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ERK2-mediated C-terminal Serine Phosphorylation of p300 Is Vital to the Regulation of Epidermal Growth Factor-induced Keratin 16 Gene Expression

2007; Elsevier BV; Volume: 282; Issue: 37 Linguagem: Inglês

10.1074/jbc.m700264200

1083-351X

Yun‐Ju Chen, Ying‐Nai Wang, Wen‐Chang Chang,

Silk-based biomaterials and applications

We previously reported that the epidermal growth factor (EGF) regulates the gene expression of keratin 16 by activating the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling which in turn enhances the recruitment of p300 to the keratin 16 promoter. The recruited p300 functionally cooperates with Sp1 and c-Jun to regulate the gene expression of keratin 16. This study investigated in detail the molecular events incurred upon p300 whereby EGF caused an enhanced interaction between p300 and Sp1. EGF apparently induced time- and dose-dependent phosphorylation of p300, both in vitro and in vivo, through the activation of ERK2. The six potential ERK2 phosphorylation sites, including three threonine and three serine residues as revealed by sequential analysis, were first identified in vitro. Confirmation of these six sites in vivo indicated that these three serine residues (Ser-2279, Ser-2315, and Ser-2366) on the C terminus of p300 were the major signaling targets of EGF. Furthermore, the C-terminal serine phosphorylation of p300 stimulated its histone acetyltransferase activity and enhanced its interaction with Sp1. These serine phosphorylation sites on p300 controlled the p300 recruitment to the keratin 16 promoter. When all three serine residues on p300 were replaced by alanine, EGF could no longer induce the gene expression of keratin 16. Taken together, these results strongly suggested that the ERK2-mediated C-terminal serine phosphorylation of p300 was a key event in the regulation of EGF-induced keratin 16 expression. These results also constituted the first report identifying the unique p300 phosphorylation sites induced by ERK2 in vivo. We previously reported that the epidermal growth factor (EGF) regulates the gene expression of keratin 16 by activating the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling which in turn enhances the recruitment of p300 to the keratin 16 promoter. The recruited p300 functionally cooperates with Sp1 and c-Jun to regulate the gene expression of keratin 16. This study investigated in detail the molecular events incurred upon p300 whereby EGF caused an enhanced interaction between p300 and Sp1. EGF apparently induced time- and dose-dependent phosphorylation of p300, both in vitro and in vivo, through the activation of ERK2. The six potential ERK2 phosphorylation sites, including three threonine and three serine residues as revealed by sequential analysis, were first identified in vitro. Confirmation of these six sites in vivo indicated that these three serine residues (Ser-2279, Ser-2315, and Ser-2366) on the C terminus of p300 were the major signaling targets of EGF. Furthermore, the C-terminal serine phosphorylation of p300 stimulated its histone acetyltransferase activity and enhanced its interaction with Sp1. These serine phosphorylation sites on p300 controlled the p300 recruitment to the keratin 16 promoter. When all three serine residues on p300 were replaced by alanine, EGF could no longer induce the gene expression of keratin 16. Taken together, these results strongly suggested that the ERK2-mediated C-terminal serine phosphorylation of p300 was a key event in the regulation of EGF-induced keratin 16 expression. These results also constituted the first report identifying the unique p300 phosphorylation sites induced by ERK2 in vivo. Keratins, which are the most prominent cytoskeletal proteins in keratinocytes, belong to a large family of ∼30 epithelially specific intermediate filament proteins that form the cytoskeleton (1Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1284) Google Scholar). Among these, keratin 16 is usually referred to as an activation- and hyperproliferation-associated keratin because it is reported to be the marker in the hyperproliferated skin diseases (2Komine M. Rao L.S. Kaneko T. Tomic-Canic M. Tamaki K. Freedberg I.M. Blumenberg M. J. Biol. Chem. 2000; 275: 32077-32088Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 3Leigh I.M. Navsaria H. Purkis P.E. Mckay I.A. Bowden P.E. Riddle P.N. Br. J. Dermatol. 