RACK1 Interacts with E1A and Rescues E1A-induced Yeast Growth Inhibition and Mammalian Cell Apoptosis
2001; Elsevier BV; Volume: 276; Issue: 29 Linguagem: Inglês
10.1074/jbc.m010346200
ISSN1083-351X
AutoresNianli Sang, Anna Severino, Patrizia Russo, Alfonso Baldi, Antonio Giordano, Anna Maria Mileo, Marco G. Paggi, Antonio De Luca,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe adenoviral E1A proteins are able to promote proliferation and transformation, inhibit differentiation, induce apoptosis, and suppress tumor growth. The extreme N terminus and conserved region one of E1A, which are indispensable for transcriptional regulation and for binding to p300/CBP, TBP, and pCAF, play essential roles in these abilities. These observations strongly suggest an intrinsic link between E1A-mediated transcriptional regulation and other effects. In this report, we show that E1A inhibits the normal growth of Saccharomyces cerevisiae HF7c, and this inhibition also depends on the domains required for transcriptional regulation. We demonstrate that E1A associates with histone acetyltransferase activity and represses the transactivation activity of transcription factor in S. cerevisiae, suggesting that E1A may suppress the expression of genes required for normal growth. Based on yeast growth rescue, we present a genetic screening strategy that identified RACK1 as an E1A antagonizing factor. Expression of human RACK1 efficiently relieves E1A-mediated growth inhibition in HF7c and protects human tumor cells from E1A-induced apoptosis. Finally, we show that RACK1 decreases E1A-associated histone acetyltransferase activity in yeast and mammalian cells, and physically interacts with E1A. Our data demonstrate that RACK1 is a repressor of E1A, possibly by antagonizing the effects of E1A on host gene transcription. The adenoviral E1A proteins are able to promote proliferation and transformation, inhibit differentiation, induce apoptosis, and suppress tumor growth. The extreme N terminus and conserved region one of E1A, which are indispensable for transcriptional regulation and for binding to p300/CBP, TBP, and pCAF, play essential roles in these abilities. These observations strongly suggest an intrinsic link between E1A-mediated transcriptional regulation and other effects. In this report, we show that E1A inhibits the normal growth of Saccharomyces cerevisiae HF7c, and this inhibition also depends on the domains required for transcriptional regulation. We demonstrate that E1A associates with histone acetyltransferase activity and represses the transactivation activity of transcription factor in S. cerevisiae, suggesting that E1A may suppress the expression of genes required for normal growth. Based on yeast growth rescue, we present a genetic screening strategy that identified RACK1 as an E1A antagonizing factor. Expression of human RACK1 efficiently relieves E1A-mediated growth inhibition in HF7c and protects human tumor cells from E1A-induced apoptosis. Finally, we show that RACK1 decreases E1A-associated histone acetyltransferase activity in yeast and mammalian cells, and physically interacts with E1A. Our data demonstrate that RACK1 is a repressor of E1A, possibly by antagonizing the effects of E1A on host gene transcription. extreme N terminus conserved region one histone acetyltransferase receptor for activated protein kinase C protein kinase C DNA-binding domain transactivation domain amino acid(s) polymerase chain reaction polyacrylamide gel electrophoresis The adenovirus E1A proteins are functionally important for viral infection and replication. The most important role they play is preparation of a favorable environment for viral replication by reprogramming the host cell processes. E1A proteins have been implicated in the promotion of cell proliferation, transformation, and inhibition of differentiation of certain cell types (1Shenk T. Flint J. Adv. Cancer Res. 1991; 57: 47-85Crossref PubMed Scopus (157) Google Scholar, 2Nevins J.R. Curr. Top. Microbiol. Immunol. 1995; 199: 25-32PubMed Google Scholar, 3Berlingieri M.T. Santoro M. Battaglia C. Grieco M. Fusco A. 