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• W2088257639 abstract "Transcription factor function can be modulated by post-translational modifications. Because the transcription factor CCAAT/enhancer-binding protein (C/EBP) β associates with the nuclear coactivator p300, which contains acetyltransferase activity, acetylation of C/EBPβ was examined to understand its regulation and function. C/EBPβ is acetylated by acetyltransferases p300 and p300/CREB-binding protein associated factor. Endogenous C/EBPβ in 3T3-F442A preadipocytes is also recognized by an acetyl-lysine-specific antibody. Analysis of truncations of C/EBPβ and peptides based on C/EBPβ sequences identified multiple lysines within C/EBPβ that can be acetylated. Among these, a novel acetylation site at lysine 39 of C/EBPβ was identified. Mutation of Lys-39 to arginine or alanine impairs its acetylation and the ability of C/EBPβ to activate transcription at the promoters for C/EBPα and c-fos. Different C/EBPβ-responsive promoters require different patterns of acetylated lysines in C/EBPβ for transcription activation. Furthermore, C/EBPβ acetylation was increased by growth hormone, and mutation of Lys-39 impaired growth hormone-stimulated c-fos promoter activation. These data suggest that acetylation of Lys-39 of C/EBPβ, alone or in combination with acetylation at other lysines, may play a role in C/EBPβ-mediated transcriptional activation. Transcription factor function can be modulated by post-translational modifications. Because the transcription factor CCAAT/enhancer-binding protein (C/EBP) β associates with the nuclear coactivator p300, which contains acetyltransferase activity, acetylation of C/EBPβ was examined to understand its regulation and function. C/EBPβ is acetylated by acetyltransferases p300 and p300/CREB-binding protein associated factor. Endogenous C/EBPβ in 3T3-F442A preadipocytes is also recognized by an acetyl-lysine-specific antibody. Analysis of truncations of C/EBPβ and peptides based on C/EBPβ sequences identified multiple lysines within C/EBPβ that can be acetylated. Among these, a novel acetylation site at lysine 39 of C/EBPβ was identified. Mutation of Lys-39 to arginine or alanine impairs its acetylation and the ability of C/EBPβ to activate transcription at the promoters for C/EBPα and c-fos. Different C/EBPβ-responsive promoters require different patterns of acetylated lysines in C/EBPβ for transcription activation. Furthermore, C/EBPβ acetylation was increased by growth hormone, and mutation of Lys-39 impaired growth hormone-stimulated c-fos promoter activation. These data suggest that acetylation of Lys-39 of C/EBPβ, alone or in combination with acetylation at other lysines, may play a role in C/EBPβ-mediated transcriptional activation. Acetylation of nuclear proteins was first detected in histones and is viewed as a part of a mechanism allowing DNA to become accessible to transcription regulatory machinery (1Allfrey V.G. Faulkner R. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Scopus (1728) Google Scholar, 2Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1586) Google Scholar). It is now recognized that many cellular proteins are acetylated. In the nucleus, acetylation of several transcription factors is reported to have broad impact on their function. For example, acetylation of p53 stabilizes it by preventing its ubiquitination by Mdm2, allowing p53 to enter the nucleus to activate target genes (3Li M. Luo J. Brooks C.L. Gu W. J. Biol. Chem. 2002; 277: 50607-50611Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). Acetylation of p53 at lysines 320, 373, and 382 increases its binding to cognate DNA (4Liu L. Scolnick D.M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (650) Google Scholar, 5Gu W.G. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2160) Google Scholar). Acetylation is also reported to increase nuclear localization of NF-κB (6Chen L. Fischle W. Verdin E. Greene W.C. Science. 2001; 293: 1653-1657Crossref PubMed Scopus (1040) Google Scholar), which is essential for its transcription factor function. Acetylation of GATA-1 was found to increase its binding to DNA, thereby stimulating GATA-1-dependent transcription (7Boyes J. Byfield P. Nakatani Y. Ogryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (633) Google Scholar). Other functional consequences of acetylation include promoting interaction of the nuclear import factor importin-α with importin-β (8Bannister A.J. Miska E.A. Gorlich D. Kouzarides T. Curr. Biol. 2000; 10: 467-470Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Ku70 is unable to bind and sequester pro-apoptotic BAX when Ku70 is acetylated (9Cohen H.Y. Lavu S. Bitterman K.J. Hekking B. Imahiyerobo T. Miller C. Frye R. Ploegh H. Kessler B.M. Sinclair D.A. Mol. Cell. 2004; 13: 627-638Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). Acetylation in the DNA-binding domain of HMGI(Y) is inhibitory, decreasing its DNA-binding ability and weakening its transcriptional potency (10Kouzarides T. EMBO J. 2000; 19: 1176-1179Crossref PubMed Scopus (1002) Google Scholar). Thus, acetylation modifies the function of a variety of cellular proteins. CCAAT/enhancer-binding protein (C/EBP) 3The abbreviations used are: C/EBPβ, CCAAT/enhancer-binding protein β; CHO, Chinese hamster ovary cells; GH, growth hormone; GHR, growth hormone receptor; NAM, nicotinamide; TSA, trichostatin A; WT, wild type; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; anti-Ac-K, anti-acetyl-lysine; GST, glutathione S-transferase. 3The abbreviations used are: C/EBPβ, CCAAT/enhancer-binding protein β; CHO, Chinese hamster ovary cells; GH, growth hormone; GHR, growth hormone receptor; NAM, nicotinamide; TSA, trichostatin A; WT, wild type; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; anti-Ac-K, anti-acetyl-lysine; GST, glutathione S-transferase. β is a basic ZIP transcription factor that is expressed in adipose, hepatic, and immune tissues and a variety of other tissues (11Descombes P. Chojkier M. Lichtsteiner S. Falvey E. Schibler U. Genes Dev. 1990; 4: 1541-1551Crossref PubMed Scopus (419) Google Scholar, 12Poli V. Mancini F.P. Cortese R. Cell. 1990; 63: 643-653Abstract Full Text PDF PubMed Scopus (457) Google Scholar, 13Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1206) Google Scholar, 14Chang C.J. Chen T.T. Lei H.Y. Chen D.S. Lee S.C. Mol. Cell. Biol. 1990; 10: 6642-6653Crossref PubMed Scopus (200) Google Scholar, 15Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1338) Google Scholar, 16Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar). C/EBPβ is present in cells in three forms as follows: LAP1 (full-length liver-enriched activating protein 1, residues 1–296) (17Eaton E.M. Hanlon M. Bundy L. Sealy L. J. Cell. Physiol. 2001; 189: 91-105Crossref PubMed Scopus (62) Google Scholar), LAP2 (residues 22–296), and the inhibitory form LIP (residues 151–296). C/EBPβ plays an important role in the gluconeogenic pathway (18Croniger C. Trus M. Lysek-Stupp K. Cohen H. Liu Y. Darlington G.J. Poli V. Hanson R.W. Reshef L. J. Biol. Chem. 1997; 272: 26306-26312Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), liver regeneration (19Diehl A.M. J. Biol. Chem. 1998; 273: 30843-30846Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), and the hematopoietic system (20Scott L.M. Civin C.I. Rorth P. Friedman A.D. Blood. 