Centrosomal P4.1-associated Protein Is a New Member of Transcriptional Coactivators for Nuclear Factor-κB
2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês
10.1074/jbc.m410420200
ISSN1083-351X
AutoresMichiyo Koyanagi, Makoto Hijikata, Koichi Watashi, Osamu Masui, Kunitada Shimotohno,
Tópico(s)Plant Disease Resistance and Genetics
ResumoNuclear factor-κB (NF-κB) is a transcription factor important for various cellular events such as inflammation, immune response, proliferation, and apoptosis. In this study, we performed a yeast two-hybrid screening using the N-terminal domain of the p65 subunit (RelA) of NF-κB as bait and isolated centrosomal P4.1-associated protein (CPAP) as a candidate for a RelA-associating partner. Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments followed by Western blotting also showed association of CPAP with RelA. When overexpressed, CPAP enhanced NF-κB-dependent transcription induced by tumor necrosis factor-α (TNFα). Reduction of the protein level of endogenous CPAP by RNA interference resulted in decreased activation of NF-κB by TNFα. After treatment with TNFα, a portion of CPAP was observed to accumulate in the nucleus, although CPAP was found primarily in the cytoplasm without any stimulation. Moreover, CPAP was observed in a complex recruited to the transcriptional promoter region containing the NF-κB-binding motif. One hybrid assay showed that CPAP has the potential to activate gene expression when tethered to the transcriptional promoter. These data suggest that CPAP functions as a coactivator of NF-κB-mediated transcription. Since a physiological interaction between CPAP and the coactivator p300/CREB-binding protein was also observed and synergistic activation of NF-κB-mediated transcription was achieved by these proteins, CPAP-dependent transcriptional activation is likely to include p300/CREB-binding protein. Nuclear factor-κB (NF-κB) is a transcription factor important for various cellular events such as inflammation, immune response, proliferation, and apoptosis. In this study, we performed a yeast two-hybrid screening using the N-terminal domain of the p65 subunit (RelA) of NF-κB as bait and isolated centrosomal P4.1-associated protein (CPAP) as a candidate for a RelA-associating partner. Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments followed by Western blotting also showed association of CPAP with RelA. When overexpressed, CPAP enhanced NF-κB-dependent transcription induced by tumor necrosis factor-α (TNFα). Reduction of the protein level of endogenous CPAP by RNA interference resulted in decreased activation of NF-κB by TNFα. After treatment with TNFα, a portion of CPAP was observed to accumulate in the nucleus, although CPAP was found primarily in the cytoplasm without any stimulation. Moreover, CPAP was observed in a complex recruited to the transcriptional promoter region containing the NF-κB-binding motif. One hybrid assay showed that CPAP has the potential to activate gene expression when tethered to the transcriptional promoter. These data suggest that CPAP functions as a coactivator of NF-κB-mediated transcription. Since a physiological interaction between CPAP and the coactivator p300/CREB-binding protein was also observed and synergistic activation of NF-κB-mediated transcription was achieved by these proteins, CPAP-dependent transcriptional activation is likely to include p300/CREB-binding protein. Nuclear factor-κB (NF-κB) 1The abbreviations used are: NF-κB, nuclear factor-κB; TNFα, tumor necrosis factor-α; TAD, transactivation domain; CREB, cAMP response element-binding protein; CBP, cAMP response element-binding protein-binding protein; CPAP, centrosomal P4.1-associated protein; STAT, signal transducer and activator of transcription; GST, glutathione S-transferase; siRNA, small interfering RNA; DBD, DNA-binding domain; SRC-1, steroid receptor coactivator-1; C/H, cysteine/histidine-rich. is a Rel transcription factor that regulates the expression of a wide variety of genes involved in cellular events such as inflammation, immune response, proliferation, and apoptosis (1.Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5572) Google Scholar, 2.Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4605) Google Scholar, 3.Karin M. Cao Y. Greten F.R. Li Z.W. Nat. Rev. Cancer. 2002; 2: 301-310Crossref PubMed Scopus (2257) Google Scholar). Rel family members form hetero- and homodimers that possess distinct specificities and functions. In mammals, five Rel family members have been identified: c-Rel, RelA/p65, RelB, NF-κB1 (p50/p105), and NF-κB2 (p52/p100). In the canonical NF-κB signaling pathway, the prototypical NF-κB complex composed of p50 and RelA subunits is sequestered in the cytoplasm through its assembly with a family of NF-κB inhibitors (IκB) at steady state. When cells are stimulated by signals such as tumor necrosis factor-α (TNFα) and interleukin-1, IκB is phosphorylated by the IκB kinase complex, marking it for ubiquitination and subsequent degradation. The liberated NF-κB heterodimer rapidly translocates into the nucleus and activates target genes by binding directly to κB regulatory elements present in the target loci. Although these cytoplasmic signaling events are understood in detail, the subsequent nuclear events that regulate the strength and duration of NF-κB-mediated transcriptional activation remain poorly defined (4.Chen L.F. Greene W.C. Nat. Rev. Mol. Cell. Biol. 2004; 5: 392-401Crossref PubMed Scopus (1038) Google Scholar). RelA contains a transactivation domain (TAD) in its C-terminal region that is known to be responsible for transcriptional activation. TAD has so far been reported to interact with various transacting and basal transcription factors that recruit RNA polymerase II, including TATA-binding protein, transcription factor IIB, TAFII105 (TATA-binding protein-associated factor II105), and TLS (translocated in liposarcoma) (5.Schmitz M.L. Stelzer G. Altmann H. Meisterernst M. Baeuerle P.A. J. Biol. Chem. 1995; 270: 7219-7226Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 6.Yamit-Hezi A. Dikstein R. EMBO J. 1998; 17: 5161-5169Crossref PubMed Scopus (69) Google Scholar, 7.Yamit-Hezi A. Nir S. Wolstein O. Dikstein R. J. Biol. Chem. 2000; 275: 18180-18187Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 8.Uranishi H. Tetsuka T. Yamashita M. Asamitsu K. Shimizu M. Itoh M. Okamoto T. J. Biol. Chem. 2001; 276: 13395-13401Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In addition, general coactivators such as cAMP response element-binding protein (CREB)-binding protein (p300/CBP) (9.Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (715) Google Scholar, 10.Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar), p300/CBP-associated factor, and ACTR (coactivator for nuclear hormone receptors) are recruited to the NF-κB transcriptional complex and enhance NF-κB-mediated transcriptional activation (11.Sheppard K.A. Rose D.W. Haque Z.K. Kurokawa R. McInerney E. Westin S. Thanos D. Rosenfeld M.G. Glass C.K. Collins T. Mol. Cell. Biol. 1999; 19: 6367-6378Crossref PubMed Google Scholar, 12.Werbajh S. Nojek I. Lanz R. Costas M.A. FEBS Lett. 2000; 485: 195-199Crossref PubMed Scopus (104) Google Scholar). The N-terminal domain of RelA is also known to play important roles in the regulation of NF-κB-mediated transcriptional activation. For example, stimulus-coupled phosphorylation of RelA is known to change its transcriptional activity (4.Chen L.F. Greene W.C. Nat. Rev. Mol. Cell. Biol. 2004; 5: 392-401Crossref PubMed Scopus (1038) Google Scholar, 10.Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar, 13.Zhong H. May M.J. Jimi E. Ghosh S. Mol. Cell. 2002; 9: 625-636Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar, 14.Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, 15.Vermeulen L. De Wilde G. Van Damme P. Vanden Berghe W. Haegeman G. EMBO J. 2003; 22: 1313-1324Crossref PubMed Scopus (640) Google Scholar, 16.Duran A. Diaz-Meco M.T. Moscat J. EMBO J. 2003; 22: 3910-3918Crossref PubMed Scopus (269) Google Scholar), and two of the four serine phosphoacceptor sites present in RelA are in the N-terminal domain. In addition, association with p300/CBP has been reported to occur not only via TAD, but also through the N-terminal domain of RelA. RelA phosphorylation at Ser276 by the catalytic subunit of cAMP-dependent protein kinase (14.Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar) or mitogen- and stress-activated protein kinase-1 (15.Vermeulen L. De Wilde G. Van Damme P. Vanden Berghe W. Haegeman G. EMBO J. 2003; 22: 1313-1324Crossref PubMed Scopus (640) Google Scholar) or at Ser311 by protein kinase Cζ (16.Duran A. Diaz-Meco M.T. Moscat J. EMBO J. 2003; 22: 3910-3918Crossref PubMed Scopus (269) Google Scholar) was shown to enhance the binding of p300/CBP to RelA. Moreover, p300/CBP has also been reported to acetylate RelA at three sites in the N-terminal domain: Lys218, Lys221, and Lys310. Acetylation is thought to regulate the transcriptional activity of RelA by increasing its DNA-binding affinity for the κB enhancer or by preventing its association with IκBα (4.Chen L.F. Greene W.C. Nat. Rev. Mol. Cell. Biol. 2004; 5: 392-401Crossref PubMed Scopus (1038) Google Scholar, 17.Chen L. Fischle W. Verdin E. Greene W.C. Science. 2001; 293: 1653-1657Crossref PubMed Scopus (1049) Google Scholar, 18.Chen L.F. Mu Y. Greene W.C. EMBO J. 2002; 21: 6539-6548Crossref PubMed Scopus (635) Google Scholar, 19.Chen L.F. Greene W.C. J. Mol. Med. 2003; 81: 549-557Crossref PubMed Scopus (244) Google Scholar, 20.Kiernan R. Bres V. Ng R.W. Coudart M.P. El Messaoudi S. Sardet C. Jin D.Y. Emiliani S. Benkirane M. J. Biol. Chem. 2003; 278: 2758-2766Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). Finally, BRCA1 also associates with the N-terminal domain of RelA as well as CBP and functions as a scaffolding protein by tethering together the RelA-CBP-BRCA1 complex, thereby supporting the transacting function of CBP (21.Benezra M. Chevallier N. Morrison D.J. MacLachlan T.K. El-Deiry W.S. Licht J.D. J. Biol. Chem. 2003; 278: 26333-26341Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Thus, not only TAD, but also the N-terminal region of RelA appears to contribute to NF-κB target gene induction. However, little is known about the factors that interact with the N-terminal region. Here, we sought to clarify the mechanism of NF-κB-dependent transcriptional activation by identifying factors that interact with the N-terminal region of RelA. In a yeast two-hybrid screen using the N terminus of RelA as bait, we identified a novel RelA-interacting factor, centrosomal P4.1-associated protein (CPAP). CPAP was previously identified by virtue of its interaction with the cytoskeletal protein 4.1R-135 (22.Hung L.Y. Tang C.J. Tang T.K. Mol. Cell. Biol. 2000; 20: 7813-7825Crossref PubMed Scopus (140) Google Scholar). Although CPAP appears to be a component of the centrosomal complex, the majority of CPAP is found in soluble fractions, mainly in the cytoplasm and a small portion in the nucleus (22.Hung L.Y. Tang C.J. Tang T.K. Mol. Cell. Biol. 2000; 20: 7813-7825Crossref PubMed Scopus (140) Google Scholar, 23.Peng B. Sutherland K.D. Sum E.Y. Olayioye M. Wittlin S. Tang T.K. Lindeman G.J. Visvader J.E. Mol. Endocrinol. 