Transactivation Ability of p53 Transcriptional Activation Domain Is Directly Related to the Binding Affinity to TATA-binding Protein
1995; Elsevier BV; Volume: 270; Issue: 42 Linguagem: Inglês
10.1074/jbc.270.42.25014
ISSN1083-351X
AutoresJun Chang, Do‐Hyung Kim, Seung Woo Lee, Kwan Yong Choi, Young Chul Sung,
Tópico(s)Virus-based gene therapy research
ResumoTumor suppressor protein p53 is a potent transcriptional activator and regulates cell growth negatively. To characterize the transcriptional activation domain (TAD) of p53, various point mutants were constructed in the context of Gal4 DNA binding domain and tested for their transactivation ability. Our results demonstrated that the positionally conserved hydrophobic residues shared with herpes simplex virus VP16 and other transactivators are essential for transactivation. Also, the negatively charged residues and proline residues are necessary for full activity, but not essential for the activity of p53 TAD. Deletion analyses showed that p53 TAD can be divided into two subdomains, amino acids 1-40 and 43-73. An in vitro glutathione S-transferase pull-down assay establishes a linear correlation between p53 TAD-mediated transactivation in vivo and the binding activity of p53 TAD to TATA-binding protein (TBP) in vitro. Mutations that diminish the transactivation ability of Gal4-p53 TAD also impair the binding activity to TBP severely. Our results suggest that at least TBP is a direct target for p53 TAD and that the binding strength of TAD to TBP (TFIID) is an important parameter controlling activity of p53 TAD. In addition, circular dichroism spectroscopy has shown that p53 TAD peptide lacks any regular secondary structure in solution and that there is no significant difference between the spectra of the wild type TAD and that of the transactivation-deficient mutant type. Tumor suppressor protein p53 is a potent transcriptional activator and regulates cell growth negatively. To characterize the transcriptional activation domain (TAD) of p53, various point mutants were constructed in the context of Gal4 DNA binding domain and tested for their transactivation ability. Our results demonstrated that the positionally conserved hydrophobic residues shared with herpes simplex virus VP16 and other transactivators are essential for transactivation. Also, the negatively charged residues and proline residues are necessary for full activity, but not essential for the activity of p53 TAD. Deletion analyses showed that p53 TAD can be divided into two subdomains, amino acids 1-40 and 43-73. An in vitro glutathione S-transferase pull-down assay establishes a linear correlation between p53 TAD-mediated transactivation in vivo and the binding activity of p53 TAD to TATA-binding protein (TBP) in vitro. Mutations that diminish the transactivation ability of Gal4-p53 TAD also impair the binding activity to TBP severely. Our results suggest that at least TBP is a direct target for p53 TAD and that the binding strength of TAD to TBP (TFIID) is an important parameter controlling activity of p53 TAD. In addition, circular dichroism spectroscopy has shown that p53 TAD peptide lacks any regular secondary structure in solution and that there is no significant difference between the spectra of the wild type TAD and that of the transactivation-deficient mutant type. INTRODUCTIONTranscriptional activators have been shown to stimulate in vitro the assembly of transcriptional preinitiation complexes (1Choy B. Green M.R. Nature. 1993; 366: 531-536Crossref PubMed Scopus (235) Google Scholar, 2Lin Y.S. Green M.R. Cell. 1991; 64: 971-981Abstract Full Text PDF PubMed Scopus (366) Google Scholar) as well as transcriptional elongation by RNA polymerase II(3Yankulov K. Blau J. Purton T. Roberts S. Bentley D.L. Cell. 1994; 77: 749-759Abstract Full Text PDF PubMed Scopus (207) Google Scholar). This stimulation is thought to depend on direct or indirect protein-protein interactions between transcriptional activators and the general transcriptional machinery and/or on relieving the inhibitory effects of chromatin(4Felsenfeld G. Nature. 1992; 355: 219-224Crossref PubMed Scopus (714) Google Scholar, 5Laybourn P.J. Kadonaga J.T. Science. 