1995; 133: 501-511Crossref PubMed Scopus (259) Google Scholar). Furthermore, whereas 10% of the invasive breast carcinomas are positive, in either a diffuse or focal pattern, with the keratin 16 antibodies, normal breast tissue and noninvasive breast carcinomas are nearly negative with the keratin 16 antibodies (4Wetzels R.H. Kuijpers H.J. Lane E.B. Leigh I.M. Troyanovsky S.M. Holland R. van Haelst U.J. Ramaekers F.C. Am. J. Pathol. 1991; 138: 751-763PubMed Google Scholar). A recent study indicates that keratin 16 is induced in human papilloma virus-infected tissues at the transcriptional level, and more importantly, the induction leads to the proliferation of keratinocytes, a known characteristic of human papilloma virus infection (5McClowry T.L. Shors T. Brown D.R. J. Med. Virol. 2002; 66: 96-101Crossref PubMed Scopus (6) Google Scholar). Therefore, a full understanding of the gene regulation of keratin 16 might contribute to therapies beneficial to those hyperproliferated diseases. It is reported that p44/p42 mitogen-activated protein kinases (MAPKs), 2The abbreviations used are:MAPKmitogen-activated protein kinaseEGFepidermal growth factorWTwild typeERKextracellular signal-regulated kinasePKCprotein kinase CCBPcAMP-response element-binding protein-binding proteinaaamino acidPBSphosphate-buffered salineHAThistone acetyltransferaseHAhemagglutininsiRNAshort interfering RNAGSTglutathione S-transferaseDAPADNA affinity precipitation assay also known as extracellular signal-regulated kinase 1 and 2 (ERK1/2), phosphorylate specific substrates, including transcription factors like Elk-1 (6Janknecht R. Ernst W.H. Pingoud V. Nordheim A. EMBO J. 1993; 12: 5097-5104Crossref PubMed Scopus (508) Google Scholar, 7Gille H. Kortenjann M. Thomae O. Moomaw C. Slaughter C. Cobb M.H. Shaw P.E. EMBO J. 1995; 14: 951-962Crossref PubMed Scopus (590) Google Scholar) and coactivators such as p300/CBP (8Liu Y.Z. Thomas N.S. Latchman D.S. Neuroreport. 1999; 10: 1239-1243Crossref PubMed Scopus (48) Google Scholar, 9Sang N. Stiehl D.P. Bohensky J. Leshchinsky I. Srinivas V. Caro J. J. Biol. Chem. 2003; 278: 14013-14019Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Once activated, MAPKs directly phosphorylate proteins containing the minimal consensus phosphoacceptor motif Ser/Thr-Pro (10Yang S.H. Sharrocks A.D. Whitmarsh A.J. Gene (Amst.). 2003; 320: 3-21Crossref PubMed Scopus (424) Google Scholar). In addition to the MAPK phosphoacceptor consensus sites, it is becoming increasingly clear that in many cases, additional determinants, known as docking domains, are required to specify for the MAPK substrates (11Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (443) Google Scholar). MAPKs bind directly to the docking domain on the substrate or bind indirectly to the substrate through an adaptor possessing the docking domain (10Yang S.H. Sharrocks A.D. Whitmarsh A.J. Gene (Amst.). 2003; 320: 3-21Crossref PubMed Scopus (424) Google Scholar). mitogen-activated protein kinase epidermal growth factor wild type extracellular signal-regulated kinase protein kinase C cAMP-response element-binding protein-binding protein amino acid phosphate-buffered saline histone acetyltransferase hemagglutinin short interfering RNA glutathione S-transferase DNA affinity precipitation assay p300, as well as its related protein CBP, are well known transcriptional coactivators. Both play pivotal roles in coordinating and integrating multiple signal-dependent events with the transcription machinery and participate in diverse physiological processes, including development, proliferation, differentiation, and apoptosis (12Chan H.M. La Thangue N.B. J. Cell Sci. 2001; 114: 2363-2373Crossref PubMed Google Scholar, 13Janknecht R. Hunter T. Curr. Biol. 1996; 6: 951-954Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Several functional domains of p300/CBP, such as CH1 and CH3 domains, are important for protein-protein interactions (12Chan H.M. La Thangue N.B. J. Cell Sci. 2001; 114: 2363-2373Crossref PubMed Google Scholar). To date, the versatile coactivators p300/CBP enhance