2002; 16: 2019-2033Crossref PubMed Scopus (42) Google Scholar). In addition, it was previously reported that CPAP interacts with STAT5 and enhances STAT5-mediated transcription (23.Peng B. Sutherland K.D. Sum E.Y. Olayioye M. Wittlin S. Tang T.K. Lindeman G.J. Visvader J.E. Mol. Endocrinol. 2002; 16: 2019-2033Crossref PubMed Scopus (42) Google Scholar), although the mechanism by which this occurs remains unclear. In this study, we show evidence suggesting that CPAP is a novel coactivator of NF-κB that binds to the N-terminal region of RelA, possibly activating transcription through CBP. Plasmid Construction—pEFr-FLAG-CPAP was a kind gift from Dr. J. E. Visvader (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) (23.Peng B. Sutherland K.D. Sum E.Y. Olayioye M. Wittlin S. Tang T.K. Lindeman G.J. Visvader J.E. Mol. Endocrinol. 2002; 16: 2019-2033Crossref PubMed Scopus (42) Google Scholar). The cDNA fragment consisting of the entire open reading frame of CPAP was generated by PCR using a human spleen cDNA library (Clontech, Palo Alto, CA) as the template and primers 5′-CGCGGATCCATGTTCCTGATGCCAACCTC-3′ and 5′-TTTTCCTTTTGCGGCC GCATCGTCACAGCTCCGTGTCC-3′. The fragment was inserted into the BamHI-NotI sites of the pcDNA3-Myc vector (24.Watashi K. Hijikata M. Tagawa A. Doi T. Marusawa H. Shimotohno K. Mol. Cell. Biol. 2003; 23: 7498-7509Crossref PubMed Scopus (52) Google Scholar) to construct pcDNA3-Myc-CPAP and blunt-end cloned into the pCMV-FLAG vector (24.Watashi K. Hijikata M. Tagawa A. Doi T. Marusawa H. Shimotohno K. Mol. Cell. Biol. 2003; 23: 7498-7509Crossref PubMed Scopus (52) Google Scholar) to generate pCMV-CPAP. The series of plasmids encoding deletion mutants of CPAP, pcDNA3-Myc-CPAP-(1–1149), pcDNA3-Myc-CPAP-(1150–1338), and pcDNA3-Myc-CPAP-(967–1338), was constructed by inserting fragments generated by PCR using appropriate synthetic oligonucleotides as primers and pcDNA3-Myc-CPAP as the template. pcDNA3-HA-RelA (where HA is hemagglutinin), pcDNA3-HA-RelA-(1–427), pcDNA3-HA-RelA-(428–551), pcDNA3-HA-RelA-(1–312), pcDNA3-HA-RelA-(313–427), and pcDNA3-HA-RelA-(201–427) were generated in a similar manner using pcDNA3-RelA (25.Masui O. Ueda Y. Tsumura A. Koyanagi M. Hijikata M. Shimotohno K. Int. J. Mol. Med. 2002; 9: 489-493PubMed Google Scholar) as the template. pGEX-2TK-RelA was created by inserting the RelA BamHI-MfeI fragment into the BamHI-EcoRI sites of the pGEX-2TK vector (Clontech). pFastBac1-RelA was constructed by inserting the glutathione S-transferase (GST)-RelA fragment of pGEX-2TK-RelA into the BamHI-XbaI sites of the pFastBac1 vector (Invitrogen). pGEX-CPAP-C was created by inserting the EcoRI-NotI fragment of pcDNA3-Myc-CPAP-(967–1338) into the EcoRI-NotI sites of the pGEX-6P-1 vector (Clontech). pM-CPAP was generated by inserting the BamHI-XbaI fragment, which was PCR-amplified from pcDNA3-Myc-CPAP using primers 5′-CGCGGATCCCAATGTTCCTGATGCCAACCTC-3′ and 5′-GCTCTAGAATCGTCACAGCTCCGTGTCC-3′, into the BamHI-XbaI sites of the pM vector (Clontech). pM-CPAP-(967–1338) was obtained by inserting the EcoRI-XbaI fragment of pcDNA3-Myc-CPAP-(967–1338) into