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• W2005429042 abstract "N-CoR and SMRT are corepressor paralogs that partner with and mediate transcriptional repression by a wide variety of metazoan transcription factors, including nuclear hormone receptors. Although encoded by distinct genetic loci, N-CoR and SMRT share substantial sequence interrelatedness, form analogous assemblies with histone deacetylases and auxiliary factors, can interact with overlapping sets of transcription factor partners, and exert overlapping functions in cells. SMRT is subject to negative regulation by MAPK signaling pathways operating downstream of growth factor and stress signaling pathways. We report here that whereas activation of MEKK1 leads to phosphorylation of SMRT, its dissociation from its transcription factor partners in vivo and in vitro, and its redistribution from the cell nucleus to a cytoplasmic compartment, N-CoR is refractory to all these forms of regulation. In contrast to this MAPK cascade, other signal transduction pathways operating downstream of growth factor/cytokine receptors appear able to affect both corepressor paralogs. Our results indicate that SMRT and N-CoR are embedded in distinct regulatory networks and that the two corepressors interpret growth factor, cytokine, differentiation, and prosurvival signals differently. N-CoR and SMRT are corepressor paralogs that partner with and mediate transcriptional repression by a wide variety of metazoan transcription factors, including nuclear hormone receptors. Although encoded by distinct genetic loci, N-CoR and SMRT share substantial sequence interrelatedness, form analogous assemblies with histone deacetylases and auxiliary factors, can interact with overlapping sets of transcription factor partners, and exert overlapping functions in cells. SMRT is subject to negative regulation by MAPK signaling pathways operating downstream of growth factor and stress signaling pathways. We report here that whereas activation of MEKK1 leads to phosphorylation of SMRT, its dissociation from its transcription factor partners in vivo and in vitro, and its redistribution from the cell nucleus to a cytoplasmic compartment, N-CoR is refractory to all these forms of regulation. In contrast to this MAPK cascade, other signal transduction pathways operating downstream of growth factor/cytokine receptors appear able to affect both corepressor paralogs. Our results indicate that SMRT and N-CoR are embedded in distinct regulatory networks and that the two corepressors interpret growth factor, cytokine, differentiation, and prosurvival signals differently. Many transcription factors display bimodal regulatory properties and can confer both repression and activation on their target genes. This functional dualism reflects the ability of these transcription factors to recruit two alternative classes of auxiliary proteins, denoted corepressors and coactivators, that determine the polarity of the transcriptional response (1Chen J.D. Li H. Crit. Rev. Eukaryotic Gene Expression. 1998; 8: 169-190Crossref PubMed Scopus (102) Google Scholar, 2Ito M. Roeder R.G. Trends Endocrinol. Metab. 2001; 12: 127-134Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 3Lee J.W. Lee Y.C. Na S.Y. Jung D.J. Lee S.K. Cell. Mol. Life Sci. 2001; 58: 289-297Crossref PubMed Scopus (119) Google Scholar, 4McKenna N.J. O'Malley B.W. Endocrinology. 2002; 143: 2461-2465Crossref PubMed Scopus (279) Google Scholar, 5Ordentlich P. Downes M. Evans R.M. Curr. Top. Microbiol. Immunol. 2001; 254: 101-116PubMed Google Scholar, 6Privalsky M.L. Annu. Rev. Physiol. 2004; 66: 315-360Crossref PubMed Scopus (261) Google Scholar, 7Rachez C. Freedman L.P. Curr. Opin. Cell Biol. 2001; 13: 274-280Crossref PubMed Scopus (236) Google Scholar, 8Shibata H. Spencer T.E. Onate S.A. Jenster G. Tsai S.Y. Tsai M.J. O'Malley B.W. Recent Prog. Horm. Res. 1997; 52 (Discussion 164-165): 141-164PubMed Google Scholar, 9Xu L. Glass C.K. Rosenfeld M.G. Curr. Opin. Genet. Dev. 1999; 9: 140-147Crossref PubMed Scopus (815) Google Scholar). Nuclear receptors, for example, are a family of ligand-regulated transcription factors that regulate key aspects of metazoan development, differentiation, and homeostasis (10Beato M. Klug J. Hum. Reprod. Update. 