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In fact, E1A has been used as a tool to identify important factors that are involved in fundamental cellular processes through their functional interaction with E1A (24Pines J. Hunter T. Nature. 1990; 346: 760-763Crossref PubMed Scopus (529) Google Scholar,27Schaeper U. Boyd J.M. Verma S. Uhlmann E. Subramanian T. Chinnadurai G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10467-10471Crossref PubMed Scopus (309) Google Scholar, 28Ewen M.E. Xing Y.G. Lawrence J.B. Livingston D.M. Cell. 1991; 66: 1155-1164Abstract Full Text PDF PubMed Scopus (346) Google Scholar, 29Mayol X. Graña X. Baldi A. Sang N. Hu Q. Giordano A. Oncogene. 1993; 8: 2561-2566PubMed Google Scholar, 30Li Y. Graham C. Lacy S. Duncan A.M. Whyte P. Genes Dev. 1993; 7: 2366-2377Crossref PubMed Scopus (302) Google Scholar, 31Lundblad J.R. Kwok R.P.S. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (531) Google Scholar). Recently, we showed that the extreme N terminus (ENT)1 (aa 1–37) and the conserved region one (CR1) (aa 40–80) of E1A possess transactivation activity in both yeast and mammalian cells (32Sang N. Claudio P.P. Fu Y. Horikoshi N. Graeven U. Weinmann R. Giordano A. DNA Cell Biol. 1997; 16: 1321-1333Crossref PubMed Scopus (6) Google Scholar). In addition, we observed that these regions are able to regulate host cell gene expression, either positively or negatively, in a pRb/E2F-independent manner. The mechanisms underlying the transcriptional regulation may involve the direct interaction with p300/CBP, two nuclear histone acetyltransferases (33Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1535) Google Scholar, 34Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar), and with components of the basal transcriptional machinery, such as TBP (32Sang N. Claudio P.P. Fu Y. Horikoshi N. Graeven U. Weinmann R. Giordano A. DNA Cell Biol. 1997; 16: 1321-1333Crossref PubMed Scopus (6) Google Scholar, 35Sang N. Avantaggiati M.L. Giordano A. J. Cell. Biochem. 1997; 66: 277-285Crossref PubMed Scopus (36) Google Scholar). Furthermore, we reported that the extreme N terminus of E1A interacts with unidentified cellular proteins (36Sang N. Giordano A. J. Cell. Physiol. 1997; 170: 182-191Crossref PubMed Scopus (14) Google Scholar). More recently, it has been shown that these regions directly interact with pCAF, another histone acetyltransferase (26Reid J.L. Bannister A.J. Zegerman P. Martinez-Balbas M.A. Kouzarides T. EMBO J. 1998; 17: 4469-4477Crossref PubMed Scopus (108) Google Scholar). Interestingly, these regions also are required for E1A-induced apoptosis (37White E. Cipriani R. Sabbatini P. Denton A. J. Virol. 1991; 65: 2968-2978Crossref PubMed Google Scholar, 38Mymryk J.S. Shire K. Bayley S.T. Oncogene. 1994; 9: 1187-1193PubMed Google Scholar) and for tumor suppression (16Deng J. Xia W. Hung M.C. Oncogene. 1998; 17: 2167-2175Crossref PubMed Scopus (64) Google Scholar) in mammalian cells, suggesting that E1A induces apoptosis and tumor suppression by disrupting the regulation of host gene expression. Yeast is used widely as a model system to study cell growth regulation, DNA replication, and transcriptional regulation, since most of the essential regulatory mechanisms are well conserved between yeast and mammals (39Albright S.R. Tjian R. Gene (Amst.). 2000; 242: 1-13Crossref PubMed Scopus (275) Google Scholar, 40Okayama H. Nagata A. Jinno S. Murakami H. Tanaka K. Nakashima N. Adv. Cancer Res. 1996; 69: 17-62Crossref PubMed Google Scholar, 41Lee M.G. Norbury C.J. Spurr N.K. Nurse P. Nature. 1988; 333: 676-679Crossref PubMed Scopus (133) Google Scholar). Genetic complementation approaches in yeast have been used to identify a variety of mammalian genes involved in growth regulation. In addition, identification of the basal transcriptional complexes reveals astonishing similarities between yeast and mammals, providing strong evidence that transcriptional regulation is a highly conserved process between evolutionarily distant species. Particularly, the yeast system has been successfully used for functional and genetic analysis of E1A effects (42Miller M.E. Engel D.A. Smith M.M. Oncogene. 