the EcoRI-XbaI sites of the pM vector. pCMV-CBP was a kind gift from Dr. I. Talianidis (Institute of Molecular Biology and Biotechnology, Crete, Greece) (26.Soutoglou E. Katrakili N. Talianidis I. Mol. Cell. 2000; 5: 745-751Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). The expression plasmids for a series of CBP deletion mutants (CBP1–CBP5) were kindly provided by Dr. A. Fukamizu (Center for Tsukuba Advanced Research Alliance, Tsukuba, Japan) (27.Yoshida E. Aratani S. Itou H. Miyagishi M. Takiguchi M. Osumu T. Murakami K. Fukamizu A. Biochem. Biophys. Res. Commun. 1997; 241: 664-669Crossref PubMed Scopus (94) Google Scholar). The reporter plasmids pNF-κB-luc and pFR-luc were obtained from Stratagene. The construction of the reporter plasmid pNF-κB-mt-luc was described previously (28.Marusawa H. Hijikata M. Chiba T. Shimotohno K. J. Virol. 1999; 73: 4713-4720Crossref PubMed Google Scholar). Yeast Two-hybrid Screening—The DNA fragment encoding amino acids 1–427 of RelA was subcloned into the pHybLex-Zeo vector (Invitrogen). This plasmid was used as a bait construct to screen a human leukemia cDNA library (Clontech) according to the manufacturer's instructions (Invitrogen). A total of 1.6 × 106 transformants were selected based on histidine prototrophy and β-galactosidase activity. GST Pull-down Assay—GST and the GST-RelA fusion protein, encoded by pFastBac1 and pFastBac1-RelA, respectively, were produced in Sf9 cells using the Bac-to-Bac baculovirus expression system (Invitrogen). GST and the GST-CPAP-(967–1338) fusion protein, encoded by pGEX-6P-1 and pGEX-CPAP-(967–1338), respectively, were produced in BL21 cells (Amersham Biosciences) exposed to 0.1 mm isopropyl β-d-thiogalactopyranoside. After affinity separation of the proteins from cell lysates using glutathione-Sepharose (Amersham Biosciences), proteins bound to the resin were mixed and incubated with in vitro transcription/translation products at 4 °C for 2 h. The in vitro transcription/translation product was prepared with the TnT T7 quick coupled transcription/translation system (Promega) using 0.25 μg of each expression plasmid in the presence of l-[35S]methionine (Amersham Biosciences) according to the manufacturer's instructions. After being washed five times in binding buffer (50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride), resin-bound radiolabeled proteins were fractionated by SDS-PAGE and detected by autoradiography. Cell Culture and Transfection—293T and MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (Nissui) supplemented with 10% fetal bovine serum and l-glutamine. Transfection of plasmids into cells was performed with FuGENE 6 transfection reagent (Roche) according to the manufacturer's recommendations. Immunoprecipitation—Cells were lysed in immunoprecipitation buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, and 0.1% Nonidet P-40). After centrifugation, the supernatant was incubated with anti-FLAG antibody M2 (Sigma), anti-RelA antibody F-6 (Santa Cruz Biotechnology, Inc.), anti-c-Myc antibody 9E10 (Santa Cruz Biotechnology, Inc.), or normal mouse IgG (Zymed Laboratories Inc.) for at least 1 h. Immunocomplexes were recovered by adsorption to protein G-Sepharose (Amersham Biosciences). After being washed five times in immunoprecipitation buffer, the immunoprecipitates were analyzed by immunoblotting. Immunoblot Analysis—Immunoblot analysis was performed essentially as described previously (29.Watashi K. Hijikata M. Marusawa H. Doi T. Shimotohno K. Virology. 