2000; 6: 225-236Crossref PubMed Scopus (487) Google Scholar, 11Hager G.L. Prog. Nucleic Acid Res. Mol. Biol. 2001; 66: 279-305Crossref PubMed Google Scholar, 12Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6110) Google Scholar, 13Zhang J. Lazar M.A. Annu. Rev. Physiol. 2000; 62: 439-466Crossref PubMed Scopus (581) Google Scholar). In the absence of hormone ligand, nuclear receptors can recruit a corepressor complex containing the SMRT protein, leading to repression of target gene expression (14Chen J.D. Evans R.M. Nature. 1995; 377: 454-457Crossref PubMed Scopus (1715) Google Scholar, 15Chen J.D. Umesono K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7567-7571Crossref PubMed Scopus (222) Google Scholar, 16Ordentlich P. Downes M. Xie W. Genin A. Spinner N.B. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2639-2644Crossref PubMed Scopus (140) Google Scholar, 17Park E.J. Schroen D.J. Yang M. Li H. Li L. Chen J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3519-3524Crossref PubMed Scopus (109) Google Scholar, 18Sande S. Privalsky M.L. Mol. Endocrinol. 1996; 10: 813-825Crossref PubMed Scopus (210) Google Scholar, 19Zamir I. Harding H.P. Atkins G.B. Horlein A. Glass C.K. Rosenfeld M.G. Lazar M.A. Mol. Cell. Biol. 1996; 16: 5458-5465Crossref PubMed Scopus (200) Google Scholar). Conversely, binding of hormone agonist causes the release of the SMRT corepressor complex and the recruitment of coactivator complexes that enhance target gene expression (20Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 21Privalsky M.L. Curr. Top. Microbiol. Immunol. 2001; 254: 117-136Crossref PubMed Google Scholar). Analogous corepressor and coactivator complexes partner with a broad assortment of other transcriptional regulators, including NF-κB, serum response factor, AP-1 proteins, Smad proteins, CCAAT binding factor, c-Myb, PLZF, Bcl-6, Pbx/Hox proteins, ETO-1 and ETO-2, aryl hydrocarbon receptor, and MyoD, among others (reviewed in Ref. 6Privalsky M.L. Annu. Rev. Physiol. 2004; 66: 315-360Crossref PubMed Scopus (261) Google Scholar). Corepressors and coactivators modulate gene expression by modifying the chromatin template and by making inhibitory or stimulatory contacts with the general transcriptional machinery (22Ayer D.E. Trends Cell Biol. 1999; 9: 193-198Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 23Berger S.L. Science. 2001; 292: 64-65PubMed Google Scholar, 24Chen H. Tini M. Evans R.M. Curr. Opin. Cell Biology. 2001; 13: 218-224Crossref PubMed Scopus (152) Google Scholar, 25Hassig C.A. Schreiber S.L. Curr. Opin. Chem. Biol. 1997; 1: 300-308Crossref PubMed Scopus (339) Google Scholar, 26Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7709) Google Scholar, 27Muscat G.E. Burke L.J. Downes M. Nucleic Acids Res. 1998; 26: 2899-2907Crossref PubMed Scopus (111) Google Scholar, 28Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (772) Google Scholar, 29Rice J.C. Allis C.D. Curr. Opin. Cell Biol. 2001; 13: 263-273Crossref PubMed Scopus (571) Google Scholar, 30Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6679) Google Scholar, 31Turner B.M. BioEssays. 2000; 22: 836-845Crossref PubMed Scopus (978) Google Scholar, 32Wong C.W. Privalsky M.L. Mol. Cell. Biol. 1998; 18: 5500-5510Crossref PubMed Scopus (119) Google Scholar, 33Workman J.L. Kingston R.E. Annu. Rev. Biochem. 1998; 67: 545-579Crossref PubMed Scopus (975) Google Scholar, 34Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Many coactivators possess histone acetyltransferase activity, whereas the SMRT corepressor recruits histone deacetylases, such as HDAC3 (1Chen J.D. Li H. Crit. Rev. Eukaryotic Gene Expression. 1998; 8: 169-190Crossref PubMed Scopus (102) Google Scholar, 3Lee J.W. Lee Y.C. Na S.Y. Jung D.J. Lee S.K. Cell. Mol. Life Sci. 2001; 58: 289-297Crossref PubMed Scopus (119) Google Scholar, 4McKenna N.J. O'Malley B.W. Endocrinology. 2002; 143: 2461-2465Crossref PubMed Scopus (279) Google Scholar, 5Ordentlich P. Downes M. Evans R.M. Curr. Top. Microbiol. Immunol. 