1991; 254: 238-245Crossref PubMed Scopus (292) Google Scholar). Transcriptional activators can be divided into at least two discrete functional domains(6Ptashne M. Nature. 1988; 335: 683-689Crossref PubMed Scopus (1167) Google Scholar); a DNA binding/targeting domain is required to direct the activator to the appropriate DNA sequence element and then the transcriptional activation domain (TAD) 1The abbreviations used are: TADtranscriptional activation domainTBPTATA-binding proteinTAFTBP-associated factorbpbase pair(s)GSTglutathione S-transferasePAGEpolyacrylamide gel electrophoresisTFEtrifluoroethanol. can induce the enhanced transcription of target genes. TADs have been divided into three major classes according to a predominance of particular amino acid residues: acidic, proline-rich, or glutamine-rich(7Mitchell P. Tjian R. Science. 1988; 245: 371-378Crossref Scopus (2185) Google Scholar). Of these classes, the acidic TADs appear to be unique in that they can apparently function universally in all eukaryotes tested from yeast to human(8Sadowski I. Ma J. Triezenberg S. Ptashne M. Nature. 1988; 335: 563-564Crossref PubMed Scopus (971) Google Scholar).Like other transcriptional activators, tumor suppressor protein p53 appears to have a modular domain structure; it contains an NH2-terminal region which functions as a TAD when coupled to a heterologous DNA binding domain(9Fields S. Jang S. Science. 1990; 249: 1046-1049Crossref PubMed Scopus (656) Google Scholar, 10Raycroft L. Wu H. Lozano G. Science. 1990; 249: 1049-1051Crossref PubMed Scopus (494) Google Scholar), a central site-specific DNA binding domain(11Bargonetti J. Friedman P.N. Kern S.E. Vogelstein B. Prives C. Cell. 1991; 65: 1063-1091Abstract Full Text PDF PubMed Scopus (282) Google Scholar, 12Kern S.E. Kinzler K.W. Bruskin A. Jarosz D. Friedman P. Prives C. Vogelstein B. Science. 1991; 252: 1708-1711Crossref PubMed Scopus (934) Google Scholar), an oligomerization domain(13Sturzbecher H.-W. Brain R. Addison C. Rudge K. Remm M. Grimaldi M. Keenan E. Jenkins J.R. Oncogene. 1992; 7: 1513-1523PubMed Google Scholar, 14Iwabuchi K. Li B. Bartel P. Fields S. Oncogene. 1993; 8: 1693-1696PubMed Google Scholar), and a basic COOH-terminal nuclear localization domain(15Addison C. Jenkins J. Sturzbecher H.-W. Oncogene. 1990; 5: 423-426PubMed Google Scholar). The NH2-terminal TAD of p53 is similar in size, net negative charge, and transactivating potency to the well defined TAD of herpes simplex virus virion protein 16 (HSV VP16)(16Cress D. Triezenberg S.J. Gene (Amst.). 1991; 103: 235-238Crossref PubMed Scopus (18) Google Scholar). This region is also rich in proline residues which are conserved through evolution(17Soussi T. Caron de Fromental C. May P. Oncogene. 1990; 5: 945-952PubMed Google Scholar). Like VP16 and a number of other transactivators, p53 is thought to be a transactivator of the acidic type(9Fields S. Jang S. Science. 1990; 249: 1046-1049Crossref PubMed Scopus (656) Google Scholar, 18Truant R. Xiao H. Ingles C.J. Greenblatt J. J. Biol. Chem. 1993; 268: 2284-2287Abstract Full Text PDF PubMed Google Scholar).Early studies suggested TFIID as the target for various activators(19Hirikoshi M. Carey M.F. Kakidani H. Roeder R.G. Cell. 1988; 54: 665-669Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 20Hirikoshi M. Hai T. Lin Y.S. Green M.R. Roeder R.G. Cell. 1988; 54: 1033-1042Abstract Full Text PDF PubMed Scopus (268) Google Scholar). Subsequently, the TATA-binding proteins (TBP) of yeast and human were shown to bind in vitro to the strong TADs of such viral and cellular activators as VP16(21Stringer K.F. Ingles J. Greenblatt J. Nature. 1990; 345: 783-786Crossref PubMed Scopus (407) Google Scholar), E1A(22Lee W.S. Kao C.C. Bryant G.O. Liu X. Berk A.J. Cell. 1991; 67: 365-376Abstract Full Text PDF PubMed Scopus (272) Google Scholar), Zta(23Lieberman P.M. Berk A.J. Genes & Dev. 1991; 5: 2441-2454Crossref PubMed Scopus (162) Google Scholar), and p53(18Truant R. Xiao H. Ingles C.J. Greenblatt J. J. Biol. Chem. 1993; 268: 2284-2287Abstract Full Text PDF PubMed Google Scholar, 24Liu X. Miller C.W. Koettler D.H. Berk A.J. Mol. Cell. Biol. 1993; 13: 3291-3300Crossref PubMed Scopus (232) Google Scholar, 25Seto E. Usheva A. Zambetti G.P. Momand J. Hirikoshi N. Weinmann R. Levine A.J. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12028-12032Crossref PubMed Scopus (464) Google Scholar). It has also been shown that another general transcription factor, TFIIB, interacts with various transactivators such as VP16 (26Lin Y.S. Ha I. Maldonado E. Reinberg D. Green M.R. Nature. 1991; 353: 569-571Crossref PubMed Scopus (261) Google Scholar), Rel oncogene product(27Kerr L.D. Ransone L.J. Wamsley P. Schmitt M.J. Boyer T.G. Zhou Q. Berk A.J. Verma I.M. Nature. 1993; 365: 412-419Crossref PubMed Scopus (131) Google Scholar), and CTF(28Kim T.K. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4170-4174Crossref PubMed Scopus (98) Google Scholar). Recent report showed that VP16 TAD and p53 TAD can also bind to TFIIH(29Xiao H. Pearson A. Coulombe B. Truant R. Zhang S. Regier J.L. Triezenberg S.J. Reinberg D. Flores O. Ingles J. Greenblatt G. Mol. Cell. Biol. 1994; 14: 7013-7024Crossref PubMed Scopus (327) Google Scholar). In addition to general transcription factors, coactivators or adaptors are required for transactivation in the in vitro transcription system. The best characterized proteins among adaptors are the TBP-associated factors (TAFs) of the Drosophila melanogaster and humans (30Dynlacht B.D. Hoey T. Tjian R. Cell. 1991; 66: 563-576Abstract Full Text PDF PubMed Scopus (483) Google Scholar, 31Pugh B.F. Tjian R. Genes & Dev. 1991; 5: 1935-1945Crossref PubMed Scopus (476) Google Scholar, 32Tanese N. Pugh B.F. Tjian R. Genes & Dev. 1991; 5: 2212-2224Crossref PubMed Scopus (241) Google Scholar, 33Zhou Q. Lieberman P.M. Boyer T.G. Berk A.J. Genes & Dev. 1992; 6: 1964-1974Crossref PubMed Scopus (288) Google Scholar). Recently, it was reported that p53 TAD can also interact with two subunits of the TFIID, TAFII40, and TAFII60(34Catherine J.T. Chen J.-L. Klemm R. Tjian R. Science. 1995; 267: 100-104Crossref PubMed Scopus (406) Google Scholar). Clearly, transcriptional activation appears to be more complicated than originally envisioned (6Ptashne M. Nature. 1988; 335: 683-689Crossref PubMed Scopus (1167) Google Scholar) and may involve multiple targets that make direct or indirect contacts in different spatial and temporal arrangements with TADs and the transcriptional machinery.Here, we demonstrate that p53 TAD is a complex activation domain composed of two subdomains, in which positionally conserved hydrophobic residues are critical for activating function. The negatively charged residues and proline residues are also necessary for full activity, but not essential for the activity of p53 TAD. Mutations that severely impair the function of p53 TAD in vivo have been shown to diminish binding activity to TBP in vitro, indicating that the observed in vitro interaction is biologically relevant. Circular dichroism (CD) spectroscopy demonstrates that p53 TAD peptide does not have any detectable secondary structure at physiological condition.MATERIALS AND METHODSPlasmid Constructions and MutagenesisGal4 DNA-binding domain expression plasmid, Gal4D, was constructed by inserting the 450-bp HindIII-XmaI fragment of pSG424 (8Sadowski I. Ma J. Triezenberg S. Ptashne M. Nature. 1988; 335: 563-564Crossref PubMed Scopus (971) Google Scholar) into the HindIII-BamHI site of pcDNA (Invitrogen) following by flushing XmaI and BamHI overhangs. A DNA fragment encoding amino acids 1-73 of p53 was amplified from the human cDNA of p53 with two primers (5′-GGTCGGATCCATGGAGGAGCCGCAGTCA and 3′-GGTGAAGCTTACACGGGGGGAGCAGCCTC; BamHI and HindIII sites are underlined) and digested with BamHI and HindIII. The resulting DNA fragment was ligated into the BamHI-HindIII site of pSK(-) (Stratagene), yielding pSK-p53 TAD. Gal4D-p53 TAD was generated by inserting the 210-bp BamHI-HindIII fragment of pSK-p53 TAD into the BamHI-EcoRV site of Gal4D after flushing the HindIII overhang (Fig. 1). Oligonucleotide-directed mutagenesis was performed as described (35Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4543) Google Scholar) using single-stranded DNA of pSK-p53 TAD. Mutations were identified by restriction endonuclease digestion and dideoxy sequencing. The specific amino acid changes introduced by mutagenic primers are listed in Table 1. The BamHI-HindIII DNA