1995; 11: 1623-1630PubMed Google Scholar, 43Zieler H.A. Walberg M. Berg P. Mol. Cell. Biol. 1995; 15: 3227-3237Crossref PubMed Scopus (34) Google Scholar). We investigated here the E1A-mediated growth inhibition in yeast cells and the roles of the ENT and CR1 regions. We found that the domains required for yeast growth inhibition also are required for transcriptional regulation, apoptosis, and tumor suppression in mammalian cells. We also found that E1A associated with a HAT activity in yeast, and that expression of E1A repressed transactivation activity of yeast transcription factors. Based on the rescue of yeast growth, we designed a genetic screening strategy and found that human RACK1 successfully antagonizes E1A-mediated growth inhibition in yeast. Finally, we observed that co-transfection of RACK1 protects human tumor cells from undergoing E1A-induced apoptosis and that RACK1 physically interacts with E1A in vitro and in vivo. Oligonucleotides used as primers for PCR were synthesized by the Nucleic Acid Facility at Kimmel Cancer Institute, Thomas Jefferson University. The pGBT9, pGBKT7, pGAD424 vectors, and pCL1 were purchased from CLONTECH Laboratories. The pEG202 vector and pSH18–34 (URA3, LexAop-LacZ reporter plasmid) were described previously (32Sang N. Claudio P.P. Fu Y. Horikoshi N. Graeven U. Weinmann R. Giordano A. DNA Cell Biol. 1997; 16: 1321-1333Crossref PubMed Scopus (6) Google Scholar). Plasmids pcDNA3 and pcDNA3T7Tag were obtained from Invitrogen. DNA sequences of PCR-generated fragments were confirmed by sequencing. The wild-type 243R E1A fusion construct and most of the E1A mutant constructs in pGBT9 were as previously described (32Sang N. Claudio P.P. Fu Y. Horikoshi N. Graeven U. Weinmann R. Giordano A. DNA Cell Biol. 1997; 16: 1321-1333Crossref PubMed Scopus (6) Google Scholar, 44Sang N. Condorelli G. De Luca A. MacLachlan T.K. Giordano A. Anal. Biochem. 1996; 233: 142-144Crossref PubMed Scopus (25) Google Scholar). To express 243R E1A alone in yeast, a KpnI-PstI fragment of 243R E1A was generated by PCR. This fragment was then used to replace the KpnI-PstI fragment encoding the GAL4 transactivation domain in the pGAD424 vector, thus generating the construct pG-E1A. The LexA-E1A fusion constructs, pEG202-E1A and pG-LexA-E1A with a Trp selection marker were described previously. To express LexA alone in HF7c, the E1A part was removed by restriction digestion with EcoRI and PstI followed by blunting and re-ligation that resulted in pG-LexA. To express 243R E1A in EGY48 the smaller SnaBI-PstI fragment from pG-E1A was used to replace the smallerSnaBI-PstI fragment in pGBT9, and to generate pE1A. Plasmid pGBT9-Rb2/p130 was constructed by PCR. Plasmids pGBT9-Rb and pGBT9-p107 were described previously (32Sang N. Claudio P.P. Fu Y. Horikoshi N. Graeven U. Weinmann R. Giordano A. DNA Cell Biol. 1997; 16: 1321-1333Crossref PubMed Scopus (6) Google Scholar). Human RACK-1 cDNA was obtained by reverse transcriptase-PCR using total RNA from the 293 cell line as a template. Primers were 5′-GATCCCCGGGCATGACTGAGCAGATGACC-3′ and 5′ GATCCTGCAGCTAGCGTGTGCCAATGGTC-3′. The amplified fragment was then cloned into a pGAD424 vector fused to GAL4 activation domain. For mammalian cell transfection assays, all the cDNAs encoding E1A, E1A mutants, DBD, DBD-E1A, DBD-E1A mutants, GST, GST-E1A, and GST-E1A mutants were driven by the cytomegalovirus promoter. The plasmid pON260 that constitutively expresses full-length GAL4 as a control of transfection efficiency was described previously (32Sang N. Claudio P.P. Fu Y. Horikoshi N. Graeven U. Weinmann R. Giordano A. DNA Cell Biol. 1997; 16: 1321-1333Crossref PubMed Scopus (6) Google Scholar). For SAOS-2 (human osteosarcoma cell line) transfection, RACK1 was cloned in pcDNA3T7Tag (Stratagene). Saccharomyces cerevisiae strains HF7c (MATα, ura3–52, his3–200, lys2–801, ade2–101, trp1–901, leu2–3112, gal4–542, gal80–538, LYS2::GAL1-HIS3, URA3::(GAL4 17mers)3-CYC1-lacZ), PJ69–2A (MATa, trp1–901, leu2–3, 112, ura3–52, his3–200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2) were purchased fromCLONTECH Laboratories and the EGY48 strain (MATa, ura3, his3, trp1, LexAop-LEU2) was described previously (32Sang N. Claudio P.P. Fu Y. Horikoshi N. Graeven U. Weinmann R. Giordano A. DNA Cell Biol. 1997; 16: 1321-1333Crossref PubMed Scopus (6) Google Scholar). All