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• W2032299946 abstract "The tumor suppressor protein p53 functions as a transcriptional factor that activates genes controlling cell cycle arrest and apoptosis. Here, we report that protein inhibitor of activated Stat1 (PIAS1) interacts with the tetramerization and C-terminal regulatory domains of p53 in yeast two-hybrid analyses. Endogenous PIAS1 is also associated with endogenous p53 in mammalian cells. Ectopic expression of PIAS1 activates p53-mediated expression in mouse embryonic fibroblast cells (p53 −/−) as well as a variety of other cell lines. Furthermore, ectopic expression of PIAS1 induces p53-mediated expression of cyclin-dependent kinase inhibitor p21 and G1arrest of the cell cycle in H1299 cells. In addition, a PIAS1 mutant without the RING-finger domain required for sumoylation could still activate p53-mediated gene expression, indicating that activation of p53 by PIAS1 does not require the RING-finger domain. Taken together, our results suggest that PIAS1 is a novel activator of p53. The tumor suppressor protein p53 functions as a transcriptional factor that activates genes controlling cell cycle arrest and apoptosis. Here, we report that protein inhibitor of activated Stat1 (PIAS1) interacts with the tetramerization and C-terminal regulatory domains of p53 in yeast two-hybrid analyses. Endogenous PIAS1 is also associated with endogenous p53 in mammalian cells. Ectopic expression of PIAS1 activates p53-mediated expression in mouse embryonic fibroblast cells (p53 −/−) as well as a variety of other cell lines. Furthermore, ectopic expression of PIAS1 induces p53-mediated expression of cyclin-dependent kinase inhibitor p21 and G1arrest of the cell cycle in H1299 cells. In addition, a PIAS1 mutant without the RING-finger domain required for sumoylation could still activate p53-mediated gene expression, indicating that activation of p53 by PIAS1 does not require the RING-finger domain. Taken together, our results suggest that PIAS1 is a novel activator of p53. protein inhibitor of activated Stat1 signal transducer and activator of transcription small ubiquitin-related modifier-1 glutathione S-transferase mouse embryonic fibroblast fetal bovine serum phosphate-buffered saline wash buffer fluorescein isothiocyanate c-Jun NH2-terminal kinase The tumor suppressor protein p53 is normally very short-lived and remains at very low levels through proteasomal degradation in unstressed mammalian cells. In response to damaged DNA, nucleotide depletion, hypoxia, oncogenes, and other genotoxic stresses, p53 accumulates dramatically in nucleus and functions as a transcriptional activator (1Prives C. Hall P.A. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1234) Google Scholar, 2Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar, 3Appella E. Anderson C.W. Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (916) Google Scholar, 4Ryan K.M. Phillips A.C. Vousden K.H. Curr. Opin. Cell Biol. 2001; 13: 332-337Crossref PubMed Scopus (583) Google Scholar). Activation of p53 leads to cell cycle arrest or apoptosis by inducing a number of genes including cyclin-dependent kinase inhibitor p21 and apoptotic genes such as Bax. Although the exact mechanisms by which some of these stress signals are transduced to p53 are not known, signaling to p53 is thought to be mediated by upstream p53 regulators in at least three distinguishable pathways (5Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5850) Google Scholar). The first pathway transduces signals of DNA damage induced by ionizing radiation, which activates ATM, Chk2, and subsequently p53. The second pathway is mediated by p14ARF-MDM2 interaction (6Sherr C.J. Weber J.D. Curr. Opin. Genet. Dev. 2000; 10: 94-99Crossref PubMed Scopus (572) Google Scholar), which is triggered by oncogenic signals such as Ras and Myc. The third pathway is turned on by a wide array of chemotherapeutic drugs, UV light, and protein kinase inhibitors, which requires kinases ATR and casein kinase II. There are additional proteins that can activate p53, but the significance of some of these interactions remains to be elucidated (1Prives C. Hall P.A. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1234) Google Scholar). The key negative regulator of the p53 network is MDM2. Transcription of MDM2 is itself activated by p53. However, MDM2 protein can both inhibit the transcriptional activity of p53 and target p53 for ubiquitin-mediated protein degradation (7Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3750) Google Scholar, 8Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1604) Google Scholar, 9Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2860) Google Scholar), forming a negative regulatory loop for p53 function. Interference of the p53 and MDM2 interaction appears to be a main convergent point for many of the stress signals, resulting in stabilization and activation of p53 (10Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). The discovery of p63 and p73, the p53 family members, added a new level of complexity to understanding the p53 network (11Yang A. McKeon F. Nat. Rev. Mol. Cell. Biol. 2000; 1: 199-207Crossref PubMed Scopus (426) Google Scholar). Bothp63 and p73 produce multiple transcripts, which could either activate or inhibit p53-mediated gene expression. In contrast to p53, p63 and p73 do not interact with most viral proteins and do not undergo MDM2-mediated degradation. Furthermore, mutations of p63 and p73 are rarely detected in cancers, whereas mutation of p53 is the most common defect in cancer cells (for a recent review see Ref.11Yang A. McKeon F. Nat. Rev. Mol. Cell. Biol. 2000; 1: 199-207Crossref PubMed Scopus (426) Google Scholar). To identify novel upstream regulators of p53 and its relatives, we have undertaken a systematic two-hybrid screening of p53, p63, p73, and the known components of the p53 network. Here we report the identification of PIAS11 as a novel activator of p53. pcDNA3PIAS1-(c-Flag) and pcDNA3 PIAS1 deletions expressed PIAS1 and PIAS1 truncations that were Flag-tagged at their C termini. The following reagents were generous gifts: PG13-Luc, MG15-Luc, pcDNA-p53, WWP-Luc, and p53(R175H) (B. Vogelstein); p40 (D. Sidransky); Stat1 (J. Darnell); pCMV5-PIAS1 (J. Liao and K. Shui); Tera-1 cells (testicular carcinoma) (V. Jin); Primary mouse embryonic fibroblasts (MEFp53−/−, 3–4 passages; Pier Paolo Pandolfi). H1299 cells (human lung large cell carcinoma) were purchased from ATCC. Anti-PIAS1 antibody was produced by immunizing New Zealand White rabbits with GST-PIAS1 (residues 47–180) as described previously (12Xu C.W. Hines J.C. Engel M.L. Russell D.G. Ray D.S. Mol. Cell. Biol. 1996; 16: 564-576Crossref PubMed Scopus (68) Google Scholar). Other antibodies used were anti-p21 antibody (Upstate Biotechnology), anti-p53 DO-1 (Santa Cruz Biotechnology), anti-Flag (M2), and anti-actin antibodies (Sigma). cDNA encoding p63/p40 was cloned at the BamHI and XhoI sites of pCWX200 for two-hybrid screening with a human HeLa cDNA library (13Xu C.W. Mendelsohn A.R. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12473-12478Crossref PubMed Scopus (52) Google Scholar). For mapping the interaction domains, deletions of p53 and PIAS1 were cloned into pEG202 and pJG4–5 or pJG4–5AAT, respectively (14Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1324) Google Scholar). p53 (residues 1–50) was not included. The interactions were scored by the expression of β-galactosidase in the two-hybrid mating assay (15Finley Jr., R.L. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12980-12984Crossref PubMed Scopus (241) Google Scholar). MEFs and Tera-1 cells were cultured in Dulbecco's modified Eagle's (high glucose) medium with 10% FBS, and H1299 cells were maintained in RPMI medium with 10% FBS (5% CO2 at 37 οC). Cell transfections were carried out with FuGene 6 according to the manufacturer's instruction (Roche). For luciferase assays, cells were plated at 2 × 105cell/60-mm dish and grown overnight before transfection. The total amount of DNA transfected was adjusted with pcDNA3. For cell cycle and Western blotting analyses, cells were plated at a lower density (4 × 105 cells/100-mm dish), and the total amount of DNA was adjusted with salmon sperm DNA (Sigma). Luciferase assays were performed according to the manufacturer's instruction (Tropix). Relative luciferase activity was normalized to protein concentration as determined by the Bradford method (Bio-Rad). Logarithmic Tera-1 cells were treated for 8 h with or without 10 μm etoposide. Cell pellets were rotated in 0.5 ml lysis buffer (50 mm Hepes, pH 7.4, 350 mm NaCl, 1% Triton, 0.2% Sarcosyl, 0.5 mmdithiothreitol, 1 mm NaF, 1 mmNaVO3, 1 tablet of protease inhibitor with EDTA) for 30 min at 4 °C and were subsequently passed through a 26.5-gauge syringe six times to break nuclei. After centrifuged at 14,000 rpm for 10 min at 4 °C, aliquots (120 μl, 12 μg of protein) were incubated with protein G-beads (Pharmacia) coated with anti-PIAS1 or normal IgG antibodies for 1 h at 4 °C. Immunoprecipitates were washed four times with 400 μl of the lysis buffer and Western-blotted with anti-p53 DO-1 antibody. Transfected H1299 cells were washed with ice-cold PBS, detached with 5 mm EDTA in PBS, and collected by centrifugation at 4 °C. The cells were extracted in 250 μl of lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm EDTA, 2 mm EGTA, 2 mm dithiothreitol, 14 mm β-mercaptoethanol, 0.5% Triton X-100, and one protease inhibitor mixture (Roche Molecular Biochemicals)) for 30 min on ice, and kept at −80 °C until use. Soluble protein (60 μg) was used for Western blotting analysis. Transfected H1299 cells were washed with PBS, treated with trypsin-EDTA for 1 min at 37 οC, placed immediately on ice, and washed twice with 5 ml of ice-cold WB (10% FBS and 1% BSA in PBS). The cells were resuspended in 2 ml of ice-cold 75% methanol with simultaneous mixing and kept overnight or for several days at 4 °C. The cells were washed with WB, permeablized for 5 min in 1 ml of WB containing 0.25% Triton X-100, and subsequently washed with 5 ml of WB. For labeling, the cells were resuspended in 80 μl of WB and incubated for 60 min at 4οC with 20 μl of fluorescein isothiocyanate (FITC)-conjugated anti-p53 antibody (B&D PharMingen). The cells were washed with 5 ml of WB and stained overnight with PBS containing 10 μg/ml propidium iodide (Sigma), 200 Kunitz units/ml RNase A (Sigma), and 0.1% Nonidet P-40. The dual labeled cells were analyzed with an automated bench top flow cytometer (FACSCalibur) equipped with CELLQuest software (Becton Dickinson). Cell cycle phases of FITC-positive cells were determined with MultiCycle software. Our original goal was to identify cellular proteins that discriminate the tumor suppressor protein p53 from its newly discovered relative p40 (16Trink B. Okami K. Wu L. Sriuranpong V. Jen J. Sidransky D. Nat. Med. 1998; 4: 747-748Crossref PubMed Scopus (225) Google Scholar), an alternative splice form of p63 (17Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1851) Google Scholar). We performed a two-hybrid screening for p40 interacting proteins. About 140 positive clones were identified from 4 × 107 library transformants. One-third of the positive clones encoded the carboxyl half of the protein inhibitor of activated Stat1 (PIAS1) (18Liu B. Liao J. Rao X. Kushner S.A. Chung C.D. Chang D.D. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10626-10631Crossref PubMed Scopus (636) Google Scholar). Another third of the positive clones encoded UBC9. Among the rest of the positive clones was a cellular protein that specifically interacted with p40/p63 but not with p53 (data not shown). Both PIAS1 and UBC9 interacted with p53 in the yeast two-hybrid assay (data not shown). In this report, we have focused on the interaction between PIAS1 and p53. The tumor suppressor protein p53 comprises distinct functional domains (2Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar, 4Ryan K.M. Phillips A.C. Vousden K.H. Curr. Opin. Cell Biol. 2001; 13: 332-337Crossref PubMed Scopus (583) Google Scholar, 19Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar). They include the transcriptional activation domain (residues 1–100), the central DNA binding core domain (100–300), the linker sequence (301–320), the tetramerization domain (320–360) and the C-terminal regulatory domain (363–393). To map which p53 domains are involved in the interaction with the carboxyl and amino deletions of PIAS1, we employed a two-hybrid mating assay (15Finley Jr., R.L. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12980-12984Crossref PubMed Scopus (241) Google Scholar). In this assay, yeast strains expressing PIAS1 or p53 were mated, and interacting domains were scored by the expression of β-galactosidase (Fig. 1). We found that PIAS1(full-length, residues 1–651), PIAS1-(10–651), PIAS1-(1–560), and PIAS1-(306–651) interacted with both p53 and p40/p63. However, neither PIAS1-(1–400) nor PIAS1-(1–480) interacted with p53-(50–393) and p40/p63. We further determined that the tetramerization and C-terminal regulatory domains of p53 were sufficient for PIAS1 interaction. Therefore, the carboxyl half of PIAS1 interacted with the tetramerization and the C-terminal regulatory domains of p53 (Fig. 1 B). To confirm the interaction between PIAS1 and p53, we tested whether endogenous PIAS1 was associated with endogenous p53 by co-immunoprecipitation. Using purified anti-PIAS1 antibodies raised against PIAS1-(47–180), we immunoprecipitated endogenous PIAS1 from extracts of testicular carcinoma cells (Tera-1) that express both endogenous p53 and endogenous PIAS1. The immunoprecipitates were subsequently washed under stringent conditions with a high concentration of NaCl and detergents. The presence of p53 was detected by Western blotting with the anti-p53 antibody DO-1 in the anti-PIAS1 precipitates but not in the normal IgG precipitates (Fig.1 C). We further demonstrated the association between endogenous PIAS1 and endogenous p53 in human hepatoma cells (HepG2) (data not shown). DNA damages and other stress signals lead to the increased levels and activation of p53. To address the question of whether endogenous PIAS1 was associated with endogenous activated p53 under these conditions, logarithmic Tera-1 cells were treated for 8 h with 10 μm etoposide, a DNA-damaging agent. Endogenous co-immunoprecipitation experiments were performed as described above. As shown in Fig. 1 C, PIAS1 interacted with p53 under the stress condition. These data indicate that PIAS1 interacts with p53 in the presence or absence of stress signals. p53 functions as a sequence-specific transcriptional activator. Because PIAS1 and p53 interacted with each other in vivo, we investigated the effects of PIAS1 on p53-mediated gene expression. The PIAS1-expressing vector pcDNA3PIAS1-(c-Flag), alone or in combination with a vector encoding p53 (pcDNA3-p53), was co-transfected into mouse embryonic fibroblast cells (MEF, p53 −/−) together with a reporter containing a synthetic p53 binding site placed upstream of the luciferase reporter gene (PG13-Luc). Ectopic expression of p53 produced a marked stimulation of transcription. A significant stimulation (2-fold) of the reporter above the levels observed with p53 alone was specifically induced by coexpression of PIAS1 in a dose-dependent manner (Fig.2 A). In contrast, ectopic expression of PIAS1 did not activate the expression of the luciferase reporter containing a synthetic mutant p53 binding site (MG15-Luc) in the same MEF cells (p53 −/− ) (Fig.2 A). We next evaluated the effects of PIAS1 on p53-dependent transcription in H1299 cells lacking endogenous p53. As shown in Fig. 2 B, ectopic expression of PIAS1 activated p53-mediated expression of PG13-Luc in a dose-dependent manner but did not stimulate