fragments of mutant derivatives were ligated into the same site of Gal4D except for M41 and M241 in which HincII site was used instead of HindIII site. The carboxyl-terminal deletion mutant, Gal4D-p53 (1-40), was generated by ligating the 120-bp BamHI-HindIII DNA fragment of pSK-p53 TAD M41 into the same site of Gal4D. Gal4D-p53 (43-73) was obtained by inserting the 90-bp HindIII-HindIII DNA fragment of pSK-p53 TAD M41 into the EcoRI site of Gal4D after filling in cohesive ends with Klenow fragment of DNA polymerase I. Gal4D-p53 (1-40) M22 was generated by introducing M22 mutation into Gal4D-p53 (1-40).Tabled 1View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab The glutathione S-transferase (GST) fusion plasmids were made by using pGEX-KG which contains a GST gene under the control of tac promoter and a flanked polycloning site(36Gaun K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1637) Google Scholar). pGEX-p53 TAD was constructed by inserting the 210-bp BamHI-HindIII DNA fragment of pSK-p53 TAD into the BamHI-HindIII site of pGEX-KG (Fig. 1). pGEX-p53 M2, M12, M19, M22, M23, M25, M31, M34, and M1234 were generated by the same method. pGEX-p53 M41 and M241 were made by inserting the BamHI-XhoI DNA fragments of pSK-p53 TAD M41 and M241 into the BamHI-XhoI sites of pGEX-KG, respectively. pGEX-p53(1-40) was generated by inserting the 120-bp BamHI-HindIII DNA fragment of pSK-p53 TAD M41 into the BamHI-HindIII site of pGEX-KG. The reporter plasmid, G5E1bCAT, was described previously(37Lee C.Q. Yun Y. Hoeffler J.P. Habener J.F. EMBO J. 1990; 9: 4455-4465Crossref PubMed Scopus (183) Google Scholar).Transfection and Chloramphenicol Acetyltransferase AssaysBHK-21 and COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Plasmid transfections were carried out by a DEAE-dextran method(38Queen C. Baltimore D. Cell. 1983; 33: 741-748Abstract Full Text PDF PubMed Scopus (389) Google Scholar). Cells (106) were seeded on a 100-mm dish 24 h before transfection and transfected with 1 μg of each of the reporter and activator plasmids. At 48 h after transfection, cells were harvested and chloramphenicol acetyltransferase activity was measured as described previously(39Lee C.W. Chang J. Lee K.J. Sung Y.C. J. Virol. 1994; 68: 2708-2719Crossref PubMed Google Scholar). To determine expression levels of the Gal4 fusions, COS-7 cells were transfected in parallel with 2 μg of activator plasmids. Nuclear extracts were prepared as described previously (40Schreiber E. Matthias P. Müller M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3908) Google Scholar) and electrophoretic mobility shift assays were performed as described (41Gill G. Pascal E. Tseng Z. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 192-196Crossref PubMed Scopus (469) Google Scholar) with DNA fragment containing five Gal4 binding sites. The amount of probes shifted by each derivative was quantitated using a Fuji BAS2000 photoimager. The difference in transfection efficiency was normalized by using a second reporter plasmid, pGL2 (Promega), containing a luciferase gene. Luciferase activity was measured by using the luciferase assay system (Promega) according to supplier's recommendation. All chloramphenicol acetyltransferase assay data reported in this article were from points in the linear range of the assay.GST Pull-down ExperimentGST fusion proteins were expressed in Escherichia coli DH5α and were purified by using glutathione-Sepharose beads (Pharmacia Biotech Inc.) in accordance with the supplier's recommendation. 35S-Labeled human TBP was generated by using a coupled transcription-translation reticulocyte lysate (TNT system, Promega) with linearized pETHIID plasmid (42Kao C.C. Lieberman P.M. Schmidt M.C. Zhou Q. Pei R. Berk A.J. Science. 1990; 248: 1646-1650Crossref PubMed Scopus (224) Google Scholar) as a template. 