2001; 286: 391-402Crossref PubMed Scopus (28) Google Scholar). The antibodies used in these experiments were specific for FLAG, RelA (antibody F-6), and α-tubulin (antibody-1, Calbiochem). The rabbit antiserum against CPAP was kindly provided from Dr. T. K. Tang (Institute of Biomedical Sciences, Taipei, Taiwan, Republic of China) (22.Hung L.Y. Tang C.J. Tang T.K. Mol. Cell. Biol. 2000; 20: 7813-7825Crossref PubMed Scopus (140) Google Scholar). Reporter Assay—Cell extracts were prepared in reporter lysis buffer (Promega) 48 h after transfection. After removal of cell debris, the luciferase activity in the extracts was measured with a luciferase assay kit (Promega) and a Berthold Lumat LB 9507 luminometer according to the manufacturers' instructions. RNA Interference Technique—A 21-nucleotide small interfering RNA (siRNA) duplex (5′-AAUGGAAUGCACGUGACGAUG-3′) containing 3′-dTdT overhanging sequences was synthesized (Qiagen Inc.). siRNA transfection was performed using Oligofectamine reagent (Invitrogen) according to the manufacturer's instructions. RNA Isolation and Reverse Transcription-PCR—Total RNA was isolated from cultured cells using Sepasol RNA I Super (Nacalai Tesque, Kyoto, Japan) according to the manufacturer's instructions. The relative expression of each mRNA was evaluated by semiquantitative reverse transcription-PCR using a One-Step RNA PCR kit (Takara). Glyceraldehyde-3-phosphate dehydrogenase mRNA was used as an internal control. The primers used were as follows: CPAP, 5′-AAAGGGACCACAGGTAGCGG-3′ and 5′-TGAATTCACTCGCACGATCTGGGATG; interferon-β, 5′-CACGACAGCTCTTTCCATGA-3′ and 5′-AGCCAGTGCTCGATGAATCT-3′; and TNF receptor-associated factor-1, 5′-GCCCCTGATGAGAATGAGTT-3′ and 5′-CTCATGCTCTTGCACAGACT-3′. Indirect Immunofluorescence Analysis—Indirect immunofluorescence analysis was performed as described previously (29.Watashi K. Hijikata M. Marusawa H. Doi T. Shimotohno K. Virology. 2001; 286: 391-402Crossref PubMed Scopus (28) Google Scholar). Cells were permeabilized with 0.05% Triton X-100 after fixation and treated with anti-RelA primary antibody F-6 or rabbit antiserum against CPAP (22.Hung L.Y. Tang C.J. Tang T.K. Mol. Cell. Biol. 2000; 20: 7813-7825Crossref PubMed Scopus (140) Google Scholar). Secondary antibodies conjugated to Alexa 488 and Alexa 568 (Molecular Probes, Inc.) were used to visualize primary antibody distribution. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma). DNA-Protein Complex Immunoprecipitation Assay—293T cells treated with 10 nm TNFα were transfected with plasmids. After cross-linking with 1% formaldehyde for 15 min, cells were lysed; sonicated; and subjected to immunoprecipitation using anti-FLAG or anti-RelA antibody or normal mouse IgG. Recovered immunocomplexes were incubated at 65 °C for 16 h and then digested with proteinase K for 2 h. DNA was extracted from the immunocomplexes with phenol and precipitated with ethanol. The primers used for detection of recovered DNA were 5′-ACCGAAACGCGCGAGGCAGGATCAGCCATA-3′ and 5′-GCTCTCCAGCGGTTCCATC-3′ for pNF-κB-luc and 5′-CTAGCAAAATAGGCTGTCCC-3′ and 5′-CTTTATGTTTTTGGCGTATTCCA-3′ for pNF-κB-mt-luc. Identification of CPAP as a Factor That Interacts with RelA—To identify cellular factors that interact with the N-terminal region of RelA, a yeast two-hybrid screen was performed using a human leukemia cDNA library as bait and the N-terminal 427-amino acid region of RelA as prey. From 1.6 × 106 L40 yeast transformants, 64 clones were obtained that appeared to interact with RelA. Among them, three independent clones were revealed to encode portions of CPAP. To confirm the interaction of CPAP with RelA, we performed an immunoprecipitation