effect was observed in human RKO cells, although a little ubiquitinated p53 could be observed in the untreated cells in this case. UV irradiation reduced the basal levels of ubiquitination in RKO cells, and treatment with H7 blocked ubiquitination completely. Because phorbol ester activates PKC, we tested its effect on p53. Pretreatment of mouse (12)1/CA fibroblasts with phorbol ester for 6 h partially inhibited the accumulation of p53 in response to stress without affecting the basal level (Ref. 44Chernov M.V. Ramana C.V. Adler V.V. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2284-2289Crossref PubMed Scopus (74) Google Scholar and Fig.6A; compare lane 2with lanes 7 and 8). There was no detectable increase in the ubiquitination of p53 in cells in which the UV-induced accumulation of p53 was inhibited by phorbol ester (Fig. 6 A,lanes 7 and 8). We detected a very faint band that migrates at the same position as ubiquitinated p53 only after immunoprecipitation of p53 from approximately five times more cell extract than was used in the direct Western analysis (Fig.6 B, lanes 2 and 8). The capacity of the degradation machinery seems to be high enough to deal with the increased amount of p53 targeted for degradation. Because the ubiquitinated forms of p53 were detected in (12)1/CA cells only after proteosomes were inhibited by LLnL, we used LLnL to expose the effect of phorbol ester on p53 ubiquitination and in this way detected an increase in the amount of ubiquitinated p53 in cells co-treated with UV and phorbol ester (Fig. 6 B, lanes 5 and6). This result suggests that activated PKC might stimulate ubiquitination, thus down-regulating the accumulation of p53 in response to stress. It is well established that post-translational modification is the major mechanism regulating the stabilization and activation of p53 in response to DNA damage (9Oren M. J. Biol. Chem. 1999; 274: 36031-36034Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). Several stress-induced pathways mediate phosphorylation of the N-terminal domain of p53, leading to inhibition of the p53-MDM2 interaction, simultaneously stabilizing p53 and facilitating its activation as a transcription factor, making these two aspects of stress-induced p53 activation mutually interdependent. However, Hupp et al. (40Hupp T.R. Sparks A. Lane D.P. Cell. 1995; 83: 237-245Abstract Full Text PDF PubMed Scopus (448) Google Scholar) demonstrated that low doses of UV could induce p53-dependent transcription without stabilizing the protein, and we have found that sodium salicylate can inhibit the ability of p53 to transactivate without affecting its stability (45Chernov M.V. Stark G.R. Oncogene. 1997; 14: 2503-2510Crossref PubMed Scopus (43) Google Scholar). Furthermore, inhibition of PKC can stabilize a latent form of p53 that is incapable of inducing transcription (44Chernov M.V. Ramana C.V. Adler V.V. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2284-2289Crossref PubMed Scopus (74) Google Scholar). The mechanisms involved in regulating p53 can be divided into those that stabilize and activate p53 in cells exposed to stress and those that operate in unstressed cells, and it is relevant to note that we have recently shown that the basal level of p53 mRNA is maintained through a RAS-dependent mechanism in unstressed cells (49Agarwal M.L. Chilakamarti V.R. 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). Modifications of the C-terminal domains of p53 play a major role in regulating its ability to bind to DNA. There are two well characterized phosphorylation sites in this domain. One includes Ser-392, which is phosphorylated by casein kinase II, and another, containing Ser-376 and Ser-378, is phosphorylated by PKC. Phosphorylation of both sites in vitro by the appropriate kinases stimulates the DNA binding activity of p53 (41Hupp T.R. Lane D.P. Cold Spring Harb. Symp. Quant. Biol. 1994; 59: 195-206Crossref PubMed Scopus (106) Google Scholar). However, although the phosphorylation of Ser-392 is induced in response to DNA damage (50Lu H. Taya Y. Ikeda M. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6399-6402Crossref PubMed Scopus (151) Google Scholar), phosphorylation of the PKC site has never been observed in vivo, even when cells were treated with phorbol ester or other activators of PKC (51Milne D.M. McKendrick L. Jardine L.J. Deacon E. Lord J.M. Meek D.W. Oncogene. 1996; 13: 205-211PubMed Google Scholar). On the other hand, Waterman et al. (52Waterman M.J. Stavridi E.S. Waterman J.L. Halazonetis T.D. Nat. Genet. 1998; 19: 175-178Crossref PubMed Scopus (404) Google Scholar) have shown that in response to ionizing radiation, the PKC site is dephosphorylated, creating binding sites for 14-3-3 proteins. The data suggest that, despite the similar effects of these modifications on p53 in vitro, their in vivo roles may be quite different. As mentioned above, inhibiting PKC stabilizes p53 and activating it destabilizes p53, indicating that the constitutive phosphorylation of the PKC site is likely to play a role in regulating the degradation of p53 in unstressed cells. Under stress, p53 is stabilized by inhibiting its binding to MDM2. In H7-treated cells, the N-terminal domain of p53 was not phosphorylated, and the MDM2-p53 complex was still intact, although the ubiquitination of p53 was inhibited. These observations suggest an alternative mechanism for regulating the degradation of p53 in unstressed