2001; 254: 101-116PubMed Google Scholar, 6Privalsky M.L. Annu. Rev. Physiol. 2004; 66: 315-360Crossref PubMed Scopus (261) Google Scholar, 9Xu L. Glass C.K. Rosenfeld M.G. Curr. Opin. Genet. Dev. 1999; 9: 140-147Crossref PubMed Scopus (815) Google Scholar). Acetylation and deacetylation of nucleosomal histones by these coactivator and corepressor complexes, operating together with other covalent histone modifications, create a code that influences the interaction of the chromatin with additional factors and its accessibility to the general transcriptional machinery (22Ayer D.E. Trends Cell Biol. 1999; 9: 193-198Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 23Berger S.L. Science. 2001; 292: 64-65PubMed Google Scholar, 24Chen H. Tini M. Evans R.M. Curr. Opin. Cell Biology. 2001; 13: 218-224Crossref PubMed Scopus (152) Google Scholar, 25Hassig C.A. Schreiber S.L. Curr. Opin. Chem. Biol. 1997; 1: 300-308Crossref PubMed Scopus (339) Google Scholar, 26Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7709) Google Scholar, 28Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (772) Google Scholar, 29Rice J.C. Allis C.D. Curr. Opin. Cell Biol. 2001; 13: 263-273Crossref PubMed Scopus (571) Google Scholar, 30Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6679) Google Scholar, 31Turner B.M. BioEssays. 2000; 22: 836-845Crossref PubMed Scopus (978) Google Scholar, 33Workman J.L. Kingston R.E. Annu. Rev. Biochem. 1998; 67: 545-579Crossref PubMed Scopus (975) Google Scholar, 34Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Besides histone deacetylases, the SMRT complex contains additional protein components, such as TBL1/TBLR1 and GPS2, that help stabilize its overall structure and that may contribute to the release of the corepressor complex in response to hormone agonist (35Guenther M.G. Lane W.S. Fischle W. Verdin E. Lazar M.A. Shiekhattar R. Genes Dev. 2000; 14: 1048-1057Crossref PubMed Google Scholar, 36Li J. Wang J. Nawaz Z. Liu J.M. Qin J. Wong J. EMBO J. 2000; 19: 4342-4350Crossref PubMed Scopus (512) Google Scholar, 37Perissi V. Aggarwal A. Glass C.K. Rose D.W. Rosenfeld M.G. Cell. 2004; 116: 511-526Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 38Yoon H.G. Chan D.W. Huang Z.Q. Li J. Fondell J.D. Qin J. Wong J. EMBO J. 2003; 22: 1336-1346Crossref PubMed Scopus (354) Google Scholar, 39Zhang J. Kalkum M. Chait B.T. Roeder R.G. Mol. Cell. 2002; 9: 611-623Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar); other polypeptides, such as mSin3 and an assortment of additional histone deacetylases, can also interact with SMRT, but the association of these latter polypeptides with the SMRT complex in vivo and their contribution to SMRT-mediated repression remain incompletely elucidated (reviewed in Ref. 6Privalsky M.L. Annu. Rev. Physiol. 2004; 66: 315-360Crossref PubMed Scopus (261) Google Scholar). SMRT therefore acts as a molecular platform on which the remainder of the corepressor complex assembles and serves as the principal contact between the corepressor complex and its transcription factor partners. Regulatory events that cause a dissociation of SMRT also cause the release of the remainder of the corepressor complex and a loss of repression (20Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 21Privalsky M.L. Curr. Top. Microbiol. Immunol. 2001; 254: 117-136Crossref PubMed Google Scholar). Notably, a second corepressor protein, denoted N-CoR, is widely distributed in vertebrates and performs similar or identical functions compared with SMRT (40Hörlein A.J. Näär A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Söderström M. Glass C.K. Rosenfeld G.M. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1714) Google Scholar, 41Seol W. Mahon M.J. Lee Y.K. Moore D.D. Mol. Endocrinol. 1996; 10: 1646-1655PubMed Google Scholar). Although encoded by a distinct genetic locus, N-CoR shares the same overall molecular architecture and significant amino acid identity with SMRT (see Fig. 1A); interacts with many of the same transcription factors partners (although, in some cases, with different affinities); and assembles into similar or identical complexes with TBL1, TBLR1, and GPS2 and with other known or suspected corepressor components (reviewed in Ref. 6Privalsky M.L. Annu. Rev. Physiol. 