1992; 80: 1725-1735Crossref PubMed Google Scholar). Among the many genes responsive to C/EBPβ, the proto-oncogene c-fos is one prominent example (21Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 22Piwien-Pilipuk G. Huo J.S. Schwartz J. J. Pediatr. Endocrinol. Metab. 2002; 15: 771-786Crossref PubMed Scopus (78) Google Scholar). Another example, C/EBPβ, an early mediator of the differentiation of adipocytes (15Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1338) Google Scholar, 23Lin F.-T. Lane M.D. Genes Dev. 1992; 6: 533-544Crossref PubMed Scopus (274) Google Scholar, 24Tanaka T. Yoshida N. Kishimoto T. Akira S. EMBO J. 1997; 16: 7432-7443Crossref PubMed Scopus (637) Google Scholar, 25Darlington G.J. Ross S.E. MacDougald O.A. J. Biol. Chem. 1998; 273: 30057-30060Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar), is well known to activate expression of genes for C/EBPα and peroxisome proliferator-activated receptor-γ, which in turn mediate adipogenic differentiation (25Darlington G.J. Ross S.E. MacDougald O.A. J. Biol. Chem. 1998; 273: 30057-30060Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 26Tang Q.Q. Zhang J.W. Lane M.D. Biochem. Biophys. Res. Commun. 2004; 318: 213-218Crossref PubMed Scopus (63) Google Scholar, 27Clarke S.L. Robinson C.E. Gimble J.M. Biochem. Biophys. Res. Commun. 1997; 240: 99-103Crossref PubMed Scopus (188) Google Scholar, 28Zhu Y. Qi C. Korenberg J.R. Chen X.N. Noya D. Rao M.S. Reddy J.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7921-7925Crossref PubMed Scopus (598) Google Scholar). The function of C/EBPβ is modulated by its phosphorylation at several sites. For example, phosphorylation of human C/EBPβ at a mitogen-activated protein kinase (MAPK) substrate site at Thr-235 (which corresponds to Thr-188 in mouse C/EBPβ) (29Nakajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Crossref PubMed Scopus (514) Google Scholar) alters its ability to activate transcription of a variety of downstream target genes, including c-fos (21Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 30Piwien-Pilipuk G. MacDougald O.A. Schwartz J. J. Biol. Chem. 2002; 277: 44557-44565Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The phosphorylation of C/EBPβ at Thr-188 is rapidly increased in an extracellular signal-regulated kinase (ERK)-dependent manner by factors such as growth hormone (GH) (30Piwien-Pilipuk G. MacDougald O.A. Schwartz J. J. Biol. Chem. 2002; 277: 44557-44565Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), interleukin-1 β (31Raymond L. Eck S. Mollmark J. Hays E. Tomek I. Kantor S. Elliott S. Vincenti M. J. Cell. Physiol. 2006; 207: 683-688Crossref PubMed Scopus (48) Google Scholar), and interferon-γ (32Ghosh A.K. Bhattacharyya S. Mori Y. Varga J. J. Cell. Physiol. 2006; 207: 251-260Crossref PubMed Scopus (30) Google Scholar). Phosphorylation of C/EBPβ at Thr-188 is also increased during adipogenesis in preadipocytes and NIH-3T3 cells (33Park B.H. Qiang L. Farmer S.R. Mol. Cell. Biol. 2004; 24: 8671-8680Crossref PubMed Scopus (164) Google Scholar, 34Tang Q.Q. Jiang M.S. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13571-13575Crossref PubMed Scopus (36) Google Scholar, 35Tang Q.Q. Gronborg M. Huang H. Kim J.W. Otto T.C. Pandey A. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9766-9771Crossref PubMed Scopus (261) Google Scholar). Murine C/EBPβ has been observed to relocalize to heterochromatin within the nucleus in response to GH in a manner dependent on its phosphorylation at Thr-188 (36Piwien-Pilipuk G. Galigniana M.D. Schwartz J. J. Biol. Chem. 2003; 278: 35668-35677Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Phosphorylation of rat C/EBPβ at Ser-105 or of mouse C/EBPβ at Thr-217 by p90 ribosomal S6 kinase stimulates proliferation in differentiated hepatocytes induced by transforming growth factor-α (37Buck M. Poli V. van der Geer P. Chojkier M. Hunter T. Mol. Cell. 1999; 4: 1087-1092Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Protein kinase A- or protein kinase C-mediated phosphorylation at Ser-240 of rat C/EBPβ is reported to attenuate DNA binding (38Trautwein C. van der Geer P. Karin M. Hunter T. Chojkier M. J. Clin. Investig. 1994; 93: 2554-2561Crossref PubMed Scopus (128) Google Scholar). Furthermore, GH induces a delayed de-phosphorylation at a GSK-3 site at Ser-184 of mouse C/EBPβ, which may interfere with its binding to the c-fos promoter (39Piwien-Pilipuk G. Van Mater D. Ross S.E. MacDougald O.A. Schwartz J. J. Biol. Chem. 2001; 276: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Phosphorylation of C/EBPβ at Ser-184 also contributes to adipogenesis and activation of adipocyte genes such as C/ebpα and aP2 (35Tang Q.Q. Gronborg M. Huang H. Kim J.W. Otto T.C. Pandey A. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9766-9771Crossref PubMed Scopus (261) Google Scholar). C/EBPβ has been shown to interact with p300 (40Mink S. Haenig B. Klempnauer K.H. Mol. Cell. Biol. 1997; 17: 6609-6617Crossref PubMed Google Scholar), a nuclear coactivator with intrinsic acetyltransferase activity (41Ogryzko V.V. Schlitz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2377) Google Scholar). C/EBPβ also associates with cAMP response-element-binding protein-binding protein, a coactivator and acetyltransferase homologous to p300 (42Kovacs K.A. Steinmann M. Magistretti P.J. Halfon O. Cardinaux J.R. J. Biol. Chem. 2003; 278: 36959-36965Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 43Duong D.T. Waltner-Law M.E. Sears R. Sealy L. Granner D.K. J. Biol. Chem. 2002; 277: 32234-32242Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Because of the association between C/EBPβ and these acetyltransferases, this study investigates the acetylation of C/EBPβ and its functional consequences. This report indicates that C/EBPβ is acetylated, in agreement with several recent reports (44Joo M. Park G.Y. Wright J.G. Blackwell T.S. Atchison M.L. Christman J.W. J. Biol. Chem. 2004; 279: 6658-6665Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 45Xu M. Nie L. Kim S.H. Sun X.H. EMBO J. 2003; 22: 893-904Crossref PubMed Scopus (130) Google Scholar). Multiple acetylation sites are identified, including a novel acetylation site on C/EBPβ at Lys-39 (numbering is based on the murine sequence of C/EBPβ unless indicated otherwise). Importantly, mutation of Lys-39 decreases the ability of C/EBPβ to mediate transcriptional activation of target gene promoters. Using an antiacetyl-lysine antibody that recognizes acetylated Lys-39, endogenous C/EBPβ was found to be acetylated in preadipocytes, in which C/EBPβ is an important factor during adipogenesis. C/EBPβ is a critical mediator of GH-regulated transcription of c-fos (46Clarkson R.W.E. Chen C.M. Harrison S. Wells C. Muscat G.E.O. Waters M.J. Mol. Endocrinol. 1995; 9: 108-120Crossref PubMed Scopus (83) Google Scholar, 47Cui T.X. Piwien-Pilipuk G. Huo J.S. Kaplani J. Kwok R. Schwartz J. Mol. Endocrinol. 