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• W2069409615 abstract "Posttranslational modifications may alter the biochemical functions of a protein by modifying associations with other macromolecules, allosterically altering intrinsic catalytic activities, or determining subcellular localization. The adenovirus-transforming protein E1A is acetylated by its cellular targets, the co-activators CREB-binding protein, p300, and p300/CREB-binding protein-associated factor in vitro and also in vivo at a single lysine residue (Lys239) within a multifunctional carboxyl-terminal domain necessary for both nuclear localization and interaction with the transcriptional co-repressor carboxyl-terminal binding protein (CtBP). In contrast to a previous report, we demonstrate that acetylation of Lys239 does not disrupt CtBP binding and that 12 S E1A-mediated repression of CREB-binding protein-dependent transcription does not require recruitment of CtBP. Instead we find that the cytoplasmic fraction of E1-transformed 293 cells is enriched for acetylated E1A with relative exclusion from the nuclear compartment. Whereas wild type 12 S E1A binds importin-α3, binding affinity was markedly reduced both by single amino acid substitution mutations and acetylation at Lys239. This is the first demonstration that acetylation may alter nuclear partitioning by direct interference with nuclear import receptor recognition. The finding that the cytoplasmic fraction of E1A is acetylated indicates that E1A may exert its pleiotropic effects on cellular transformation in part by affecting cytoplasmic processes. Posttranslational modifications may alter the biochemical functions of a protein by modifying associations with other macromolecules, allosterically altering intrinsic catalytic activities, or determining subcellular localization. The adenovirus-transforming protein E1A is acetylated by its cellular targets, the co-activators CREB-binding protein, p300, and p300/CREB-binding protein-associated factor in vitro and also in vivo at a single lysine residue (Lys239) within a multifunctional carboxyl-terminal domain necessary for both nuclear localization and interaction with the transcriptional co-repressor carboxyl-terminal binding protein (CtBP). In contrast to a previous report, we demonstrate that acetylation of Lys239 does not disrupt CtBP binding and that 12 S E1A-mediated repression of CREB-binding protein-dependent transcription does not require recruitment of CtBP. Instead we find that the cytoplasmic fraction of E1-transformed 293 cells is enriched for acetylated E1A with relative exclusion from the nuclear compartment. Whereas wild type 12 S E1A binds importin-α3, binding affinity was markedly reduced both by single amino acid substitution mutations and acetylation at Lys239. This is the first demonstration that acetylation may alter nuclear partitioning by direct interference with nuclear import receptor recognition. The finding that the cytoplasmic fraction of E1A is acetylated indicates that E1A may exert its pleiotropic effects on cellular transformation in part by affecting cytoplasmic processes. conserved region CREB-binding protein cAMP-response element-binding protein p300/CREB-binding protein-associated factor glutathione S-transferase acetylated E1A-specific monoclonal antibody nuclear localization signal chloramphenicol acetyltransferase Rous sarcoma virus carboxyl-terminal binding protein histone acetyltransferase Viral oncoproteins often target important steps cellular metabolism to exert effects on cellular proliferation and differentiation. The E1 early region of human adenoviruses is the first portion of the adenovirus genome expressed on infection of a host cell and encodes the prototypical oncoproteins E1A and E1B. Whereas both E1 region products are necessary for full viral oncogenic transformation, the E1A products alone can induce S phase, immortalize cells, and cooperate with other cellular and viral oncogenes to transform primary rodent cells (1Moran E. Mathews M.B. Cell. 1987; 48: 177-178Abstract Full Text PDF PubMed Scopus (220) Google Scholar, 2Shenk T. Flint J. Adv. Cancer Res. 1991; 57: 47-85Crossref PubMed Scopus (152) Google Scholar, 3Nevins J.R. Curr. Top. Microbiol. Immunol. 