the expression of MG15-Luc, again suggesting that PIAS1 could activate p53-mediated gene expression. Furthermore, ectopic expression of PIAS1 activated the expression of PG13-Luc in testicular carcinoma cells (Tera-1, wide type p53) (Fig. 2 C). In addition, ectopic expression of PIAS1-(10–651) activated p53-dependent transcription in additional cell lines such as U3A, Saos-2, and HeLa cells (data not shown), further suggesting that PIAS1 activates p53-mediated gene expression in a variety of cell types. p53 functions as a transcriptional activator that induces expression of cyclin-dependent kinase inhibitor p21 (5Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5850) Google Scholar). To test whether PIAS1 activates p53-dependent expression of p21, we transiently transfected a WWP luciferase construct containing the p21 promoter (WWP-Luc) and plasmids expressing PIAS1 or p53. As shown in Fig. 3 A, PIAS1 could only modestly activate p53-dependent expression of the reporter. We next examined whether PIAS1 could induce p53-dependent expression of endogenous p21 protein by transiently expressing PIAS1, wild-type p53, or mutant p53 (R175H) defective in the DNA binding in H1299 cells. Ectopic expression of PIAS1 stimulated a marked increase in the levels of endogenous p21 protein in the presence of wild-type p53 (Fig. 3 B). In contrast, PIAS1 alone or in combination with p53(R175H) had no effect on expression of the endogenous p21. In addition, ectopic expression of PIAS1 increased steady-state levels of p53 (Figs. 3 B and4 B). These data indicate that PIAS1 activates p53-dependent expression of the endogenous p21 protein.Figure 4PIAS1 -(400–651) lacking the RING-finger domain activates p53-mediated gene expression. A, PIAS1-(400–651) activates p53-dependent expression of PG13-Luc in H1299 cells. A luciferase assay was performed as described in the legend for Fig. 2 B except that 2 μg of PIAS1 constructs were used. B, PIAS1-(400–651) activates p53-dependent expression of endogenous p21 protein. The assay was as described in the legend for Fig. 3 B. PIAS1(full-length, 1–651), PIAS1-(1–480), and PIAS1-(400–651) were Flag-tagged Flag at the C terminus, but PIAS1-(10–651) was not tagged.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next investigated whether PIAS1 could also induce p53-dependent cell cycle arrest. H1299 cells were transiently transfected with plasmids expressing PIAS1 and wild-type p53 or mutant p53(R175H). Small amounts (25 ng) of pcDNA-p53 or pcDNA3-p53(R175H) were used so that p53 alone would have a minimal effect on G1 arrest of the cell cycle (about 60%). A representative experiment indicated that ectopic expression of PIAS1 induced about 8.5% more G1 arrest in cells expressing wild-type p53 than those expressing p53(R175H) (Fig. 3 C). These observations were further validated in six additional independent experiments done in duplicate. It is also noted that PIAS1 could induce G1 arrest of the cell cycle in the presence of p53(R175H). Although PIAS1 might have other roles, our data clearly demonstrate that PIAS1 can stimulate p53-dependent G1arrest of the cell cycle. It has recently been discovered that PIAS1 functions as an E3 ligase for the covalent attachment of small ubiquitin-related modifier (SUMO-1) to p53 in vitro and in transfected cells (20Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). The RING-finger domain of PIAS1-(325–382) is essential for the interaction of PIAS1 with UBC9 and SUMO-1. Deletion of this domain abolishes PIAS1-mediated sumoylation of p53 (20Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). To examine whether the RING-finger domain is required for the activation of p53 by PIAS1, we used mutant PIAS1-(400–651), which lack the RING-finger domain but still interacted with p53. Plasmids expressing p53, PG13-Luc, and PIAS1-(400–651) were co-transfected to H1299 cells. As shown in Fig. 4 A, PIAS1-(400–651) activated p53-dependent expression of PG-13 even