the yeast strains were maintained at 30 °C on YPD plates. Yeast transformation was performed with the lithium acetate (LiOAc) method described previously (32Sang N. Claudio P.P. Fu Y. Horikoshi N. Graeven U. Weinmann R. Giordano A. DNA Cell Biol. 1997; 16: 1321-1333Crossref PubMed Scopus (6) Google Scholar). Transformants were selected and maintained on synthetic medium lacking selective amino acid(s). The yeast transformants were grown in 10 ml of SD without tryptophan or other auxotrophic amino acids depending on the selective marker of the plasmids, at 30 °C with shaking (270 rpm) to stationary. The liquid culture was diluted to 0.2 (around 0.5–0.6 × 106 cells/ml)A 600 in 10 ml of fresh SD without selective amino acids or 10 ml of YPD when transformants harboring plasmids with different selective markers were tested together to prevent variations caused by using different SD medium. The freshly inoculated culture was put back to incubate at 30 °C with shaking (270 rpm). One ml of sample was taken from each culture to measureA 600 at various time points post-inoculation. The cell growth rate was measured as the ratio of theA 600 at specific time points to the initialA 600 (Growth Index). To assay the viability of the yeast transformants, 100 µl of the original freshly inoculated culture were used to make sequential dilutions (10−2, 10−3, and 10−4) and were plated out on 150-mm plates with SD-dropout agar and cultured at 30 °C for 4 days to monitor colony formation. Transformants were grown overnight in 2 ml of proper synthetic medium at 30 °C with shaking. After adding 8 ml of fresh SD, yeast cells were allowed to grow for a further 4 h. After measuring the A 600, 1.5 ml of culture was collected and washed once with Z buffer (60 mm Na2HPO4, 40 mmNaH2PO4, 10 mm KCl, 10 mm MgSO4, pH 7.0). After being re-suspended in 300 ml of Z buffer, 100 ml was transferred into a fresh sterile tube. β-Galactosidase activity was measured as described before (32Sang N. Claudio P.P. Fu Y. Horikoshi N. Graeven U. Weinmann R. Giordano A. DNA Cell Biol. 1997; 16: 1321-1333Crossref PubMed Scopus (6) Google Scholar) and corrected for cell density (A 600) and for the reaction time. The relative activity of the activators was compared with pGBT9 vector alone and expressed as relative transactivation activity. SAOS-2 (ATCC, human osteosarcoma) and HeLa (ATCC, human cervix carcinoma) cells were maintained in Dulbecco's modified Eagle's medium supplemented withl-glutamine and 10% fetal bovine serum. For transient transfection experiments, cells were seeded 24 h before the transfection to obtain a 50–70% confluence. Cells were transfected by either Transfectam (Promega) or by standard calcium phosphate protocols. Transfection efficiency was monitored by measuring β-galactosidase activity derived from the co-transfected pON260 plasmid (32Sang N. Claudio P.P. Fu Y. Horikoshi N. Graeven U. Weinmann R. Giordano A. DNA Cell Biol. 1997; 16: 1321-1333Crossref PubMed Scopus (6) Google Scholar). To use Transfectam, the instructions for serum-free transfection procedures provided by the manufacturer were followed. Three hours after transfection, fresh medium with serum was added, and the cells then were cultured for 48 h before harvest. When using calcium phosphate transfection, 18 h after transfection, the medium was changed and cells were cultured for an additional 24 h. Transfection efficiency obtained from this method was around 20–25%. For stable transfection, the calcium phosphate method was used to transfect 10 µg of linearized plasmids. Forty-eight hours after transfections, the cells were trypsinized and selected in G418 medium for 4 weeks. Cell labeling with [35S]methionine and GST pull-down assays were performed as previously described (36Sang N. Giordano A. J. Cell. Physiol. 