2005; 19: 2175-2186Crossref PubMed Scopus (37) Google Scholar). A nonacetylatable mutation at Lys-39 of C/EBPβ impaired GH-stimulated c-fos promoter activation, and GH was found to increase acetylation of C/EBPβ. Taken together, this study identifies acetylation at Lys-39 as novel modification of C/EBPβ that is regulated and contributes, alone and in combination with other acetylatable lysines, to C/EBPβ-mediated transcription. Plasmids and Antibodies—The numbering used to designate residues in C/EBPβ is based on the mouse C/EBPβ sequence (GenBank™ accession number NM009883). The following C/EBPβ plasmids were used. The plasmid encoding full-length C/EBPβ (LAP1) driven by the cytomegalovirus promoter (CMV-C/EBPβ) was a gift from Dr. U. Schibler (University of Geneva) and Dr. L. Sealy (Vanderbilt University). The plasmid HA-C/EBPβ encodes C/EBPβ (residues 22–296, also known as LAP2) tagged with HA at the N terminus (42Kovacs K.A. Steinmann M. Magistretti P.J. Halfon O. Cardinaux J.R. J. Biol. Chem. 2003; 278: 36959-36965Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). HA-C/EBPβ is able to activate the c-fos promoter at least as well as full-length CMV-C/EBPβ (data not shown). LAP2 is a prominent active form of C/EBPβ in GH-responsive 3T3-F442A cells (21Liao J. Piwien-Pilipuk G. Ross S.E. Hodge C.L. Sealy L. MacDougald O.A. Schwartz J. J. Biol. Chem. 1999; 274: 31597-31604Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Hereafter, C/EBPβ will be designated as LAP2 unless otherwise indicated. Mutations were introduced into HA-C/EBPβ at indicated residues using Stratagene QuikChange XL site-directed mutagenesis kit. All mutations were confirmed by sequencing. The mutated forms of C/EBPβ are referred to as follows: K39R, K39A, K39Q, K117R, and K215R/K216R; TM refers to combined mutations K39R/K117R/K215R/K216R. As additional controls for mutation of Lys-39 in the transcriptional activation domain of C/EBPβ, arginine 42 was mutated to alanine (R42A) and lysine 98 was mutated to arginine (K98R). HA-C/EBPβ mutated to alanine at phosphorylation sites Thr-188 (T188A) or Ser-184 (S184A) was similarly generated. GST-C/EBPβ plasmids encode fusion proteins of GST with truncated forms of C/EBPβ (residues 22–227, 22–193, and 22–103) as described previously (42Kovacs K.A. Steinmann M. Magistretti P.J. Halfon O. Cardinaux J.R. J. Biol. Chem. 2003; 278: 36959-36965Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). HIS-C/EBPβ encodes C/EBPβ tagged with six histidine residues at the N terminus (48Kovacs K.A. Steinmann M. Magistretti P.J. Halfon O. Cardinaux J.R. J. Neurochem. 2006; 98: 1390-1399Crossref PubMed Scopus (23) Google Scholar). Recombinant HIS-C/EBPβ was expressed and purified on a nickel-nitrilotriacetic acid-agarose column (Qiagen). Plasmids for full-length, N-terminally FLAG-tagged p300 (p300) and N-terminally FLAG-tagged P/CAF (P/CAF) were prepared, expressed, and purified as described previously (49Kashanchi F. Duvall J.F. Kwok R.P. Lundblad J.R. Goodman R.H. Brady J.N. J. Biol. Chem. 1998; 273: 34646-34652Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 50Lu Q. Hutchins A.E. Doyle C.M. Lundblad J.R. Kwok R.P. J. Biol. Chem. 2003; 278: 15727-15734Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The plasmid 5XC/EBP-luc encodes a luciferase reporter gene driven by five copies of a consensus C/EBP site (42Kovacs K.A. Steinmann M. Magistretti P.J. Halfon O. Cardinaux J.R. J. Biol. Chem. 2003; 278: 36959-36965Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The plasmid C/EBPα-luc was a gift from Dr. O. MacDougald (University of Michigan) (34Tang Q.Q. Jiang M.S. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13571-13575Crossref PubMed Scopus (36) Google Scholar). The plasmid for c-fos/luciferase (c-fos-luc), which contains the mouse c-fos promoter (–379 to +1) upstream of luciferase, was a gift from Dr. W. Wharton (University of South Florida) and Dr. B. Cochran (Tufts University) (51Harvat B.L. Wharton W. Cell Growth & Differ. 1995; 6: 955-964PubMed Google Scholar). A plasmid encoding rat growth hormone receptor (GHR) was provided by Dr. C. Carter-Su (University of Michigan) (52Wang X. Moller C. Norstedt G. Carter-Su C. J. Biol. Chem. 1993; 268: 3573-3579Abstract Full Text PDF PubMed Google Scholar). RSV-β-galactosidase was provided by Dr. M. Uhler (University of Michigan). pcDNA3.1 vector, used to normalize the total amount of DNA in transfections, was purchased from Clontech. The following antibodies were used: anti-HA (Covance) and anti-C/EBPβ (specific for the C terminus of C/EBPβ; Santa Cruz Biotechnology) were used at dilutions of 1:100 for immunoprecipitations and 1:1000 for immunoblotting. Anti-acetyl-lysine (anti-Ac-K (Upstate); monoclonal antibody 4G12 that detects acetylated lysines on histones and p53) was used at a dilution of 1:500 for immunoblotting. An antibody against a peptide corresponding to human C/EBPβ phosphorylated at Thr-235 (homologous to Thr-188 of mouse C/EBPβ) (anti-pC/EBPβ, Cell Signaling) was used at a dilution of 1:1000 for immunoblotting. Cell Culture—293T cells were provided by Dr. M. Lazar (University of Pennsylvania). Murine 3T3-F442A preadipocyte fibroblasts were provided by Dr. H. Green (Harvard University) and Dr. M. Sonenberg (Sloan-Kettering). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 8% calf serum (Invitrogen) in an environment of 10% CO2, 90% air at 37 °C. Prior to use in experiments, cells were incubated overnight in serum-free DMEM containing 1% bovine serum albumin (BSA, CRG7; Serological Corp), which was also supplemented with deacetylase inhibitors trichostatin A (TSA, 1 μm, Sigma) and nicotinamide (NAM, 5 mm, Sigma). Chinese hamster ovary cells expressing rat GHR containing the N-terminal half of the cytoplasmic domain (referred to as CHO-GHR cells) were provided by Dr. G. Norstedt (Karolinska Institute, Stockholm, Sweden) and Dr. N. Billestrup (Steno Diabetes Center, Copenhagen, Denmark) (53Moller C. Hansson A. Enberg B. Lobie P.E. Norstedt G. J. Biol. Chem. 1992; 267: 23403-23408Abstract Full Text PDF PubMed Google Scholar). They were maintained in Ham's F-12 medium (Invitrogen) containing 8% fetal bovine serum and 0.5 mg/ml Geneticin (Invitrogen) in 5% CO2, 95% air at 37 °C. Prior to experiments, CHO-GHR cells were incubated overnight in medium containing 1% BSA instead of serum. All media were supplemented with 1 mm l-glutamine, 100units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin. Calcium phosphate transfections were performed as described previously (54Chen D. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4816) Google Scholar), except 50 mm HEPES-buffered saline was used instead of BES-buffered saline. Immunoprecipitation and Immunoblotting—293T cells were lysed using Lysis Buffer (420 mm NaCl, 20 mm HEPES, pH 7.9, 1 mm EDTA, 1 mm EGTA, 20% glycerol), supplemented with 150 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin and leupeptin, as well as 1 μm TSA and 5 mm NAM. For 293T cells expressing HA-C/EBPβ, lysates were precleared using protein G-Sepharose beads (G beads; Amersham Biosciences). Samples were immunoprecipitated using anti-HA antibody for 2 h at 4 °C, and immunoprecipitates were collected on beads for 1 h. To test endogenous proteins, 3T3-F442A cells were lysed in SDS lysis buffer (50 mm Tris-HCl, 1% SDS, 10 mm EDTA, 1 mm