200 ng of GST-p53 TAD and mutant derivatives coupled to 20 μl of glutathione-Sepharose beads was incubated at 4°C with 35S-labeled TBP in 600 μl of a buffer solution containing 40 mM HEPES-KOH, pH 7.5, 150 mM KCl, 0.5 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 0.1% Nonidet P-40 for 1 h. To minimize potential bead losses during subsequent washes, the buffer was mixed with glutathione beads to adjust a total bead volume of 20 μl/reaction. Following this incubation, the beads were washed five times with the same buffer and bound proteins eluted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. The proteins were separated by 10% SDS-PAGE and visualized by autoradiography. Signals were quantitated on a Fuji BAS2000 photoimager and plotted to obtain a graphical representation of the results.Purification of p53 TAD PeptidesFor large scale production of p53 TAD, E. coli DH5α cells containing pGEX-p53 TAD were induced with 0.2 mM isopropyl-1-thio-β-D-galactopyranoside and harvested 4 h after induction. The fusion protein was purified from the soluble extract by use of binding affinity to glutathione-Sepharose beads. The p53 TAD peptide was released from the GST moiety in a buffer containing 100 mM NaCl and 2.5 mM CaCl2 using 1 μg of thrombin (Sigma)/1 mg of fusion protein. The peptide was further purified by gel filtration chromatography using Superose 12 (Pharmacia). The peptide after the gel filtration step was found to be homogeneous as judged by Coomassie Blue staining of the gel after SDS-PAGE. The identity of the peptide was determined by amino acid composition analysis. The M22 mutant derivative was also purified by the same method.CD SpectroscopyCD experiments were performed with a spectropolarimeter Jasco J-720. A cuvette with 0.1-cm of path length was used for all spectral measurements. Measurements were made at room temperature in 5 mM phosphate buffer. The concentrations of peptides were determined by absorbance at 280 nm in the phosphate buffer. The used peptide concentrations were 17 μM for wild type p53 TAD and 15 μM for the M22 mutant. All spectra were corrected for background using the phosphate buffer and averaged from the spectra of at least four scans. The pH values were measured with a microelectrode calibrated at two reference pH values.RESULTSMutational Analysis of p53 TADThe preponderance of acidic amino acids within p53 TAD suggests that negative charge is a critical component of the activation domain structure. To test whether activation function is simply related to the net negative charge, we constructed Gal4D-p53 TAD and replaced, in combination, the acidic amino acids within the activation domain with uncharged or positively charged residues (Fig. 1). From the relative activities of such mutants (Table 1), we infer that negative charge is necessary for the optimal activity of p53 TAD. The M41 mutant was less active than the M2 mutant, indicating that mutations of negatively charged residues, Glu-2 and Glu-3, had a less effect on the activity than mutations on Asp-41 and Asp-42 residues. The M241 mutant was less active than the M41 mutant, showing that replacement of increasing numbers of acidic residues with other residues led to a progressive decrease in transcriptional activation. It was reported previously that the acidic residues at the amino terminus of the p53 protein may influence, but are not critical for, the transcriptional activation (43Lin J. Chen J. Elenbaas B. Levine A.J. Genes & Dev. 1994; 8: 1235-1246Crossref PubMed Scopus (577) Google Scholar).The p53 TAD is also rich in proline residues (19.2%), which is a characteristic of another class of TAD, such as CTF/NF-1(44Mermod N. O'Neill E.A. Kelley T.J. Tjian R. Cell. 1989; 58: 741-753Abstract Full Text PDF PubMed Scopus (538) Google Scholar). When M12 and M34 mutants were tested, there were about 39 and 24% reduction in p53 TAD-mediated transactivation, respectively. As expected, the M1234 mutant containing mutations in four Pro residues was shown to be about 71% reduction in the transactivation (Table 1), indicating that there was additive effect with these mutations and that proline residues are also required for the optimal activity of p53 TAD.Previous studies on the VP16 TAD have suggested that the acidic residues contribute to its activity, but intervening hydrophobic residues are more important than other residues(45Cress W.D. Triezenberg S.J. Science. 1991; 251: 87-90Crossref PubMed Scopus (323) Google Scholar). TADs of a number of transactivators exhibit a conserved pattern of hydrophobic residues (45Cress W.D. Triezenberg S.J. Science. 