gene transcription mainly via three mechanisms (12Chan H.M. La Thangue N.B. J. Cell Sci. 2001; 114: 2363-2373Crossref PubMed Google Scholar). First, they create a bridge between the transcription factors and basal transcription machinery. Second, they act as a scaffold for the assembly of multiprotein complexes, including transcription factors and cofactors through their CH1, CH3, and KIX domains, etc. The simultaneous interaction between p300/CBP and multiple transcription factors is thought to contribute to the transcriptional synergy. For example, p300 cooperates with Sp1 to regulate p21waf1/cip1 promoter activation (14Xiao H. Hasegawa T. Isobe K. J. Biol. Chem. 2000; 275: 1371-1376Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). In addition, it is reported that the acetyl-transferase region of p300 interacts with the DNA-binding domain of Sp1 to regulate the DNA binding activity of Sp1 (15Suzuki T. Kimura A. Nagai R. Horikoshi M. Genes Cells. 2000; 5: 29-41Crossref PubMed Scopus (143) Google Scholar). Third, p300/CBP possesses intrinsic histone acetyltransferase (HAT) activity, which acetylates transcription factors and histones and thus activates transcription (16Ogryzko V.V. Schiltz R.L. Russsanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar). Recently, many studies have reported that p300 and CBP are direct targets of cellular signaling, resulting in their post-translational modifications (17Legube G. Trouche D. EMBO Rep. 2003; 4: 944-947Crossref PubMed Scopus (207) Google Scholar). Of these modifications, phosphorylation is extensively studied especially in vitro. For example, activated ERK2 phosphorylates GST-p300 (aa 1572–2370) in vitro, and as a result, the transcriptional activity of p300 is enhanced (9Sang N. Stiehl D.P. Bohensky J. Leshchinsky I. Srinivas V. Caro J. J. Biol. Chem. 2003; 278: 14013-14019Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). The C-terminal phosphorylation of CBP by ERK1 stimulates its HAT activity in vitro (18Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (173) Google Scholar, 19Ait-Si-Ali S. Carlisi D. Ramirez S. Upegui-Gonzalez L.C. Duquet A. Robin P. Rudkin B. Harel-Bellan A. Trouche D. Biochem. Biophys. Res. Commun. 1999; 262: 157-162Crossref PubMed Scopus (120) Google Scholar). Nonetheless, it is still not entirely clear how phosphorylation may regulate the p300 or CBP activity in vivo. Basically, it is not known which kinases are responsible for the p300/CBP phosphorylation in vivo, nor is it known exactly where the phosphorylation sites may take place. Most important of all, the functional links between the specific phosphorylation events and the downstream gene regulation remain largely unknown. Up to this point, very few phosphorylation sites on p300/CBP have been identified in vivo. These identified phosphorylation sites from in vivo studies are described as follows. The PKC or AMP-activated protein kinase-mediated Ser-89 phosphorylation of p300 attenuates its HAT activity or its interaction with individual nuclear receptors (20Yuan L.W. Gambee J.E. J. Biol. Chem. 2000; 275: 40946-40951Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 21Yuan L.W. Soh J.W. Weinstein I.B. Biochim. Biophys. Acta. 2002; 1592: 205-211Crossref PubMed Scopus (63) Google Scholar, 22Yang W. Hong Y.H. Shen X.Q. Frankowski C. Camp H.S. Leff T. J. Biol. Chem. 2001; 276: 38341-38344Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The growth factor-induced Ser-436 phosphorylation of CBP through phosphatidylinositol 3-kinase-PKC pathway controls the recruitment of CBP to the transcription complex (23Zanger K. Radovick S. Wondisford F.E. Mol. Cell. 2001; 7: 551-558Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The Ser-301 phosphorylation of CBP by calmodulin kinase IV mediates the CBP-dependent transcriptional activation (24Impey S. Fong A.L. Wang Y. Cardinaux J.R. Fass D.M. Obrietan K. Wayman G.A. Storm D.R. Soderling T.R. Goodman R.H. Neuron. 