1995; 199: 25-32PubMed Google Scholar, 4Gallimore P.H. Turnell A.S. Oncogene. 2001; 20: 7824-7835Crossref PubMed Google Scholar). The cellular effects of E1A are thought to be mediated primarily by effects on gene transcription, modulating both cellular and viral gene expression through physical associations with cellular transcriptional regulatory proteins (5Flint J. Shenk T. Annu. Rev. Genet. 1997; 31: 177-212Crossref PubMed Scopus (184) Google Scholar). The E1A region of adenovirus encodes two major mRNAs (12 and 13 S), generated through alternative splicing of two exons from a single primary transcript. For the C-type adenovirus (serotypes 2 and 5), these mRNAs encode proteins of 243 and 289 amino acids, respectively (Fig. 1 A). Amino acid sequences of E1A isoforms from each of the adenovirus serotypes differ substantially, except within CRs1 that act as interaction motifs for E1A target proteins. Both forms interact with cellular proteins including pRB and the transcriptional co-activators CBP and p300 through CR2, and CR1 and N-terminal regions, respectively. The E1A 13 S form (E1A 289R) differs from the 12 S form (E1A 243R) in the presence of an additional first exon-derived sequence (CR3), which serves as an interaction domain for ATF-2 and TBP/TFIID (5Flint J. Shenk T. Annu. Rev. Genet. 1997; 31: 177-212Crossref PubMed Scopus (184) Google Scholar). Whereas both of the two major forms of E1A are necessary for productive infection and viral transformation, the CR3 region is dispensable for immortalization and cellular transformation in conjunction with cellular oncogenes (1Moran E. Mathews M.B. Cell. 1987; 48: 177-178Abstract Full Text PDF PubMed Scopus (220) Google Scholar, 2Shenk T. Flint J. Adv. Cancer Res. 1991; 57: 47-85Crossref PubMed Scopus (152) Google Scholar, 6Williams J. Williams M. Liu C. Telling G. Curr. Top. Microbiol. Immunol. 1995; 199: 149-175PubMed Google Scholar). The 12 S E1A protein inhibits CBP- and p300-dependent transcription by interacting with and repressing the activities of these co-activators, although the mechanism of repression remains controversial. E1A repression of the co-activator function of CBP depends on a direct physical interaction between the N terminus of E1A and at least one domain of the CBP and p300 co-activators (7Wang H.G. Rikitake Y. Carter M.C. Yaciuk P. Abraham S.E. Zerler B. Moran E. J. Virol. 1993; 67: 476-488Crossref PubMed Google Scholar, 8Lundblad J.R. Kwok R.P. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (529) Google Scholar, 9Arany Z. Newsome D. Oldread E. Livingston D.M. Eckner R. Nature. 1995; 374: 81-84Crossref PubMed Scopus (489) Google Scholar). This region of CBP/p300 serves as an interaction site for a large number of transcription factors, including c-Fos, E2F, MyoD, and the basal factor TFIIB (10Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar). Models proposed for inhibition of CBP/p300 function have included sequestration of CBP/p300 from active transcription complexes (11Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1306) Google Scholar, 12Nakajima T. Uchida C. Anderson S.F. Parvin J.D. Montminy M. Genes Dev. 1997; 11: 738-747Crossref PubMed Scopus (210) Google Scholar, 13Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar) and displacement of positive transcription factors by competitive binding (14O'Connor M.J. Zimmermann H. Nielsen S. Bernard H.U. Kouzarides T. J. Virol. 1999; 73: 3574-3581Crossref PubMed Google Scholar, 15Felzien L.K. Farrell S. Betts J.C. Mosavin R. Nabel G.J. Mol. Cell. Biol. 1999; 19: 4241-4246Crossref PubMed Scopus (53) Google Scholar). Whereas some reports suggest that E1A may repress the HAT activity of CBP/p300 (16Hamamori Y. Sartorelli V. Ogryzko V. Puri P.L., Wu, H.Y. Wang J.Y. Nakatani Y. Kedes L. Cell. 1999; 96: 405-413Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 17Chakravarti D. Ogryzko V. Kao H.Y. Nash A. Chen H. Nakatani Y. Evans R.M. Cell. 1999; 96: 393-403Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 18Perissi V. Dasen J.S. Kurokawa R. Wang Z. Korzus E. Rose D.W. Glass C.K. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3652-3657Crossref PubMed Scopus (84) Google Scholar), others suggest that E1A may stimulate CBP/p300 HAT activityin vivo (19Ait-Si-Ali S. Ramirez S. Barre F.X. Dkhissi F. Magnaghi-Jaulin L. Girault J.A. Robin P. Knibiehler M. Pritchard L.L. Ducommun B. Trouche D. Harel-Bellan A. Nature. 1998; 396: 184-186Crossref PubMed Scopus (269) Google Scholar) and may enhance in vivo acetylation of pRB (20Chan H.M. Krstic-Demonacos M. Smith L. Demonacos C. Thangue N.B. Nat. Cell Biol. 2001; 3: 667-674Crossref PubMed Scopus (237) Google Scholar). The second exon-encoded functions of E1A are likewise functionally complex. Deletion mutations within the C terminus of E1A relieve negative effects on transformation and correlate in part with a loss of binding of E1A to CtBP, a 48-kDa phosphoprotein (21Boyd J.M. Subramanian T. Schaeper U., La Regina M. Bayley S. Chinnadurai G. EMBO J. 1993; 12: 469-478Crossref PubMed Scopus (259) Google Scholar, 22Schaeper 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 (307) Google Scholar). CtBP1 and a second isoform, CtBP2 (23Turner J. Crossley M. EMBO J. 1998; 17: 5129-5140Crossref PubMed Scopus (277) Google Scholar, 24Furusawa T. Moribe H. Kondoh H. Higashi Y. Mol. Cell. Biol. 1999; 19: 8581-8590Crossref PubMed Google Scholar), bind E1A through a conserved -PLDLS- motif near the common C terminus of both E1A isoforms (Fig.1 A). This motif and variant sequences (-PXDLS-) are also found in a number of cellular transcriptional repressor proteins important in cellular proliferation, growth, and differentiation (25Chinnadurai G. Mol. Cell. 2002; 9: 213-224Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). In some of these CtBP-interacting proteins, mutation of a core -PXDLS- motif both decreases binding of CtBP and correlates with either abolished or reduced repressor activity (24Furusawa T. Moribe H. Kondoh H. Higashi Y. Mol. Cell. Biol. 1999; 19: 8581-8590Crossref PubMed Google Scholar, 26Postigo A.A. Dean D.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6683-6688Crossref PubMed Scopus (221) Google Scholar, 27Criqui-Filipe P. Ducret C. Maira S.M. Wasylyk B. EMBO J. 1999; 18: 3392-3403Crossref PubMed Scopus (135) Google Scholar, 28Meloni A.R. Smith E.J. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9574-9579Crossref PubMed Scopus (165) Google Scholar, 29van Vliet J. Turner J. Crossley M. Nucleic Acids Res. 2000; 28: 1955-1962Crossref PubMed Scopus (128) Google Scholar, 30Koipally J. Georgopoulos K. J. Biol. Chem. 2000; 275: 19594-19602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), whereas in others (31Touitou R. Hickabottom M. Parker G. Crook T. Allday M.J. J. Virol. 2001; 75: 7749-7755Crossref PubMed Scopus (81) Google Scholar), mutation of this motif has little or no effect on repression. The biochemical consequences of CtBP binding to E1A are less well defined. One model implicates CtBP in repression of CR1-dependent transactivation through E1A (32Sollerbrant K. Chinnadurai G. Svensson C. Nucleic Acids Res. 1996; 24: 2578-2584Crossref PubMed Scopus (68) Google Scholar). Alternatively, E1A may act to displace or sequester CtBP from cellular targets that contain the -PXDLS- motif (33Schaeper U. Subramanian T. Lim L. Boyd J.M. Chinnadurai G. J. Biol. Chem. 1998; 273: 8549-8552Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), a model more consistent with proposed mechanisms for E1A regulation of p300/CBP and pRB activities. A recent report suggests that CtBP may directly participate in the activity of E1A and mediate repression of CBP-dependent co-activation by E1A. Zhang et al. (34Zhang Q. Yao H., Vo, N. Goodman R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14323-14328Crossref PubMed Scopus (107) Google Scholar) report that p300- or P/CAF-dependent acetylation of 12 S E1A at Lys239 within the -PLDLSCK 239- motif attenuates E1A repression of CBP activity by impairing the interaction of CtBP with E1A. In this model, recruitment of the CtBP co-repressor complex is essential for the repression of CBP-dependent co-activation by E1A. Whereas several reports have indicated that sequences outside of the -PLDLS- might influence the interaction of CtBP with its targets, in E1A this lysine has been shown to be dispensable in peptide competition studies (35Molloy D.P. Milner A.E. Yakub I.K. Chinnadurai G. Gallimore P.H. Grand R.J. J. Biol. Chem. 1998; 273: 20867-20876Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and examination of -PXDLS- and surrounding sequences of the known probable CtBP cellular targets indicates that this lysine is not invariably conserved (36Turner J. Crossley M. Bioessays. 