cells. Phosphorylation of the C-terminus plays an important role in the allosteric regulation of p53 activity (40Hupp T.R. Sparks A. Lane D.P. Cell. 1995; 83: 237-245Abstract Full Text PDF PubMed Scopus (448) Google Scholar, 53Hupp T.R. Lane D.P. Curr. Biol. 1994; 4: 865-875Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). According to the current model, the unphosphorylated C-terminal domain blocks the DNA-binding domain (40Hupp T.R. Sparks A. Lane D.P. Cell. 1995; 83: 237-245Abstract Full Text PDF PubMed Scopus (448) Google Scholar). The same interaction can protect multiple lysine residues that are located near the PKC site and are required for ubiquitination and degradation (54Nakamura S. Roth J.A. Mukhopadhyay T. Mol. Cell. Biol. 2000; 20: 9391-9398Crossref PubMed Scopus (162) Google Scholar, 55Rodriguez M.S. Desterro J.M. Lain S. Lane D.P. Hay R.T. Mol. Cell. Biol. 2000; 20: 8458-8467Crossref PubMed Scopus (309) Google Scholar). We have found that, in HeLa cells, a human adenocarcinoma cell line in which the level of p53 is low because of the viral oncoprotein E6, the induction of p53 by UV and H7 is severely impaired. In contrast, treatment with LLnL still induces the accumulation of p53 (Ref. 44Chernov M.V. Ramana C.V. Adler V.V. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2284-2289Crossref PubMed Scopus (74) Google Scholar and data not shown). E6 binds to two distinct sites in p53, and the one in the DNA binding domain (56Li X. Coffino P. J. Virol. 1996; 70: 4509-4516Crossref PubMed Google Scholar) is required to stimulate the degradation of p53. The binding of E6 to the core domain probably inhibits its interaction with the C-terminal domain, making the latter available for ubiquitination by E6AP even when the PKC site is not phosphorylated. Interestingly, deletion of 16 C-terminal amino acids protected p53 against MDM2-dependent degradation, but the same protein could still be degraded when E6 was overexpressed (43Kubbutat M.H. Ludwig R.L. Ashcroft M. Vousden K.H. Mol. Cell. Biol. 1998; 18: 5690-5698Crossref PubMed Scopus (172) Google Scholar). This observation correlates very well with the suggested allosteric mechanism of the regulation of p53 degradation by the C-terminal domain. In response to DNA damage, p53 is regulated not only by phosphorylation but also by acetylation (57Liu L. Scolnick D.M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (655) Google Scholar). Primarily, acetylation is involved in regulating the DNA binding activity of p53 (42Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar), but because both acetylation and ubiquitination occur on the same C-terminal lysine residues, acetylation may also be involved indirectly in stabilizing p53 (58Kobet E. Zeng X. Zhu Y. Keller D. Lu H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12547-12552Crossref PubMed Scopus (147) Google Scholar). Phosphorylation of Ser-378 in the PKC site negatively regulates acetylation of the C-terminal domain (59Sakaguchi 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 (1025) Google Scholar), suggesting that deacetylation can enhance the ubiquitination of p53 in unstressed cells. The p53 bound to the p21 promoter in UV-irradiated cells reacts with antibody PAb421 (60Shaulian E. Schreiber M. Piu F. Beeche M. Wagner E.F. Karin M. Cell. 2000; 103: 897-907Abstract Full Text Full Text PDF PubMed Google Scholar), suggesting that it is not phosphorylated at the PKC site because phosphorylation of this site inhibits recognition by PAb421 (41Hupp T.R. Lane D.P. Cold Spring Harb. Symp. Quant. Biol. 1994; 59: 195-206Crossref PubMed Scopus (106) Google Scholar). An involvement of PKC-dependent phosphorylation in the degradation of p53 would also explain why it is difficult to detect phosphorylation of p53 at the C-terminal site even after treating cells with phorbol ester (51Milne D.M. McKendrick L. Jardine L.J. Deacon E. Lord J.M. Meek D.W. Oncogene. 1996; 13: 205-211PubMed Google Scholar). Our data suggest that the inhibition of p53 function by phorbol ester is due to increased degradation, indicating that p53 is targeted for degradation before it can be activated by stress-dependent pathways through N-terminal phosphorylation and explaining why the PKC site has to be dephosphorylated to allow p53 to be activated in response to DNA damage. All together, our data show that the phosphorylation of the C-terminal domain of p53 by PKC represents an important mechanism of negative regulation in unstressed cells, preventing the acetylation of this domain and blocking its interaction with factors such as 14-3-3, which stimulate the ability of p53 to bind to DNA. This phosphorylation by PKC also regulates the constitutive degradation of p53 in unstressed cells by stimulating ubiquitination. The vital role of tight negative regulation of p53 in unstressed cells is highlighted by the dramatic effect of the deletion of mdm2 on mouse development, apparent in cells that have p53 although not in p53-null cells (16Montes de Oca L.R. Wagner D.S. Lozano G. Nature. 1995; 378: 203-206Crossref PubMed Scopus (1211) Google Scholar). We thank Arnold Levine and Mary E. Perry for a generous supply of monoclonal antibodies." @default.
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• W2000450562 title "Regulation of Ubiquitination and Degradation of p53 in Unstressed Cells through C-terminal Phosphorylation" @default.
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