2002; 34: 235-244Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Moreover, the Akt-mediated Ser-1834 phosphorylation of p300 increases the p300 recruitment to the ICAM-1 promoter and enhances its associated HAT activity, resulting in the transcriptional activation of ICAM-1 gene (25Huang W.C. Chen C.C. Mol. Cell. Biol. 2005; 25: 6592-6602Crossref PubMed Scopus (221) Google Scholar). We have previously proposed a model for the transcriptional regulation of keratin 16 (26Wang Y.N. Chang W.C. J. Biol. Chem. 2003; 278: 45848-45857Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27Wang Y.N. Chen Y.J. Chang W.C. Mol. Pharmacol. 2006; 69: 85-98Crossref PubMed Scopus (20) Google Scholar). We suggest that the epidermal growth factor (EGF) up-regulates the recruitment of coactivator p300 to the promoter of keratin 16 through the activation of ERK, the last being of critical importance for the EGF regulation of keratin 16. We also show that the recruited p300 functionally cooperates with Sp1 and c-Jun to enhance the gene expression of keratin 16. Furthermore, the HAT domain of p300 is absolutely required for the keratin 16 promoter activation incurred upon EGF treatment. Therefore, the aim of this work was to study how the ERK activation regulated the recruitment of p300, thereby activating the keratin 16 gene expression. We found that the ERK2-mediated C-terminal serine (Ser-2279, Ser-2315, and Ser-2366) phosphorylations of p300 were the major targets by EGF, resulting in the stimulation of its HAT activity and its interaction with Sp1. These ERK2-mediated serine phosphorylation sites on p300 apparently controlled the p300 recruitment to the keratin 16 promoter, thus explaining the activation of the transcriptional activity of keratin 16 caused by EGF. Furthermore, this study was the first to identify the exact ERK2 phosphorylation sites on p300 in vivo. Materials—Human EGF (natural, culture grade) was purchased from PePro Technology (Rocky Hill, NJ). U0126, an inhibitor of MEK1, and the luciferase assay system were from Promega (Madison, WI). TRIzol RNA extraction kit, SuperScript™III, Dulbecco's modified Eagle's medium, Opti-MEM medium, Sp1-specific siRNA, and control siRNA were obtained from Invitrogen. Recombinant p300 was purchased from Active Motif (Carlsbad, CA). Polyclonal antibodies against ERK1/2, monoclonal antibody against phosphoserine/threonine-proline (phospho-Ser/Thr-Pro), recombinant histone H3, HA-p300 expression vector, protein A-agarose/salmon sperm DNA, and HAT assay kit were from Upstate Biotechnology, Inc. (Lake Placid, NY). Recombinant activated ERK2 and inactivated ERK2 were obtained from Biomol (Plymouth Meeting, PA). Biotinylated oligonucleotides were purchased from MDBio, Inc. (Taipei, Taiwan). Arrest-In reagent was from Open Technology (Taipei, Taiwan). Pfu-turbo polymerase was obtained from Stratagene (La Jolla, CA). Streptavidin-agarose was purchased from Sigma. Monoclonal antibodies against ERK2, c-Jun, and p300 were obtained from BD Transduction Laboratories. Agarose conjugated to p300 and Sp1, polyclonal antibodies against Sp1, monoclonal antibody against GST, normal mouse IgG, and normal rabbit IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies directed against acetyl-lysine, phospho-ERK1/2, and agarose conjugated to phospho-ERK1/2 were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against HA and affinity matrix-conjugated to HA were from Roche Applied Science. All other reagents used were of the highest purity obtainable. Cell Culture and EGF Treatment—HaCaT cells, a spontaneously immortalized human epidermal keratinocyte cell line, were grown at 37 °C under 5% CO2 in 10-cm plastic dishes containing 8 ml of Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 100 μg/ml streptomycin, and 100 IU/ml penicillin. A431 cells, a human epidermoid carcinoma cell line, were grown in the same condition as HaCaT cells. In this series of experiments, both cells were treated with 30 ng/ml EGF in optimal serum-free conditions, unless stated otherwise. Immunoprecipitation Assay—Nuclear extracts and cell lysates were prepared as described before (27Wang Y.N. Chen Y.J. Chang W.C. Mol. Pharmacol. 