2001; 23: 683-690Crossref PubMed Scopus (146) Google Scholar). Furthermore, previous studies demonstrate that a sequence carboxyl-terminal to the -PLDLS- motif, including Lys239, is essential for nuclear partitioning of E1A (37Lyons R.H. Ferguson B.Q. Rosenberg M. Mol. Cell. Biol. 1987; 7: 2451-2456Crossref PubMed Scopus (114) Google Scholar, 38Douglas J.L. Quinlan M.P. J. Virol. 1995; 69: 8061-8065Crossref PubMed Google Scholar, 39Douglas J.L. Quinlan M.P. Cell Growth Differ. 1994; 5: 475-483PubMed Google Scholar, 40Douglas J.L. Quinlan M.P. Virology. 1996; 220: 339-349Crossref PubMed Scopus (7) Google Scholar). In this report, we demonstrate that like P/CAF and p300, CBP acetylates lysine 239 of 12S E1A in vitro and that a small fraction of E1A is acetylated at this lysine in vivo in E1-transformed HEK293 cells and E1A-transfected cells. In our experiments, single amino acid substitution mutations and bona fide acetylation of full-length E1A at Lys239 do not disrupt binding to CtBPin vitro. Whereas substitution mutations at this position abrogate 12 S E1A repression of phospho-CREB-dependent, CBP-mediated co-activation of transcription due to impaired nuclear localization, mutations within the -PLDLS- motif that completely disrupt CtBP binding have no effect on E1A-mediated repression of CBP function, suggesting that the association of CtBP with E1A is not necessary for repression of CBP activity. Instead, consistent with a previous study (39Douglas J.L. Quinlan M.P. Cell Growth Differ. 1994; 5: 475-483PubMed Google Scholar), we find that the lysine at position 239 is invariably essential for the nuclear localization of 12 S E1A, and further that E1A acetylated at this lysine resides predominantly in the cytoplasm of E1A-expressing cells. 12 S E1A binds strongly to importin-α3 in vitro, while Lys239substitution mutations and acetylation of E1A by CBP abrogate this interaction. These results indicate that acetylation of E1A determines subcellular localization by interfering with nuclear import. Acetylation of E1A may act to either attenuate the nuclear functions of E1A or redirect a portion of 12 E1A to cytoplasmic targets. These results raise the possibility that E1A may exert its pleiotropic effects on cellular transformation in part by affecting cytoplasmic processes. Mammalian expression pRc/RSV-derived expression plasmids for wild type adenovirus type 2 12 S E1A protein (243R), CBP, CREB341, and the catalytic subunit of protein kinase A have been previously described (8Lundblad J.R. Kwok R.P. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (529) Google Scholar). Individual cDNAs encoding wild-type adenovirus type 5 13 S E1A protein (289R) and deletion mutants mCR1 (deletion of amino acids 38–65), mCR2 (deletion of amino acids 125–133), and mCR3 (deletion of amino acids 140–185), obtained from Dr. Ganes Sen (Cleveland Clinic), were inserted into pRc/RSV (Invitrogen) by PCR (41Kalvakolanu D.V. Liu J. Hanson R.W. Harter M.L. Sen G.C. J. Biol. Chem. 1992; 267: 2530-2536Abstract Full Text PDF PubMed Google Scholar). Site-directed mutagenesis at lysine 239 in 12 S E1A or the corresponding lysine 285 in 13 S E1A was performed by using the QuikChangeTM system (Stratagene). Eukaryotic Rc/RSV expression vectors for wild type 12 S E1A and K239A 12 S E1A with the carboxyl-terminal fusion of the SV40 T-antigen nuclear localization signal (-PKKKRKV) (42Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1843) Google Scholar) were produced by PCR. A plasmid for expression of histidine-tagged E1A in Escherichia coli was constructed by cloning the 12 S E1A cDNA in-frame into pET23d (Novagen) by PCR. A bacterial expression plasmid for expression of a GST fusion protein with full-length CtBP1 (pGST-CtBP1) was constructed