2004; 66: 315-360Crossref PubMed Scopus (261) Google Scholar). Despite these many parallels between SMRT and N-CoR, these corepressor paralogs were established and subsequently maintained as distinct gene products from the beginning of the vertebrate evolutionary radiation and perform distinct functions in cells (reviewed in Ref. 6Privalsky M.L. Annu. Rev. Physiol. 2004; 66: 315-360Crossref PubMed Scopus (261) Google Scholar). What differences do N-CoR and SMRT therefore manifest at the molecular level to account for their distinct biological and evolutionary properties? We have shown that growth factor receptors are important regulators of SMRT function and operate through a MAPK 1The abbreviations used are: MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; T3R, thyroid hormone receptor; T3, triiodothyronine; IL-1β, interleukin-1β; PBS, phosphate-buffered saline; GST, glutathione S-transferase; BSA, bovine serum albumin; MOPS, 4-morpholinepropanesulfonic acid; EMSA, electrophoretic mobility shift assay; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; Gal4DBD, Gal4 DNA-binding domain; Gal4AD, Gal4 activation domain; RARα, retinoic acid receptor-α; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase. cascade (42Hong S.H. Wong C.W. Privalsky M.L. Mol. Endocrinol. 1998; 12: 1161-1171Crossref PubMed Google Scholar, 43Hong S.H. Privalsky M.L. Mol. Cell. Biol. 2000; 20: 6612-6625Crossref PubMed Scopus (163) Google Scholar). Activation of the epidermal growth factor (EGF) receptor or its downstream mediator, MEKK1, leads to inhibition of the ability of SMRT to interact with its transcription factor partners and a redistribution of SMRT from the nucleus to the cytoplasm (42Hong S.H. Wong C.W. Privalsky M.L. Mol. Endocrinol. 1998; 12: 1161-1171Crossref PubMed Google Scholar, 43Hong S.H. Privalsky M.L. Mol. Cell. Biol. 2000; 20: 6612-6625Crossref PubMed Scopus (163) Google Scholar). These effects of MEKK1 on SMRT represent an important nexus between growth factor signaling and nuclear receptor function and contribute to the differentiation-promoting effects of arsenic trioxide treatment in acute promyelocytic leukemia (44Hong S.H. Yang Z. Privalsky M.L. Mol. Cell. Biol. 2001; 21: 7172-7182Crossref PubMed Scopus (56) Google Scholar). We report here that direct phosphorylation of SMRT by MEKK1 is sufficient to inhibit the SMRT/thyroid hormone receptor (T3R) interaction in vitro and that the relocalization of SMRT to the cytoplasm in cells expressing MEKK1 occurs unaccompanied by the T3R partner (which is retained in the nucleus). More important, we also report that N-CoR is unexpectedly resistant to these inhibitory effects of MEKK1 under conditions in which SMRT function is strongly suppressed. Unlike SMRT, N-CoR is refractory to MEKK1 phosphorylation, does not release from nuclear receptor partners in vitro or in vivo, and does not detectably change in its subcellular distribution in response to MEKK1 signaling. Taken together with the observations by other investigators, these results indicate that the SMRT and N-CoR corepressor paralogs are subject to distinct forms of regulation. We suggest that these divergent forms of control help account for the establishment and retention of these two distinct forms of corepressor during vertebrate evolution. Plasmid Constructs—The construction of the mammalian expression plasmids pSG5-Gal4AD, pSG5-Gal4AD-T3Rα, pSG5-Gal4DBD, pSG5-Gal4DBD-SMRTτ-(1773-2471), pSG5-Gal4DBD-N-CoR-(1946-2435), pSG5-Gal4AD-RARα, and pSG5-Gal4DBD-RARα was described previously (32Wong C.W. Privalsky M.L. Mol. Cell. Biol. 1998; 18: 5500-5510Crossref PubMed Scopus (119) Google Scholar, 43Hong S.H. Privalsky M.L. Mol. Cell. Biol. 2000; 20: 6612-6625Crossref PubMed Scopus (163) Google Scholar, 45Hauksdottir H. Farboud B. Privalsky M.L. Mol. Endocrinol. 2003; 17: 373-385Crossref PubMed Scopus (55) Google Scholar, 46Hong S.H. David G. Wong C.W. Dejean A. Privalsky M.