2006; 69: 85-98Crossref PubMed Scopus (20) Google Scholar). Nuclear extracts (80–1000 μg of protein of each) or lysates (1000–1800 μg of protein of each) were immunoprecipitated with 20 μl of p300 antibodies agarose-conjugated, phospho-ERK1/2 antibodies agarose-conjugated, Sp1 antibodies agarose-conjugated, or HA antibodies affinity matrix-conjugated individually in immunoprecipitation buffer (20 mm Hepes (pH 7.9), 2 mm MgCl2, 0.2 mm EDTA, 0.1 mm KCl, 10% (v/v) glycerol, and 1 mm dithiothreitol) under gentle shaking at 4 °C overnight. Immunoprecipitated beads were pelleted and washed three times with 500 μl of washing buffer (1× PBS containing 0.5% Nonidet P-40). Protein was removed from the beads by boiling in 2× SDS loading buffer for 5 min and separated by SDS-PAGE, followed by Western blot analysis, as described before (27Wang Y.N. Chen Y.J. Chang W.C. Mol. Pharmacol. 2006; 69: 85-98Crossref PubMed Scopus (20) Google Scholar), probed with phospho-Ser/Thr-Pro antibodies, or as indicated. In Vitro Kinase Assay—The commercially obtained full length of recombinant p300 protein was incubated at 30 °C for 30 min with recombinant activated or inactivated ERK2 in the presence of 23.3 μm cold ATP in MAPK buffer (25 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 5 mm glycerol phosphate, 2 mm dithiothreitol, and 0.1 mm Na3VO4). The reaction was stopped by adding an equal volume of 5× SDS loading buffer and by heating at 95 °C for 5 min. After separation through 10% SDS-PAGE, the gel was transferred and followed by Western blot analysis probed with phospho-Ser/Thr-Pro antibodies. In Vitro Acetylation Assay and Nonradioactive Histone Acetyltransferase Activity Assay—The commercially obtained full length of recombinant p300 protein was subjected to an in vitro kinase assay, as described previously, and then mixed with purified 0.5 μg of histone H3 and 2 μg of acetyl-CoA in the presence of acetylation assay buffer (50 mm Tris-HCl (pH 8.0), 10% glycerol, 0.1 mm EDTA, and 1 mm dithiothreitol) and incubated at 30 °C for 30 min. In addition, the immunoprecipitates of HA-p300-WT or HA-p300-S3A with or without EGF treatment were subjected to this in vitro acetylation assay. The reaction was stopped by adding an equal volume of 5× SDS loading buffer and by heating at 95 °C for 5 min. After separation through a 12% SDS-PAGE, the gel was followed by Western blot analysis probed with acetyl-lysine antibodies. Nonradioactive HAT activity assay was performed by using a HAT assay kit according to the manufacturer's instruction. The commercially obtained full length of recombinant p300 protein was subjected to an in vitro kinase assay as described previously, and nuclear extracts with HA-p300-WT (wild type) or HA-p300-S3A (with Ser-2279, Ser-2315, and Ser-2366 all replaced by alanine) expression treated with or without EGF were subjected to this assay. Briefly, each reaction mixed with 100 μm acetyl-CoA, and 1× HAT assay buffer was incubated on an enzyme-linked immunosorbent assay plate pre-coated with histone H3 at 30 °C for 30 min. After washing with 1× Tris-buffered saline for several times, acetylated histones were detected with acetyl-lysine antibodies, followed by the horseradish peroxidase-based colorimetric assay. Transfection with Arrest-In and Reporter Gene Assay—Cells were transfected with plasmids by lipofection using Arrest-In according to the manufacturer's instruction with a slight modification. Cells were replated 24 h before transfection at an optimal cell density (60–80% of confluence) in 2 ml of fresh culture medium in a 3.5-cm plastic dish. For use in transfection, 1 μl of Arrest-In (DNA:Arrest-In = 1:5) was incubated with 0.2 μg of pXK-5-1 luciferase plasmid that contained the EGF-responsive region of the keratin 16 gene (26Wang Y.N. Chang W.C. J. Biol. Chem. 2003; 278: 45848-45857Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) or the indicated plasmids as described in each experiment in 60 μl of Opti-MEM medium for 10 min at room temperature. Total DNA concentration for each transfection was matched with HA-pcDNA3.0. Cells were transfected by changing the medium with 1 ml of Opti-MEM medium containing the plasmids and Arrest-In, followed by incubation at 37 °C in a humidified atmosphere of 5% CO2 for 5 h. After a change of Opti-MEM medium to 2 ml of fresh culture medium and overnight incubation, cells were stimulated with EGF if necessary and then incubated for an additional 24 h. For cells transfected in a 6-cm plastic dish, 2 μg of indicated plasmids as described in each experiment were transfected in 2 ml of Opti-MEM medium, and the residual