1997; 170: 182-191Crossref PubMed Scopus (14) Google Scholar). Yeast protoplasts were prepared by standard methods (45Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2 Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Briefly, overnight grown yeast culture was washed three times with ice-cold phosphate-buffered saline. After being incubated for 10 min at 30 °C with stabilization buffer A (1 m sorbitol, 10 mm MgCl2, 2 mm dithiothreitol, 50 mm potassium phosphate, pH 7.8, 100 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin), the yeast was incubated at 30 °C for 5 min with stabilization buffer B (1m sorbitol, 10 mm MgCl2, 2 mm dithiothreitol, 25 mm potassium phosphate, pH 7.8, 25 mm sodium succinate, pH 5.5, 100 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin). Protoplasts then were generated by enzymatic digestion with 25 units/ml lyticase (Sigma) for 15 min at room temperature in stabilization buffer B. The protoplasts were washed with phosphate-buffered saline three times and lysed in IPH buffer as previously described (26Reid J.L. Bannister A.J. Zegerman P. Martinez-Balbas M.A. Kouzarides T. EMBO J. 1998; 17: 4469-4477Crossref PubMed Scopus (108) Google Scholar, 33Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1535) Google Scholar). Cultured human cells were directly lysed in IPH buffer as described. For immunoprecipitation, lysates containing 250 µg of protein were first incubated with a nonspecific mouse serum and cleared by mixing with killed whole Staphylococcus aureus cells (Life Technologies, Inc.). The cleared supernatant was incubated with anti-E1A monoclonal antibody and immunoprecipitation was performed as previously described to obtain E1A complexes (35Sang N. Avantaggiati M.L. Giordano A. J. Cell. Biochem. 1997; 66: 277-285Crossref PubMed Scopus (36) Google Scholar). The anti-E1A monoclonal antibodies M73 and M37 were described elsewhere (46Harlow E. Franza Jr., B.R. Schley C. J. Virol. 1985; 55: 533-546Crossref PubMed Google Scholar, 47Giordano A. Whyte P. Harlow E. Franza Jr., B.R. Beach D. Draetta G. Cell. 1989; 58: 981-990Abstract Full Text PDF PubMed Scopus (167) Google Scholar). E1A complexes then were separated by SDS-PAGE followed by Western blotting analysis, or used for HAT assays. The procedure for Western blotting was described previously (32Sang N. Claudio P.P. Fu Y. Horikoshi N. Graeven U. Weinmann R. Giordano A. DNA Cell Biol. 1997; 16: 1321-1333Crossref PubMed Scopus (6) Google Scholar, 35Sang N. Avantaggiati M.L. Giordano A. J. Cell. Biochem. 1997; 66: 277-285Crossref PubMed Scopus (36) Google Scholar). HAT assays were performed with the HAT-check kit (Pierce). Briefly, the immunoprecipitated complexes were incubated with a synthetic substrate corresponding to the first 23 amino acids of histone H4 and coupled to biotin at 30 °C for 1 h with 3H-labeled acetyl-CoA in 1 × HAT buffer. Washed immobilized streptavidin-agarose, pre-equilibrated in 1 × HAT buffer, was then added to each assay tube. The samples were rotated at room temperature for 30 min. After 3 washes with IPH buffer, the matrix was resuspended in 1 × HAT buffer and transferred to a scintillation vial. The incorporated3H-acetyl was measured in a scintillation counter. One microgram of the plasmid encoding for RACK1 was used to program a TnT rabbit reticulocyte lysate (Promega) under control of the T7 polymerase in the presence of [35S]methionine. Aliquots of the reaction mixture were added to glutathione-Sepharose beads coupled with 4 µg of GST chimerized with E1A. Incubation was carried out in NENT buffer (20 mm Tris, pH 8.0, 100 mm NaCl, 1 mmEDTA, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin) for 60 min at 4 °C with gentle rocking. Beads were washed three times in NENT buffer, and then electrophoresis was performed on SDS-PAGE. Gel was dried and then exposed at −70 °C using Kodak Bio-Max MS film with DuPont Cronex intensifying screens. After transfection, cells were fixed in paraformaldehyde, washed in distilled water, and exposed briefly to 3% H2O2 to inactivate endogenous peroxidase. The TUNEL reaction was performed using the peroxidase-based Apoptag kit (Oncor). The TUNEL positive cells were revealed by means of diaminobenzidine and H2O2, according to the supplier's instructions. Finally, stained cells were slightly counterstained with hematoxylin. TUNEL positive cells were considered as apoptotic. We previously reported that 243R E1A possesses transactivation activity when fused to a DNA-binding domain in yeast and in mammalian cells (32Sang N. Claudio P.P. Fu Y. Horikoshi N. Graeven U. Weinmann R. Giordano A. DNA Cell Biol. 1997; 16: 1321-1333Crossref PubMed Scopus (6) Google Scholar). In that study, we consistently observed that expression of wild-type E1A in yeast strain HF7c led to a slow-growing phenotype. To further understand the E1A-mediated inhibition of yeast growth, we investigated this phenomenon in a carefully controlled study. As shown in Fig. 1 A, expression of E1A led to growth inhibition of HF7c. As controls, expression of the RB family gene products (pRb, p107, or pRb2/p130), a family of growth inhibitors in mammalian cells (23Sang N. Baldi A. Giordano A. Mol. Cell. Differ. 