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9028-9033Crossref PubMed Scopus (305) Google Scholar). The pSG5-Myc vector was created by inserting a synthetic oligonucleotide (MWG Biotech, High Point, NC) encoding a Myc epitope tag into an expanded multiple cloning site in pSG5. The pSG5-Myc-T3Rα, pSG5-Myc-SMRTτ-(1-2423), and pSG5-Myc-N-CoR-(1-2453) vectors were created using PCR to introduce approximate restriction sites on the ends of the corresponding open reading frames and by ligating the DNA products into the pSG5-Myc vector. The pCMV-GFP-SMRTτ-(1-2423) and pCMV-GFP-N-CoR-(1-2453) expression vectors were created by inserting PCR-generated DNAs containing the corresponding open reading frames into the pCMV-GFP vector (43Hong S.H. Privalsky M.L. Mol. Cell. Biol. 2000; 20: 6612-6625Crossref PubMed Scopus (163) Google Scholar). PCR-generated DNAs encoding the S1 domain of SMRTα (amino acids 2313-2517) or the S1 and S2 domains of SMRTα (amino acids 2077-2517) were cloned into pGEX-KG (47Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar) to yield the pGEX-SMRTα-S1-(2313-2517) and pGEX-SMRTα-S1/S2-(2077-2517) constructs. The pGEX-N-CoR-N1-(2211-2453) and the pGEX-N-CoR-N1/N2/N3-(1817-2453) vectors were created by inserting the HindIII-SalI or ApaI-SalI fragment of N-CoR into a pGEX-KG vector bearing an expanded multiple cloning site. All clones were confirmed by DNA sequence analysis. The origins of pCMV5-FLAG-ΔMEKK1-(817-1493), pCMV-HA-MEK1(R4F), pMT3-ERK1, pSG5-v-ErbB, pLNC-v-Raf1, and pCMV-v-Ras plasmids were described previously (42Hong S.H. Wong C.W. Privalsky M.L. Mol. Endocrinol. 1998; 12: 1161-1171Crossref PubMed Google Scholar, 43Hong S.H. Privalsky M.L. Mol. Cell. Biol. 2000; 20: 6612-6625Crossref PubMed Scopus (163) Google Scholar). A constitutively active clone of Akt, pCMV-Akt1(S473D), was the generous gift of Marty Mayo (University of Virginia). Baculovirus constructs for T3Rα and His6-ΔMEKK1 were described previously (43Hong S.H. Privalsky M.L. Mol. Cell. Biol. 2000; 20: 6612-6625Crossref PubMed Scopus (163) Google Scholar, 48Chen H.W. Privalsky M.L. Mol. Cell. Biol. 1993; 13: 5970-5980Crossref PubMed Scopus (50) Google Scholar). Cell Culture—CV-1 cells were propagated in Dulbecco's modified Eagle's medium containing high glucose, l-glutamine, and pyridoxine hydrochloride (Invitrogen) and supplemented with 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere. For expression of His6-ΔMEKK1 in the baculovirus expression system, Sf9 cells were maintained and infected in Ex-cell 420 medium (JRH Biosciences, Lenexa, KS) supplemented with 10% heat-inactivated fetal bovine serum; cells were incubated at 28°C in a humidified atmosphere. Mammalian Two-hybrid Analysis—CV-1 cells (3.0 × 104 cells/well in a 24-well plate) were transiently transfected, 24 h after plating, using Effectene transfection reagent (QIAGEN Inc.) following the manufacturer's recommended protocol. Transfection mixtures included 50 ng of the appropriate pSG5-Gal4AD vector, 12.5 ng of the appropriate pSG5-Gal4DBD vector, 50 ng of the pADH-Gal4-17-mer luciferase reporter, either 50 ng of pCH110 or 10 ng of pCMV-LacZ as an internal transfection control, appropriate expression vectors for the indicated signal transducers, and/or an empty vector, as appropriate. Twenty-four hours after transfection, the medium was replaced with fresh medium with or without 1 μm triiodothyronine (T3), 1 ng/ml interleukin-1β (IL-1β), 10 ng/ml anisomycin, and/or 1 μm U0126, as indicated. Cells were collected 48 h after transfection and lysed in lysis buffer (100 μl/well) containing 0.2% Triton X-100, 91 mm K2HPO4, and 9.2 mm KH2PO4. Luciferase and β-galactosidase activities were determined as described previously (43Hong S.H. Privalsky M.L. Mol. Cell. Biol. 2000; 20: 6612-6625Crossref PubMed Scopus (163) Google Scholar, 45Hauksdottir H. Farboud B. Privalsky M.L. Mol. Endocrinol. 