transfection procedure was similar to that as described above. The luciferase activities in cell lysates were measured by the luciferase assay system and determined as described previously (28Liu Y.W. Arakawa T. Yamamoto S. Chang W.C. Biochem. J. 1997; 324: 133-140Crossref PubMed Scopus (42) Google Scholar). Luciferase activity was normalized per μg of extract protein. DNA Affinity Precipitation Assay (DAPA)—The binding assay was performed by mixing nuclear extract proteins, 2 μg of biotinylated keratin 16-specific wild type oligonucleotide, and 20 μl of streptavidin-agarose beads (4%) with a 50% slurry. The mixture was incubated at room temperature for 1 h with rotating. Beads were pelleted and washed three times with cold 1× PBS. The binding proteins were eluted by 2× SDS loading buffer and separated by SDS-PAGE, followed by Western blot analysis probed with specific antibodies. 5′-Biotinylated wild type sequence was SpAP (where SpAP is a wild type sequence containing Sp1- and Ap1-binding sites), 5′-biotin-GTTAGGAGGGCCCCGCCTTCCCCAGG-3′ (27Wang Y.N. Chen Y.J. Chang W.C. Mol. Pharmacol. 2006; 69: 85-98Crossref PubMed Scopus (20) Google Scholar). Chromatin Immunoprecipitation Assay—Stable transfectants of HaCaT cells with HA-p300-WT or HA-p300-S3A expression were treated with or without inhibitors for 30 min, followed by EGF treatment for 24 h. Cells were cross-linked with 1% formaldehyde at room temperature for 15 min, washed once with 1× PBS, lysed with L1 buffer (50 mm Tris (pH 8.0), 2 mm EDTA, 0.1% (v/v) Nonidet P-40, and 10% (v/v) glycerol), and then resuspended with L2 buffer (50 mm Tris (pH 8.0), 5 mm EDTA, and 1% SDS). The lysates were sonicated to shear the size of DNA to 500–1000 bp. One mg of sonicated extracts was diluted 10-fold with dilution buffer (50 mm Tris (pH 8.0), 0.5 mm EDTA, 0.5% (v/v) Nonidet P-40, and 0.2 m NaCl), followed by incubation with 40 μl of protein A-agarose/salmon sperm DNA at 4 °C for 30 min for pre-cleaning. Immunoprecipitation was performed with specific antibodies and rotating at 4 °C overnight, followed by adding 40 μl of protein A-agarose/salmon sperm DNA at 4 °C for 1.5 h. Immunoprecipitated beads were pelleted and washed with high salt and low salt washing buffer each four times. DNA-protein complex was eluted in an elution buffer (1× Tris-EDTA buffer containing 1% SDS) with rotating at room temperature for 15 min, and the immune complex cross-link was reversed by heating at 65 °C overnight, followed by treatment with 100 μg/reaction proteinase K at 50 °C for 1 h. DNA was extracted once with phenol/chloroform, precipitated with ethanol, and dissolved in 45 μl of H2O. L1 buffer, L2 buffer, and dilution buffer contained 0.5 mm phenylmethylsulfonyl fluoride, 1 mm orthovanadate, 2 μg/ml pepstatin A, 2 μg/ml leupeptin, 5 mm sodium fluoride, and 1 μg/ml aprotinin. Specific sequences in the immunoprecipitates were detected by PCR amplification. The PCR product was separated by 1.5% agarose gel electrophoresis and visualized with ethidium bromide staining (27Wang Y.N. Chen Y.J. Chang W.C. Mol. Pharmacol. 2006; 69: 85-98Crossref PubMed Scopus (20) Google Scholar). Construction and Purification of GST-p300 Fusion Proteins—To create various GST-p300 fragments, PCR-generated fragments encoding amino acids 2–331, 302–670, 565–966, 952–1145, 1140–1570, 1570–1968, 1964–2197, and 2188–2375 of human p300 were inserted into the SalI/NotI sites of pGEX4T-1 individually. In addition, these constructs were subjected to QuikChange □ XL site-directed mutagenesis kit for obtaining single point or triple points mutations of GST-p300 fragments. Escherichia coli containing expression vectors of respective GST-p300 fragment were cultured in LB medium and induced with 1 mm isopropyl β-d-thiogalactopyranoside for 3–6 h at 18–37 °C as indicated. Cells were harvested, and incubated in 1× PBS buffer containing 2 μg/ml lysozyme, 1 mm EDTA, 5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 0.5 μg/ml pepstatin A at 37 °C for 30 min. After sonication, samples were centrifuged at 7,500 × g for 5 min, and the supernatant was then incubated with glutathione-Sepharose™ 4B at 4 °C for 2 h to overnight as indicated, followed by elution with elution buffer (50 mm Tris (pH 9.6), 20 mm glutathione, and 