1995; 3: 1-29Google Scholar) showed no effect on yeast growth. The fusion of E1A with the DNA-binding domain (DB) of GAL4 was not essential for this effect, because expression of E1A alone, or when fused with either the transactivation domain (TAD) of GAL4 or LexA, resulted in similar inhibition (Fig. 1 B). Similar studies were performed in the EYG48 and PJ69-2A strains. Significant growth inhibition was observed in the EYG48 strain, but expression of E1A inhibited the growth of PJ69-2A strain only slightly (data not shown), suggesting that E1A-mediated yeast growth inhibition is a strain-specific event. 243R E1A is a multifunctional oncoprotein consisting of several well defined functional domains (48Lillie J.W. Loewenstein P.M. Green M.R. Green M. Cell. 1987; 50: 1091-1100Abstract Full Text PDF PubMed Scopus (151) Google Scholar). The definition of a solid link between the specific domains and the inhibitory activity is essential for understanding the underlying mechanism and the biological significance in the virus-host interaction. We used an extensive set of E1A mutants to express mutant E1A proteins in yeast cells and compared the effects of these mutants on yeast growth with that of wild-type E1A. The mutant E1A constructs were characterized previously (44Sang N. Condorelli G. De Luca A. MacLachlan T.K. Giordano A. Anal. Biochem. 1996; 233: 142-144Crossref PubMed Scopus (25) Google Scholar) and the mutant protein expression in yeast was confirmed by Western blot analysis (Fig. 2). Two monoclonal antibodies against different epitopes of E1A were used to detect E1A proteins encoded by these constructs (46Harlow E. Franza Jr., B.R. Schley C. J. Virol. 1985; 55: 533-546Crossref PubMed Google Scholar). As shown in Fig. 2,A and B, wild-type E1A was recognized by both the M73 and M37 antibodies. Mutants were detected by M73, M37, or both, depending on the existence of intact epitopes. The scheme in Fig.2 C represents the structure of the E1A mutants employed, with the position of the epitopes recognized by M73 and M37 indicated. The data in Fig. 3 show that constitutive expression of 243R E1A in the HF7c yeast strain resulted in a markedly slower growth rate, as did the expression of E1A mutants containing aa 1–228 or 1–120. However, deletion of the N-terminal 70 or 100 aa completely abolished E1A-mediated growth inhibitory activity. While deletion of the major part of CR1 (Δ38–67) resulted in a slightly lower growth rate, deletion of ENT, while keeping CR1 intact, also led to a loss of inhibition. We concluded that the growth inhibitory domain is contained within the N-terminal aa 1–70 region and must involve aa 38–67. The slow growth was not caused by a decreased viability of yeast transformants, because the numbers of colonies formed on plates did not differ significantly (data not shown). In mammalian cells, E1A proteins function as dual regulators of gene expression. They stimulate the transcription of certain genes, but repress others (1Shenk T. Flint J. Adv. Cancer Res. 1991; 57: 47-85Crossref PubMed Scopus (157) Google Scholar, 3Berlingieri M.T. Santoro M. Battaglia C. Grieco M. Fusco A. Oncogene. 1993; 8: 249-2554PubMed Google Scholar, 49Kraus V.B. Moran E. Nevins J.R. Mol. Cell. 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Nucleic Acids Res. 1994; 22: 3053-3060Crossref PubMed Scopus (33) Google Scholar), E1A-mediated growth inhibition is not caused by the stimulation of GAL4-responsive genes as shown above. Therefore, we tested the hypothesis that overexpression of E1A might actually disrupt transcriptional regulation i
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