1991; 251: 87-90Crossref PubMed Scopus (323) Google Scholar). Since p53 TAD also shows the similar pattern of positionally conserved hydrophobic residues (Fig. 2), we generated various mutants in which conserved hydrophobic amino acids were replaced with hydrophilic ones. When these mutants were tested for transactivation activity in BHK-21 and COS-7 cells, the activities of several mutants were significantly impaired (Table 1). Mutations on both residues Leu-22 and Trp-23 reduced p53 TAD-mediated transactivation by about 95%, whereas mutations on Leu-25 and Leu-26 resulted in approximately 88% loss of the activity. Also, single amino acid change on Phe-19 reduced the activity by about 85%. In contrast, mutations on both Val-31 and Leu-32, which are not positionally conserved, did not impair the transactivation function but rather enhance the activity. Therefore, we concluded that the positionally conserved hydrophobic residues, Phe-19, Leu-22, Trp-23, Leu-25, and Leu-26 are critical for transactivation function of p53 TAD. These residues are identical in all sequences of p53 protein from several species(17Soussi T. Caron de Fromental C. May P. Oncogene. 1990; 5: 945-952PubMed Google Scholar). The effect of mutations on Leu-22 and Trp-23 is consistent with a previous report(43Lin J. Chen J. Elenbaas B. Levine A.J. Genes & Dev. 1994; 8: 1235-1246Crossref PubMed Scopus (577) Google Scholar), but those of mutations on Phe-19 and on Leu-25 and Leu-26 do not exactly coincide with their results in which human p53 mutant protein containing the double mutation on Leu-14 and Phe-19 was observed to have a 50% reduction in chloramphenicol acetyltransferase activity compared with wild type p53. In addition, the Leu-25 and Leu-26 double mutant showed either enhanced or reduced activity in Saos-2 cells, depending on p53-responsive elements either from the creatine phosphokinase gene or from the mdm-2 gene(43Lin J. Chen J. Elenbaas B. Levine A.J. Genes & Dev. 1994; 8: 1235-1246Crossref PubMed Scopus (577) Google Scholar).Figure 2Comparison of the primary amino acid sequences of different TADs. The amino acid sequences of several TADs are aligned using the bulky hydrophobic residues (boxed) as reported by Cress and Triezenberg(45Cress W.D. Triezenberg S.J. Science. 1991; 251: 87-90Crossref PubMed Scopus (323) Google Scholar). Underlined letters of p53 TAD indicate identity in all sequences of p53 from several species(17Soussi T. Caron de Fromental C. May P. Oncogene. 1990; 5: 945-952PubMed Google Scholar). The residue numbers are given for p53 TAD sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To compare the expression level among different Gal4 fusion proteins, electrophoretic mobility shift assay was performed using a labeled DNA fragment containing five Gal4 binding sites and showed that there was no significant difference among them (data not shown). The difference in the chloramphenicol acetyltransferase activity is, therefore, due to the intrinsic biological activity of different Gal4 fusion proteins, but not by the different level of Gal4 fusion proteins in the transfected cells. Although the transactivating abilities of mutants constructed in the foregoing studies were severely impaired, residual activity still remained, suggesting that p53 TAD is composed of separable subdomains just like VP16 (46Triezenberg S.J. Kingsbury R.C. McKnight S.L. Genes & Dev. 1988; 2: 718-729Crossref PubMed Scopus (587) Google Scholar) and Epstein-Barr virus Rta transactivator(47Hardwick J.M. Tse L. Applegren N. Nicholas J. Veliuona M.A. J. Virol. 1992; 66: 5500-5508Crossref PubMed Google Scholar). It was previously shown that the minimal activation domain of p53 lies within the first 42 amino acids of the protein(48Unger T. Nau M.M. Segal S. Minna J.D. EMBO J. 1992; 11: 1383-1390Crossref PubMed Scopus (227) Google Scholar). Since Gal4D-p53(1-40) consistently showed about 30-38% activity of Gal4D-p53 TAD, which contains the residues 1-73, residues 43-73 appear to be necessary for the full p53 TAD-mediated transactivation. To be certain that residues of p53 from 43 to 73 also contain an autonomous TAD, Gal4D-p53 (43-73) was constructed and tested for the transactivating ability. The resulting plasmid showed