better than PIAS1(full-length, 1–651). Furthermore, PIAS1-(400–651) induced p53-dependent expression of endogenous p21 protein to the same extent as PIAS1 and other PIAS1 deletions. PIAS1 and PIAS1 deletions alone and in combination with p53(R175H) could not induce the expression of endogenous p21. These data clearly indicate that the RING-finger domain of PIAS1 is not essential for the activation of p53 by PIAS1. We have found that endogenous PIAS1 interacts with endogenous p53 in vivo in the presence and absence of stress signals, suggesting that PIAS1 is complexed with both latent and activated endogenous p53. Ectopic expression of PIAS1 activates p53-mediated expression of cyclin-dependent kinase inhibitor p21 and stimulates p53-dependent G1arrest of the cell cycle, suggesting that PIAS1 is a novel activator of p53. PIAS1 is a multifunctional protein that was first identified as a protein inhibitor of activated Stat1 (18Liu B. Liao J. Rao X. Kushner S.A. Chung C.D. Chang D.D. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10626-10631Crossref PubMed Scopus (636) Google Scholar, 21Liao J. Fu Y. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5267-5272Crossref PubMed Scopus (94) Google Scholar, 22Betz A. Lampen N. Martinek S. Young M.W. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9563-9568Crossref PubMed Scopus (105) Google Scholar). PIAS1 also plays an important role in proper chromosomal functions in Drosophila(23Hari K.L. Cook K.R. Karpen G.H. Genes Dev. 2001; 15: 1334-1348Crossref PubMed Scopus (152) Google Scholar). PIAS1 is almost identical to RNA Gu helicase-binding protein except for the first nine amino acid residues (24Valdez B.C. Henning D. Perlaky L. Busch R.K. Busch H. Biochem. Biophys. Res. Commun. 1997; 234: 335-340Crossref PubMed Scopus (54) Google Scholar). Furthermore, PIAS1 is a putative coactivator of androgen receptors in an androgen-dependent manner and co-activator of glucocorticoid receptor in the presence of dexamethasone (25Tan J. Hall S.H. Hamil K.G. Grossman G. Petrusz P. Liao J. Shuai K. French F.S. Mol. Endocrinol. 2000; 14: 14-26Crossref PubMed Scopus (86) Google Scholar, 26Gross M. Liu B. Tan J. French F.S. Carey M. Shuai K. Oncogene. 2001; 20: 3880-3887Crossref PubMed Scopus (151) Google Scholar). In addition, PIAS1 induces apoptosis through c-Jun NH2-terminal kinase (JNK) (27Liu B. Shuai K. J. Biol. Chem. 2001; 276: 36624-36631Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). It is not known whether the p53 network cross-talks through PIAS1 with pathways mediated by Stat1, androgen receptors, RNA helicase, or JNK. Because JNK is important for p53 stabilization and transcriptional activation (28Buschmann T. Potapova O. Bar-Shira A. Ivanov V.N. Fuchs S.Y. Henderson S. Fried V.A. Minamoto T. Alarcon-Vargas D. Pincus M.R. Gaarde W.A. Holbrook N.J. Shiloh Y. Ronai Z. Mol. Cell. Biol. 2001; 21: 2743-2754Crossref PubMed Scopus (253) Google Scholar) and PIAS1 activates p53 (this study), it is possible that PIAS1-induced apoptosis through JNK involves p53. During this project, PIAS1 was independently identified as an interactor of mutant p53 in a two-hybrid screening (29Gallagher W.M. Argentini M. Sierra V. Bracco L. Debussche L. Conseiller E. Oncogene. 1999; 18: 3608-3616Crossref PubMed Scopus (50) Google Scholar). In addition, PIAS1 was identified as an E3 ligase for sumoylation of p53, which requires the RING-finger domain (residues 325–382) of PIAS1 (20Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). However, the functional role of PIAS1 in the activity of p53 has not been investigated in these studies (20Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 29Gallagher W.M. Argentini M. Sierra V. Bracco L. Debussche L. Conseiller E. Oncogene. 