2003; 17: 373-385Crossref PubMed Scopus (55) Google Scholar). Co-immunoprecipitation Assays—CV-1 cells (1.5 × 105 cells/well in a 6-well plate) were transfected with various combinations of Myc-T3Rα, Myc-SMRTτ, Myc-N-CoR, a constitutively active MEKK1 construct, or appropriate amounts of equivalent empty vectors using the Effectene protocol described above. Cells were collected 48 h after transfection and lysed by a 30-min incubation at 4°C in 300 μl of immunoprecipitation buffer consisting of phosphate-buffered saline (PBS; 137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, and 1.5 mm KH2PO4) plus 1 mm EDTA, 1.5 mg/ml iodoacetamide, 100 μm Na3VO4, 0.5% Triton X-100, 20 mm β-glycerophosphate, 1 mm NaF, 0.2 mm phenylmethylsulfonyl fluoride, 1× Complete phosphatase inhibitor mixture I (EMD Biosciences, Inc., La Jolla, CA), and 1× Complete protease inhibitor mixture (Roche Applied Science, Mannheim, Germany). The cell lysates were cleared by centrifugation at 14,000 rpm at 4°C. A 15-μl aliquot of each cell lysate was saved, and the remaining lysate was incubated at 4°C for 1 h with rabbit anti-v-ErbA polyclonal antiserum (diluted 1:100) (49Bonde B.G. Privalsky M.L. J. Virol. 1990; 64: 1314-1320Crossref PubMed Google Scholar). Next, 40 μl of protein G-Sepharose beads (50% slurry) were added, and the samples were incubated overnight at 4°C on a rotator. The Sepharose beads and any proteins bound to them were collected by centrifugation at 3000 rpm in a microcentrifuge at 4°C for 2 min. The beads were washed four times with 300 μl of immunoprecipitation buffer, and any proteins remaining bound to the beads were then eluted by boiling in SDS sample buffer; resolved by SDS-PAGE using a NuPAGE Novex Tris acetate 3-8% gradient gel system (Invitrogen); and visualized by immunoblotting using mouse anti-Myc monoclonal antibody (diluted 1:200; Gamma One Laboratories, Lexington, KY), horseradish peroxidase-conjugated goat anti-mouse IgG antibody (diluted 1:1500; Bio-Rad), and the ECL Plus Western blot detection system (Amersham Biosciences). The resulting chemiluminescent signal was detected and quantified using a Fluorchem 8900 digital detection system (Alpha Innotech, San Leandro, CA). In Vitro Kinase Assays—GST-SMRT and GST-N-CoR fusion proteins were expressed in Escherichia coli BL21 cells and purified by binding to glutathione-agarose beads as described previously (32Wong C.W. Privalsky M.L. Mol. Cell. Biol. 1998; 18: 5500-5510Crossref PubMed Scopus (119) Google Scholar). Purified GST-corepressor proteins were eluted in buffer containing 20 mm glutathione, 5% glycerol, 10 mg/ml bovine serum albumin (BSA), and 1× Complete protease inhibitor mixture in 100 mm Tris-Cl (pH 8.0). The His6-tagged ΔMEKK1 proteins were expressed by baculoviral infection of Sf9 cells. Approximately 7 × 106 Sf9 cells were infected with recombinant baculovirus encoding His6-ΔMEKK1. Infected cells were harvested 72 h after infection, washed with PBS, resuspended in 3 ml of sonication buffer (20 mm Tris-Cl (pH 8.0), 100 mm NaCl, 0.5 mm β-mercaptoethanol, and 1 μg/ml leupeptin (Sigma)), and lysed by sonication. Triton X-100 was then added to a final concentration of 0.1%; the samples were vortexed briefly; and the lysate was cleared by centrifugation at 14,000 rpm at 4°C. The His6-ΔMEKK1 protein was then purified by adding 200 μl of prewashed Talon Superflow metal affinity resin (Clontech), mixing the samples for 20 min at room temperature, and collecting the resin beads by centrifugation at 3000 rpm for 2 min at 4°C. The resin was washed four times with sonication buffer, and the protein was eluted in 200 μl of buffer containing 20 mm Tris-Cl (pH 8.0), 100 mm NaCl, 400 mm imidazole, and 1× Complete protease inhibitor mixture. The eluate was dialyzed overnight in 50 mm Tris-Cl (pH 7.5), 50 mm NaCl, 0.1% β-mercaptoethanol, and 5% glycerol. 1× Complete protease inhibitor mixture was added, and the samples were flash-frozen in liquid nitrogen and stored as aliquots at -80°C. For phosphorylation in vitro, 10 μl of the GST-SMRT or GST-N-CoR protein were incubated overnight at 30°C with 2 μl of His6-ΔMEKK1 and 1 mm ATP (Sigma) in MEKK1 assay dilution buffer (20 mm MOPS (pH 7.2), 25 mm β-glycerophosphate, 5 mm EGTA, 1 mm Na3VO4, 16.7 mm MgCl2, 1 mm sodium fluoride, 1 mm dithiothreitol, and 1× Complete phosphatase inhibitor mixture I) in a total reaction volume of 20 μl. The reactions were collected the following day for use in the appropriate electrophoretic mobility shift assays. Electrophoretic Mobility Shift Assays—An annealed