EGTA, 0.2% Triton X-100). Lysates were precleared using protein A-agarose beads (A beads; RepliGen). Samples were immunoprecipitated using anti-C/EBPβ antibody for 3 h at 4°C, and immunoprecipitates were collected on beads for 1 h. Beads were washed three times in Acetylase Buffer (10 mm Tris, pH 7.6, 150 mm NaCl, 1 mm EDTA, and 5% glycerol, 22 mg/ml sodium butyrate (Sigma), 3 mg/ml dithiothreitol (Invitrogen)). SDS protein dye (50 mm Tris, 1% SDS, 0.001% bromphenol blue, 10% glycerol, 10% β-mercaptoethanol) was then added to the beads, and samples were boiled, separated by SDS-PAGE, and immunoblotted as described previously (55Hodge C. Liao J. Stofega M. Guan K. Carter-Su C. Schwartz J. J. Biol. Chem. 1998; 273: 31327-31336Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Bands on immunoblots were visualized using IRDye 700-coupled anti-mouse IgG (1:10,000) or IRDye 800-coupled anti-rabbit IgG (1:10,000) on the Odyssey infrared scanning system (LI-COR, Inc., Lincoln, NE) as described previously (47Cui T.X. Piwien-Pilipuk G. Huo J.S. Kaplani J. Kwok R. Schwartz J. Mol. Endocrinol. 2005; 19: 2175-2186Crossref PubMed Scopus (37) Google Scholar). Molecular weight was estimated using Kaleidoscope protein molecular weight standard (Bio-Rad). In Vitro Acetylation of C/EBPβ—Acetylation assays were performed using Acetylase Buffer. To test the acetylation of C/EBPβ in vitro, purified HIS-C/EBPβ (3 μg) or the purified GST-C/EBPβ fusion proteins (3 μg each) were incubated in Acetylase Buffer alone or with added purified p300 or P/CAF (1 μg each), and 1 μl of [14C]acetyl-CoA (55 m Ci/mmol; ICN). Samples were incubated for 1 h at 30°C, separated by SDS-PAGE (8%), and analyzed by autoradiography (Kodak X-Omat Blue XB-1). To examine acetylation of expressed C/EBPβ, CMV-C/EBPβ (LAP1) was expressed in 293T cells to obtain high protein expression. C/EBPβ was immunoprecipitated using anti-C/EBPβ and used in the acetylase assay described above. Protein levels were assessed by staining the gel with Coomassie Brilliant Blue G-250 (Bio-Rad). For expressed CMV-C/EBPβ, a duplicate immunoblot was probed with anti-C/EBPβ to evaluate migration and protein loading. C/EBPβ Peptide Acetylation—The following peptides containing candidate lysines in C/EBPβ were synthesized at the University of Michigan Protein Structure Facility: DCLAYGAKAARAAPR (amino acids 32–46), FADDYGAKPSKKPADYGYV (amino acids 91–109), SLGRAGAKAAPPACF (amino acids 110–124), PPPPALLKAEPGFE (amino acids 126–139), GFEPADCKRADDAPA (amino acids 137–151), PSPADAKAAPAACF (amino acids 189–202), and PAAPAKAKAKKTVDKLSD (amino acids 206–223). A peptide based on histone H3 (amino acids 7–22) that is acetylated by p300 and P/CAF (50Lu Q. Hutchins A.E. Doyle C.M. Lundblad J.R. Kwok R.P. J. Biol. Chem. 2003; 278: 15727-15734Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) served as a positive control, ARKSTGGKAPRKQLAT. Each peptide (2 μg) was incubated in Acetylase Buffer with 1 μl of [3H]acetyl-CoA (5.8 Ci/mmol; ICN), without or with purified p300 or P/CAF (1 μg each), and incubated for 1 h at 30°C. Samples were then spotted on grade P81 cellulose paper (Whatman). Paper was rinsed with 0.1% phosphoric acid (Sigma), and acetylation, expressed as relative counts/min, was measured in CytoScint scintillation mixture (ICN Biomedicals) using a liquid scintillation counter (Packard Instrument Co.). In Vivo Acetylation of C/EBPβ—CMV-C/EBPβ (LAP1) was expressed in 293T cells, and 48 h later cells were incubated with 200 μl of [3H]sodium acetate (2.90 Ci/mmol; ICN) for 1 h. C/EBPβ was immunoprecipitated with antibodies against C/EBPβ or rabbit IgG (Sigma), which served as a control. Samples were separated by SDS-PAGE (8%) and analyzed by autoradiography. To assess the acetylation of WT HA-C/EBPβ or of HA-C/EBPβ mutated at various lysines, appropriate plasmids were coexpressed with or without plasmids for p300 (2.2 μg) or P/CAF (0.2 μg) in 293T cells. pcDNA3 was used to control for total amount of DNA transfected. 