1 mm EDTA) at 4 °C for 30 min twice. These elutions were stored at -70 °C until used. GST Pulldown Assay—Equal molar concentrations of GST or GST-Sp1 fusion protein were incubated with 250 ng of recombinant p300 protein at 4 °C overnight. The p300 protein that interacted with GST-Sp1 was precipitated with glutathione-Sepharose™ 4B. The reaction was stopped by boiling for 5 min in 2× SDS loading buffer and analyzed by Western blot probed with p300 and GST antibodies. Reverse Transcription-PCR—HaCaT cells with stable expression of HA-p300-WT or HA-p300-S3A maintained for 2 days in serum-free medium were incubated with EGF for 24 h and then harvested. Total RNA was isolated by using the TRIzol RNA extraction kit, and 2 μg of RNA were subjected to reverse transcription-PCR with SuperScript™III. Specific primers for keratin 6, 16, 17 and glyceraldehyde-3-phosphate dehydrogenase were used. The PCR products were separated by 1% agarose-gel electrophoresis and visualized with ethidium bromide staining. ERK2-dependent Phosphorylation of p300 by EGF—Several studies indicate that direct phosphorylation of p300 or CBP controls their recruitment to the transcription complex (23Zanger K. Radovick S. Wondisford F.E. Mol. Cell. 2001; 7: 551-558Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 25Huang W.C. Chen C.C. Mol. Cell. Biol. 2005; 25: 6592-6602Crossref PubMed Scopus (221) Google Scholar). In addition, our previous results indicate that the ERK activation regulates the recruitment of p300 to the keratin 16 promoter (27Wang Y.N. Chen Y.J. Chang W.C. Mol. Pharmacol. 2006; 69: 85-98Crossref PubMed Scopus (20) Google Scholar). Therefore, we studied here whether ERK caused the phosphorylation of p300. At first, we studied the interaction between p300 and ERK by coimmunoprecipitation with p300 antibodies. As shown in Fig. 1A, significant bindings of phospho-ERK2 and ERK2 to p300 in the nucleus were observed in HaCaT cells treated with EGF (IP, compare lane 2 with lane 1). Furthermore, these interactions induced by EGF were blocked by U0126, a specific MEK1 inhibitor (Fig. 1A, IP, compare lane 4 with lane 2), which presented a similar pattern of endogenous protein expressions (Fig. 1A, Nuclear extracts). Next, to confirm whether p300 was a direct substrate of ERK2, an in vitro kinase assay was performed by using phospho-Ser/Thr-Pro antibodies. The antibodies were chosen because the minimal ERK consensus phosphoacceptor motif is Ser/Thr-Pro (10Yang S.H. Sharrocks A.D. Whitmarsh A.J. Gene (Amst.). 2003; 320: 3-21Crossref PubMed Scopus (424) Google Scholar). As shown in Fig. 1B, purified full-length p300 protein was phosphorylated by recombinant activated ERK2-A in vitro (lane 2). Inactivated ERK2-I was here used as a negative control (Fig. 1B, lane 1). The inputs of p300 (Fig. 1B, lanes 3 and 4) and ERK2 (lanes 5 and 6) were shown as equal. To further confirm the phosphorylation of p300 by activated ERK2-A in vitro was not because of the different binding affinities between purified p300 protein and these two recombinant ERK2 proteins, immunoprecipitation assay with p300 antibodies was then performed. The results shown in Fig. 1C indicated that both activated ERK2-A and inactivated ERK2-I had similar binding affinities to p300 protein in vitro (middle panel, lanes 3 and 4). We then assessed whether EGF induced phosphorylation of p300 in vivo. The p300 protein was immunoprecipitated from the nuclear extracts of HaCaT cells with p300 antibodies and then immunoblotted with phospho-Ser/Thr-Pro antibodies. The results are shown in Fig. 1D. EGF induced the phosphorylation of p300 in a time-dependent manner. The induction of p300 phosphorylation by EGF was maximized at 30 min (Fig. 1D, lane 3) and sustained at least up to 90 min (lane 6) after EGF treatment. It was also observed that this EGF-induced phosphorylation of p300 was dose-dependent (see supplemental Fig. S1A, middle panel). These results showed that the phosphorylation of immunoprecipitated p300 was maximized when HaCaT cells were treated with EGF for 30 ng/ml. Similar results were observed in A431 cells (Fig. 1E). When A431 cells were transiently overexpressed with HA-p300-WT (wild type) and then the lysates were immunoprecipitated with HA antibodies