oligonucleotide probe representing a direct repeat of AGGTCA with a 4-base spacer (termed DR-4) was radiolabeled with 32P by fill-in synthesis with Klenow DNA polymerase. T3Rα was isolated from recombinant baculovirus-infected Sf9 cells (50Yoh S.M. Privalsky M.L. J. Biol. Chem. 2001; 276: 16857-16867Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). GST-SMRT-S1, GST-SMRT-S1/S2, GST-N-CoR-N1, and GST-N-CoR-N1/N2/N3 protein constructs were isolated from E. coli and incubated with or without recombinant ΔMEKK1 as described above. Electrophoretic mobility shift assays (EMSAs) were initiated by mixing the T3Rα preparation with the radiolabeled DNA probe (50,000 cpm) in binding buffer containing 10 mm Tris-Cl (pH 7.5), 2 mm MgCl2, 50 mm KCl, 2.5 mg/ml BSA, 20 μg/ml poly(dI-dC), and 1 mm dithiothreitol in a total volume of 14.5 μl. For supershift experiments, the above reactions were subsequently incubated for 15 min on ice with 5 μl of the indicated dilution of the GST-corepressor protein (either treated with ΔMEKK1 or not). The resulting DNA·protein complexes were resolved using a 5% polyacrylamide (29:1 acrylamide/bisacrylamide) gel and 0.5× 44 mm Tris base, 44 mm boric acid, and 1 mm EDTA electrophoresis system. The gels were dried, and radioactivity was visualized and quantified by PhosphorImager analysis. Fluorescence Microscopy—CV-1 cells (1.0 × 105 cells/well in a 6-well plate) were allowed to attach to 22 × 22-mm coverslips and transfected using the Effectene protocol described above. Cells were fixed 48 h after transfection in a chilled (-20°C) mixture of 50% acetone and 50% methanol for 10 m at 4°C. After aspiration of the fixing agent, cells were washed three times with PBS and incubated for 1 h at room temperature in PBS containing 2% BSA. The primary mouse anti-Myc monoclonal antibody (diluted 1:500) or a pre-absorbed control mixed with Myc-neutralizing peptide (Affinity Bioreagents, Golden, CO) was added to the coverslips in PBS containing 2% BSA and incubated for 60 min at room temperature. The coverslips were then washed three times with PBS containing 2% BSA and incubated for 1 h at room temperature with Texas Red-conjugated horse anti-mouse IgG antibody (diluted 1:1000; Vector Laboratories, Burlingame, CA) in PBS containing 2% BSA. The coverslips were washed three times with PBS containing 2% BSA and three times with PBS alone and incubated for 5 m at room temperature in PBS containing 0.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI). The coverslips were again washed three times with PBS and once with distilled water, and the excess moisture was removed by aspiration. The coverslips were mounted on slides using 25 μl of Vectashield (Vector Laboratories) and sealed with fingernail polish. The slides were visualized using a Nikon Microphot epifluorescence microscope. Digital images were captured with a Nikon Cool Pix 4500 digital camera. For quantification of the fluorescence microscopic data, 100 transfected cells were counted at random from each slide and scored for the following GFP-SMRT or GFP-N-CoR subcellular localization: nuclear, cytoplasmic, nuclear equal to cytoplasmic, or undeterminable. Phosphorylation/Dephosphorylation Assays—CV-1 cells (1.5 × 105 cells/well in a 6-well plate) were transfected with the appropriate mammalian expression vectors using the Effectene protocol described above. Cells were collected 48 h after transfection by mechanical scraping and lysed by a 30-min incubation at 4°C in 250 μl of cell extraction buffer containing 25 mm HEPES (pH 7.8), 300 mm NaCl, 1.5 mm MgCl2, 1% Triton X-100, 0.1 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, and 1× Complete protease inhibitor mixture. Lysates were clarified by centrifugation at 14,000 rpm for 30 min at 4°C, and the lysates were divided into two equal aliquots. One aliquot was treated for 30 min at 37°C with 10 units of shrimp alkaline phosphatase (Promega Corp., Madison, WI), and one aliquot was mock-treated. The samples were then resolved by SDS-PAGE using the NuPAGE Novex Tris acetate 3-8% gradient gel system. The electrophoretograms were visualized by immunoblotting using r" @default.