1999; 18: 3608-3616Crossref PubMed Scopus (50) Google Scholar). In our study, PIAS1-(400–651) lacking the RING-finger efficiently activated p53-mediated gene expression (Fig. 4), implying that p53 activation by PIAS1 is independent of PIAS1-mediated sumoylation of p53. Although earlier studies suggested that sumoylation activates p53 (30Rodriguez M.S. Desterro J.M. Lain S. Midgley C.A. Lane D.P. Hay R.T. EMBO J. 1999; 18: 6455-6461Crossref PubMed Scopus (561) Google Scholar, 31Gostissa M. Hengstermann A. Fogal V. Sandy P. Schwarz S.E. Scheffner M. Del Sal G. EMBO J. 1999; 18: 6462-6471Crossref PubMed Scopus (438) Google Scholar, 32Muller S. Berger M. Lehembre F. Seeler J.S. Haupt Y. Dejean A. J. Biol. Chem. 2000; 275: 13321-13329Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar), our data are consistent with recent studies demonstrating that the sumoylation of p53 is not essential for its transcriptional activation (33Kwek S.S. Derry J. Tyner A.L. Shen Z. Gudkov A.V. Oncogene. 2001; 20: 2587-2599Crossref PubMed Scopus (113) Google Scholar, 34Minty A. Dumont X. Kaghad M. Caput D. J. Biol. Chem. 2000; 275: 36316-36323Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). We noted that PIAS1-(10–651) was much more potent in activating p53-mediated gene expression in a variety of cell lines than PIAS1(full-length, 1–651) (for example, Fig. 4). The marked difference between PIAS1 and PIAS1-(10–651) is also evident in their inhibition of activated Stat1. Unlike PIAS1, PIAS1-(10–651) does not inhibit activated Stat1 (27Liu B. Shuai K. J. Biol. Chem. 2001; 276: 36624-36631Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Another PIAS1 mutant (1–480), which did not interact with p53 (Fig. 1 A), weakly activated the expression of p53-mediated PG13-Luc (Fig. 4 A) but strongly induced p53-dependent expression of endogenous p21 (Fig.4 B). The N-terminal half of PIAS1 is known to modulate the interaction between PIAS1 and activated Stat1 even though the N-terminal half of PIAS1 does not interact with Stat1 (21Liao J. Fu Y. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5267-5272Crossref PubMed Scopus (94) Google Scholar). It remains to be determined whether PIAS1-(1–480) plays a role in activating p53-dependent gene expression by a similar indirect mechanism. The tetramerization and the C-terminal regulatory domains of p53 is involved in the interaction with PIAS1 (Fig. 1, A andB). These p53 domains are known to regulate stability, nuclear export control, and activation of p53 (3Appella E. Anderson C.W. Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (916) Google Scholar, 4Ryan K.M. Phillips A.C. Vousden K.H. Curr. Opin. Cell Biol. 2001; 13: 332-337Crossref PubMed Scopus (583) Google Scholar, 35Chene P. Oncogene. 2001; 20: 2611-2617Crossref PubMed Scopus (205) Google Scholar, 36Liang S.H. Clarke M.F. Eur. J. Biochem. 2001; 268: 2779-2783Crossref PubMed Scopus (162) Google Scholar, 37Ahn J. Prives C. Nat. Struct. Biol. 2001; 8: 730-732Crossref PubMed Scopus (103) Google Scholar). We found that PIAS1 increased the steady-state levels of p53 (Fig.4 B) and the half-life of p53 (data not shown). It is currently under investigation as to whether PIAS1 activates the p53 function by modulating covalent and noncovalent states of p53 through the tetramerization and the C-terminal domains and/or by breaking the negative regulatory loop between MDM2 and p53. In summary, our present study suggests that PIAS1 is a novel activator of p53. We thank Pier Paolo Pandolfi, James Darnell, Bert Vogelstein, David Sidransky, Victor Jin, Jiayu Liao, Kai Shui, and Russ Finley for providing cell lines and expression constructs. We thank Raphael Nunez for cell cycle analysis and Robyn Ray for critical reading of the manuscript." @default.
• W2032299946 created "2016-06-24" @default.
• W2032299946 creator A5045243726 @default.
• W2032299946 creator A5045273968 @default.
• W2032299946 creator A5066543200 @default.
• W2032299946 date "2002-03-01" @default.
• W2032299946 modified "2023-09-28" @default.
• W2032299946 title "Activation of p53 by Protein Inhibitor of Activated Stat1 (PIAS1)" @default.
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