24 h later, cells were incubated overnight with TSA (1 μm) and NAM (5 mm) in serum-free DMEM containing 1% BSA. Cell lysates were subjected to immunoprecipitation with anti-HA; samples were separated by SDS-PAGE (4–20%), transferred to polyvinylidene difluoride membrane, and probed with either anti-Ac-K or anti-HA. Phosphorylation of WT or mutated HA-C/EBPβ was similarly analyzed, except immunoblots were probed with anti-pC/EBPβ instead of anti-Ac-K. To examine the regulation of acetylation of C/EBPβ, 293T cells were transfected with plasmids for WT HA-C/EBPβ and GHR, and then 24 h later, they were incubated overnight with TSA (1 μm) and NAM (5 mm) in serum-free DMEM containing 1% BSA. 48 h after transfection, cells were treated with 250 ng/ml (11.5 nm) human GH (recombinant GH kindly provided by Lilly) for 15 min prior to lysis. Cells were lysed in Lysis Buffer; samples were immunoprecipitated with anti-HA as described and analyzed by immunoblotting with anti-Ac-K and anti-C/EBPβ. Membranes were scanned using the Odyssey infrared scanning system (47Cui T.X. Piwien-Pilipuk G. Huo J.S. Kaplani J. Kwok R. Schwartz J. Mol. Endocrinol. 2005; 19: 2175-2186Crossref PubMed Scopus (37) Google Scholar), and bands were quantified using Odyssey software. Acetylation of C/EBPβ was calculated using values for acetylation (anti-Ac-K) divided by total C/EBPβ (anti-C/EBPβ or anti-HA). Statistical analysis of results from three or more experiments was performed using Student's t test (Excel) or 1-way analysis of variance and Bonferroni's multiple comparison test (Prism version 3). Transcription Assays—WT HA-C/EBPβ or HA-C/EBPβ mutated at various residues alone or in combination (each 400 ng/35-mm well) were coexpressed with reporter plasmids 5XC/EBP-luc, C/EBPα-luc, or c-fos-luc each (400 ng/well) in CHO-GHR cells, a reliable system to assess reporter gene activation with or without GH. β-Galactosidase (300 ng/well) was coexpressed to normalize for transfection efficiency. 24 h after transfection, cells were deprived of serum and lysed for luciferase assay 24 h after that, as described previously (56Kwok 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 (1280) Google Scholar). In some experiments, cells were treated with vehicle or GH (500 ng/ml, 23 nm) for 4 h before lysates were prepared. Transcriptional activation was determined by luciferase output as measured using an Opticomp luminometer and is expressed as relative luciferase units/β-galactosidase. Each condition was analyzed in triplicate for each experiment. Statistical analysis of results from replicate, independent experiments was performed using 1-way analysis of variance and Bonferroni's multiple comparison test (Prism version 3). C/EBPβ Is Acetylated in" @default.
• W2088257639 created "2016-06-24" @default.
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• W2088257639 date "2007-01-01" @default.
• W2088257639 modified "2023-09-28" @default.
• W2088257639 title "CCAAT/Enhancer-binding Protein (C/EBP) β Is Acetylated at Multiple Lysines" @default.
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• W2088257639 doi "https://doi.org/10.1074/jbc.m511451200" @default.
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