by cloning the full-length cDNA for human CtBP1 (derived from pT7-CtBP1; generously provided by Dr. G. Chinnadurai, St. Louis University) in frame into pGEX-GK (43Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1635) Google Scholar) by PCR. The full-length cDNA for human importin-α3 (44Kohler M. Ansieau S. Prehn S. Leutz A. Haller H. Hartmann E. FEBS Lett. 1997; 417: 104-108Crossref PubMed Scopus (204) Google Scholar), provided by Dr. Mattias Kohler, was inserted into pGEX-GK by PCR. DNA constructs and mutations were confirmed by sequencing. Wild type and mutant 12 S E1A proteins were expressed from a pET23d-derived 12 S E1A cDNA expression plasmid inE. coli BL21 (DE3) pT-Trx (45Yasukawa T. Kanei-Ishii C. Maekawa T. Fujimoto J. Yamamoto T. Ishii S. J. Biol. Chem. 1995; 270: 25328-25331Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Cells were grown to log phase, induced with 0.4 mmisopropyl-1-thio-β-d-galactopyranoside, and harvested after 3 h at 37 °C. Cells were lysed by two passages through a French pressure cell at 14,000 p.s.i. (SLM-Aminico), and lysates were cleared of insoluble cellular debris by centrifugation at 30,000 × g for 30 min. E1A partially purified by nickel-nitrilotriacetic acid resin (Qiagen Inc.) was further purified by Q-Sepharose anion exchange column chromatography. GST fusion proteins for full-length CtBP1 (GST-CtBP1) and importin-α3 (GST-importin-α3) were expressed in E. coli DH5α and purified essentially as previously described (43Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1635) Google Scholar). The full-length murine CBP cDNA (46Chrivia J.C. Kwok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1755) Google Scholar), with two copies of the FLAG epitope at the carboxyl terminus (CBP-2×FLAG), was cloned into pFastBac1, and recombinant baculovirus producing epitope-tagged full-length CBP was produced in Sf9 cells according to the manufacturer's instructions (Bac-to-Bac; Invitrogen). CBP-2×FLAG was purified by α-FLAG M2 immunoaffinity chromatography, followed by elution with FLAG peptide (Sigma) as previously described (47Brizzard B.L. Chubet R.G. Vizard D.L. BioTechniques. 1994; 16: 730-735PubMed Google Scholar). The purity of eluted CBP was greater than 95% as determined by Coomassie staining. E1A was acetylated in vitroin a reaction containing 5 μm purified 12 S E1A, 300 nm CBP-2×FLAG, 10 μm[14C]acetyl coenzyme A ([acetyl-1-14C]; 2.2 GBq/mmol; PerkinElmer Life Sciences), 50 mm Tris-HCl, pH 8.0, 10% glycerol, 10 mm sodium butyrate, 1 mmdithiothreitol, for 3 h at 30 °C. Monoclonal antibody M2 directed against the FLAG epitope coupled to agarose was obtained from Sigma. Mouse monoclonal antibodies directed against E1A, M73, M1, M37, and M29 (48Harlow E. Whyte P. Franza Jr., B.R. Schley C. Mol. Cell. Biol. 1986; 6: 1579-1589Crossref PubMed Scopus (258) Google Scholar), were a generous gift of Dr. E. Harlow. An α-tubulin monoclonal antibody (T-9026) was from Sigma. A synthetic peptide corresponding to the C terminus of Ad2 E1A, designed with an additional N-terminal cysteine (-CEDLLNESGNPLDLSCK 239RPRP243;acetylated at the ε-amino position of lysine 239) was obtained from Sigma, purified by reverse phase high pressure liquid chromatography. Peptide was coupled to maleimide-activated keyhole limpet hemocyanin (Imject; Pierce), mixed with Freund's adjuvant, and injected into mice. Sera from injected mice were screened for immunoreactivity toward recombinant E1A acetylated in vitro by CBP. Mice with high titer immunoreactivity for acetylated E1A were sacrificed to obtain splenocytes for hybridoma fusion by standard procedures (49Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988: 148-237Google Scholar). Clonal hybridoma supernatants were screened by Western blot for selective immunoreactivity toward acetylated E1A. Monolayer cultures of U2OS cells, COS-7 cells, and HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. COS-7 and U2OS cells were transfected with E1A expression vectors using the TransIT reagent (PanVera, Madison, WI). Assay of E1A-mediated repression of CBP-dependent co-activation of the somatostatin promoter was performed as previously described (8Lundblad J.R. Kwok R.P. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (529) Google Scholar). In brief, F9 teratocarcinoma cells were transfected by the calcium phosphate method (Calcium Phosphate Transfection Kit; Invitrogen) using 4 μg of p(−71) SRIF-CAT, 4 μg of pRc/RSV-FLAG-CREB341, 4 μg of pRSV-PKA, and 2 μg of pRSV-Luciferase, with or without 15 μg of pRc/RSV-CBP per 10-cm plate, with 1, 5, or 10 μg of plasmids encoding pRc/RSV-12 S E1A and 12 S E1A mutants as indicated. Results are expressed as the relative CAT activity (mean ± S.E., n = 3) compared with CAT activity in the presence of CREB and protein kinase A alone (CREB alone column). CAT activity values were normalized to luciferase activity as previously described (50Kwok R.P. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1275) Google Scholar). Transfected COS-7 cells and 293 cells were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde for 4 min at room temperature, incubated with ice-cold methanol/acetone (1:1) for 2 min, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline, and blocked with 3% bovine serum albumin in phosphate-buffered saline prior to incubation with E1A-specific antibodies. E1A was detected with either M73 alone (a carboxyl-terminal, E1A-specific monoclonal antibody), a mixture of E1A-specific antibodies (M1, M29, M37, and M73) that recognize multiple E1A epitopes (48Harlow E. Whyte P. Franza Jr., B.R. Schley C. Mol. Cell. Biol. 1986; 6: 1579-1589Crossref PubMed Scopus (258) Google Scholar), or the α-AcK-E1A monoclonal antibody and then detected with an Oregon Green 488-goat anti-mouse IgG conjugate (Molecular Probes, Inc., Eugene, OR). Nuclei were counterstained with TOPRO-3 (Molecular Probes). Coverslips were washed in phosphate-buffered saline and viewed by either conventional fluorescence microscopy or on a Nikon TE 300-inverted microscope with a Bio-Rad MRC 1024 confocal imaging system. Whole cellular extracts were prepared as previously described (8Lundblad J.R. Kwok R.P. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (529) Google Scholar). For preparation of nuclear and cytoplasmic extracts, cells were harvested and washed in phosphate-buffered saline and then resuspended in 10 mm Tris-HCl, 3 mm MgCl2 (TM) with 250 mm sucrose and lysed with a Dounce homogenizer (B pestle). Nuclei were collected by centrifugation at 250 ×g, and the supernatant (cytosol) was centrifuged at 100,000 × g for 30 min (Beckman TLA100.2) to pellet cellular membranes. Nuclei were washed twice and then brought to 0.6m sucrose and layered over a solution of 1.7 msucrose in TM and centrifuged at 28,000 rpm in a SW-28 rotor (Beckman) for 2 h. The purified nuclear pellet was washed twice in TM + 250 mm sucrose. Nuclear extracts were prepared as previously described (51Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar). Cytoplasmic and nuclear extracts were probed with a monoclonal α-tubulin antibody to confirm adequate fractionation. Total protein concentrations were determined by Bradford Reagent (Bio-Rad). Cellular proteins were fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore Corp.), and detected by incubation with antibodies as described for each experiment, by using a goat anti-mouse IgG-horseradish peroxidase conjugate (Bio-Rad) and enhanced luminol reagent (Renaissance; PerkinElmer Life Sciences). GST-CtBP1 and GST-importin-α3 binding assays were performed in 20 mm HEPES, pH 7.4, 200 mm NaCl, 0.1 mm EDTA, 0.1% Nonidet P-40, 10% glycerol, 0.5 mm dithiothreitol, 50 μg/ml nuclease-free bovine serum albumin (New England Biolabs). Each 200-μl binding reaction contained 10 nm GST-importin-α3, 20–30 nm GST-CtBP1, and 10 μl of a 50% slurry of glutathione-Sepharose beads and increasing concentrations of purified E1A protein as indicated. Reactions were incubated for 2 h at 4 °C. Glutathione-Sepharose-bound proteins were washed with binding buffer, and complexes were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride, and subjected to Western blot analysis using either a panel of E1A-specific monoclonal antibodies or the acetyl-E1A-specific monoclonal antibody. Previous studies have demonstrated that the nuclear histone acetyltransferases acetylate not only free and nucleosomal histones but also nonhistone transcriptional regulatory proteins (reviewed in Refs. 52Kouzarides T. EMBO J. 2000; 19: 1176-1179Crossref PubMed Scopus (992) Google Scholar and 53Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64: 435-459Crossref PubMed Scopus (1359) Google Scholar). E1A is also a substrate for the acetylase activity of CBP, p300, and P/CAF in vitro (17Chakravarti D. Ogryzko V. Kao H.Y. Nash A. Chen H. Nakatani Y. Evans R.M. Cell. 1999; 96: 393-403Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 18Perissi V. Dasen J.S. Kurokawa R. Wang Z. Korzus E. Rose D.W. Glass C.K. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3652-3657Crossref PubMed Scopus (84) Google Scholar,34Zhang Q. Yao H., Vo, N. Goodman R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14323-14328Crossref PubMed Scopus (107) Google Scholar). CBP-dependent acetylation of 12 S E1A shows similar substrate concentration saturation kinetics as purified H3 and H4 histones (not shown), consistent with the ability of E1A to competitively inhibit acetylation of purified histones in vitro (16Hamamori Y. Sartorelli V. Ogryzko V. Puri P.L., Wu, H.Y. Wang J.Y. Nakatani Y. Kedes L. Cell. 1999; 96: 405-413Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 17Chakravarti D. Ogryzko V. Kao H.Y. Nash A. Chen H. Nakatani Y. Evans R.M. Cell. 1999; 96: 393-403Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 18Perissi V. Dasen J.S. Kurokawa R. Wang Z. Korzus E. Rose D.W. Glass C.K. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3652-3657Crossref PubMed Scopus (84) Google Scholar). To determine the consequences of acetylation on the function of 12 S E1A, we mapped the site of acetylation by mutagenesis of three potential target lysine residues (Lys162, Lys207, Lys239). Consistent with previous results obtained with p300 and P/CAF (34Zhang Q. Yao H., Vo, N. Goodman R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14323-14328Crossref PubMed Scopus (107) Google Scholar), we find that E1A is predominantly acetylated at a single lysine residue (Lys239) in vitro at the C terminus (Fig.1 B). Recombinant wild type 12 E1A or E1A proteins with single amino acid substitutions at each of three lysine residues were incubated with baculovirus-expressed recombinant CBP in the presence of [14C]acetyl-coenzyme A, and radiolabeled E1A was detected by autoradiography. Wild type E1A, E1A K162A, and E1A K207A are acetylated by CBP in vitro, whereas E1A K239A is not significantly acetylated by CBP (Fig.1 B). Since mutation of Lys239 could impair E1A acetylation by CBP indirectly by disturbing acetylation at another site, we developed a mouse monoclonal antibody specific for E1A acetylated at Lys239 ([ε-acetyl-K239]-E1A) to confirm that Lys239 is a bona fide acetylation site bothin vitro and in vivo. Mice were immunized with a peptide corresponding to the carboxyl-terminal 16 amino acids of Ad2 E1A synthesized with ε-acetyl-lysine at a position corresponding to Lys239 (Fig. 1 C). Clonal hybridoma lines were identified that were specifically reactive to in vitroacetylated full-length 12 S E1A. Fig. 1 D shows the specificity of one of these antibodies (monoclonal antibody 327.5.1) by Western blot analysis for 12 S E1A acetylated in vitro by recombinant CBP. To demonstrate that this site is a target for acetylation of E1A in vivo, we transfected either COS-7 cells (Fig. 1 E) or U2OS cells (Fig. 1 F) with expression vectors for either wild type 12 or 13 S E1A, E1A with deletion mutations within each conserved region (mCR1, mCR2, mCR3) and the N terminus (Δ2–36), or cDNAs with point mutations in the CtBP binding motif (D235A/L236S) or at lysine 239 (K239A and K239Q). Whol" @default.
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• W2069409615 date "2002-10-01" @default.
• W2069409615 modified "2023-10-09" @default.
• W2069409615 title "Acetylation of the Adenovirus-transforming Protein E1A Determines Nuclear Localization by Disrupting Association with Importin-α" @default.
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• W2069409615 doi "https://doi.org/10.1074/jbc.m207512200" @default.
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