• W2005429042 created "2016-06-24" @default.
• W2005429042 creator A5043103834 @default.
• W2005429042 creator A5068133376 @default.
• W2005429042 date "2004-12-01" @default.
• W2005429042 modified "2023-09-27" @default.
• W2005429042 title "SMRT and N-CoR Corepressors Are Regulated by Distinct Kinase Signaling Pathways" @default.
• W2005429042 cites W1533965244 @default.
• W2005429042 cites W1588371925 @default.
• W2005429042 cites W1664472327 @default.
• W2005429042 cites W1760274621 @default.
• W2005429042 cites W1870470978 @default.
• W2005429042 cites W1968987470 @default.
• W2005429042 cites W1969097053 @default.
• W2005429042 cites W1972108825 @default.
• W2005429042 cites W1974127944 @default.
• W2005429042 cites W1978046684 @default.
• W2005429042 cites W1988565128 @default.
• W2005429042 cites W1988629919 @default.
• W2005429042 cites W1989439170 @default.
• W2005429042 cites W1996223704 @default.
• W2005429042 cites W1996890755 @default.
• W2005429042 cites W1998904806 @default.
• W2005429042 cites W2002698073 @default.
• W2005429042 cites W2012703700 @default.
• W2005429042 cites W2025175795 @default.
• W2005429042 cites W2026237709 @default.
• W2005429042 cites W2030495641 @default.
• W2005429042 cites W2035210330 @default.
• W2005429042 cites W2035915355 @default.
• W2005429042 cites W2035951027 @default.
• W2005429042 cites W2036041445 @default.
• W2005429042 cites W2038499121 @default.
• W2005429042 cites W2047809719 @default.
• W2005429042 cites W2053420149 @default.
• W2005429042 cites W2056894341 @default.
• W2005429042 cites W2057564306 @default.
• W2005429042 cites W2062599461 @default.
• W2005429042 cites W2064776404 @default.
• W2005429042 cites W2069532898 @default.
• W2005429042 cites W2072793384 @default.
• W2005429042 cites W2074687765 @default.
• W2005429042 cites W2075232957 @default.
• W2005429042 cites W2076218188 @default.
• W2005429042 cites W2079059309 @default.
• W2005429042 cites W2081509331 @default.
• W2005429042 cites W2081798747 @default.
• W2005429042 cites W2082151361 @default.
• W2005429042 cites W2087650843 @default.
• W2005429042 cites W2088086126 @default.
• W2005429042 cites W2091508315 @default.
• W2005429042 cites W2096958727 @default.
• W2005429042 cites W2097134216 @default.
• W2005429042 cites W2097724305 @default.
• W2005429042 cites W2097808377 @default.
• W2005429042 cites W2100697656 @default.
• W2005429042 cites W2107889588 @default.
• W2005429042 cites W2108984414 @default.
• W2005429042 cites W2114334107 @default.
• W2005429042 cites W2114353725 @default.
• W2005429042 cites W2115235717 @default.
• W2005429042 cites W2115446838 @default.
• W2005429042 cites W2115940794 @default.
• W2005429042 cites W2116537012 @default.
• W2005429042 cites W2117825435 @default.
• W2005429042 cites W2128003724 @default.
• W2005429042 cites W2132440338 @default.
• W2005429042 cites W2132613286 @default.
• W2005429042 cites W2133446638 @default.
• W2005429042 cites W2143658931 @default.
• W2005429042 cites W2156268135 @default.
• W2005429042 cites W2156575042 @default.
• W2005429042 cites W2162589082 @default.
• W2005429042 cites W2169457085 @default.
• W2005429042 cites W2171347864 @default.
• W2005429042 cites W2172256092 @default.
• W2005429042 cites W2172295707 @default.
• W2005429042 cites W4229958257 @default.
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