about 6% activity of Gal4D-p53 TAD (Table 1), indicating that there is an autonomous TAD in this subregion. In the case of VP16, the truncated activation domain possesses approximately 50% of wild type activity, whereas the addition of COOH-terminal subdomain restored the full activity(46Triezenberg S.J. Kingsbury R.C. McKnight S.L. Genes & Dev. 1988; 2: 718-729Crossref PubMed Scopus (587) Google Scholar). Gal4D-p53(1-40) M22, which deletes the COOH-terminal subregion from M22 mutant, completely lost the residual activity of M22 mutant (Table 1), demonstrating that the residual activity comes from the separable COOH-terminal subdomain, and that Leu-22 and Trp-23 are absolutely required for the function of minimal activating region (residues 1-40) of p53.In Vitro TBP Binding Activity of p53 TAD and MutantsPrevious studies showed that p53 TAD interacts directly and specifically with yeast and human TBP(18Truant R. Xiao H. Ingles C.J. Greenblatt J. J. Biol. Chem. 1993; 268: 2284-2287Abstract Full Text PDF PubMed Google Scholar, 24Liu X. Miller C.W. Koettler D.H. Berk A.J. Mol. Cell. Biol. 1993; 13: 3291-3300Crossref PubMed Scopus (232) Google Scholar). The binding activities of wild type p53, mutant p53(R175H)(18Truant R. Xiao H. Ingles C.J. Greenblatt J. J. Biol. Chem. 1993; 268: 2284-2287Abstract Full Text PDF PubMed Google Scholar, 25Seto E. Usheva A. Zambetti G.P. Momand J. Hirikoshi N. Weinmann R. Levine A.J. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12028-12032Crossref PubMed Scopus (464) Google Scholar), and Gal4-p53 fusion proteins (24Liu X. Miller C.W. Koettler D.H. Berk A.J. Mol. Cell. Biol. 1993; 13: 3291-3300Crossref PubMed Scopus (232) Google Scholar) to TBP were reported to correlate with their transactivation abilities in vivo, suggesting that p53 TAD activates transcription by directly interacting with TBP. In contrast, Lin et al.(43Lin J. Chen J. Elenbaas B. Levine A.J. Genes & Dev. 1994; 8: 1235-1246Crossref PubMed Scopus (577) Google Scholar) reported that wild type p53 and transactivation-deficient mutants, including R175H mutation, could bind equally well to human TBP when tested with immunoprecipitation and far-Western analysis. Thus, it remains controversial whether TBP is the target molecule of p53 TAD, and binding activity of p53 TAD to TBP is directly related to p53 TAD-mediated transactivation. To clarify this discrepancy, the residues of wild type p53 TAD from 1 to 73 and its derivatives were placed under the GST gene to generate pGEX-p53 TAD fusion constructs (Fig. 1). The GST-p53 TAD fusion protein and its derivatives were expressed in E. coli and purified by affinity chromatography (Fig. 3A). The purified fusion proteins were assayed for the activity to bind in vitro translated human TBP in a GST pull-down experiment. As shown in Fig. 3B and Table 1, the levels of TBP precipitated by GST-p53 TAD and mutant derivatives are linearly correlated with the ability of transactivation in vivo. Binding reactions were performed under nonsaturating condition, where GST-p53 TAD and mutant derivatives were a limiting factor. Under this condition, about 20% of input TBP bound to the GST-p53 TAD. We have repeated these binding assays at several times with different batches of fusion proteins and in vitro translated TBP. Relative binding activities were reproducible and resulted in the same relative order for TBP binding. This establishes a direct relationship between transactivation ability in vivo and the binding activity of the p53 TAD to TBP in vitro (Fig. 3C). The TBP binding activities of M22, M25, and M19 mutants lacking critical hydrophobic residues but bearing identical net negative charge were significantly decreased when compared with that of wild type p53 TAD, indicating that the binding of p53 TAD to TBP is not due to nonspecific ionic interaction between the positively charged region of TBP and negatively charged p53 TAD. These results do not agree well with previous report in which the binding ability of p53 protein to TBP was not affected by the mutations at residues 22 and 23 (43Lin J. Chen J. Elenbaas B. Levine A.J. Genes & Dev. 1994; 8: 1235-1246Crossref PubMed Scopus (577) Google
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