assay using 293T cells exogenously producing FLAG-tagged CPAP. FLAG-tagged CPAP was detected in cell lysates in the immunocomplex formed with anti-RelA antibody (Fig. 1A, left panels, lane 3), but not with normal IgG (lane 2). The interaction between FLAG-tagged CPAP and endogenous RelA was seen without considerable alteration both before and after treatment with TNFα (Fig. 1A, upper and lower left panels, respectively). This seemed to imply that TNFα-induced phosphorylation of RelA is not essential for the interaction with CPAP. Actually, we found that FLAG-tagged CPAP was co-immunoprecipitated with a RelA mutant in which one of the TNFα-induced phosphorylation target sites (Ser276) was replaced with alanine (data not shown). This may support the above idea. This interaction was also seen in a GST pull-down assay. Under conditions in which in vitro translated CPAP was not pulled down with GST-bound Sepharose beads (Fig. 1B, lane 2), we found it in a pellet with recombinant GST-RelA-bound Sepharose beads (lane 3). These results suggest that CPAP interacts specifically with RelA. In addition, to examine the region of CPAP responsible for interaction with RelA, we performed a GST pull-down assay as described above using several deletion mutants of CPAP. The in vitro synthesized fragments of CPAP spanning amino acids 1150–1338 and 967–1338, but not amino acids 1–1149, were co-purified with GST-RelA (Fig. 1B). This indicates that the region of CPAP responsible for interaction with RelA resides within amino acids 1150–1338, including a series of 21 nonamer repeats (G-box region). This result was also obtained with the immunoprecipitation assay. In the lysates of 293T cells producing RelA and Myc-tagged CPAP-C (C-terminal amino acids 967–1338 of CPAP), exogenous RelA was efficiently detected in immunocomplexes formed with anti-Myc antibody (Fig. 1A, right panel, lane 6), but not with normal mouse IgG (lane 5). The region of RelA that interacts with CPAP was similarly assessed. The in vitro synthesized fragments of RelA spanning amino acids 1–427 and 201–427, but not amino acids 428–551, 1–312, or 313–427, were co-purified with GST-CPAP-C (Fig. 1C). These results indicate that the central region of RelA is necessary and sufficient for interaction with CPAP. CPAP Augments NF-κB-dependent Gene Expression—Because CPAP has been reported to activate STAT5-mediated transcription (23.Peng B. Sutherland K.D. Sum E.Y. Olayioye M. Wittlin S. Tang T.K. Lindeman G.J. Visvader J.E. Mol. Endocrinol. 2002; 16: 2019-2033Crossref PubMed Scopus (42) Google Scholar), we examined the effect of this protein on RelA-mediated transcription using a reporter assay. When CPAP was ectopically expressed, NF-κB-responsive reporter gene expression was enhanced by up to 2–3-fold in a dose-dependent manner (Fig. 2A, upper panel). In contrast, reporter activity from the plasmid containing mutated NF-κB-binding sites in the promoter region was not affected by ectopically expressed CPAP (Fig. 2A, lower panel). These data suggest that CPAP can specifically up-regulate NF-κB-mediated transcription. To investigate the contribution of endogenous CPAP to transcriptional activation, we examined the effect of CPAP siRNA, which was designed to specifically knock down the expression of CPAP, on NF-κB-dependent transcriptional activation in MCF-7 breast cancer-derived cells. We confirmed that endogenous CPAP protein levels were significantly reduced by transfection with CPAP siRNA, whereas the levels of other cellular proteins such as α-tubulin were not changed (Fig. 2B). The level of NF-κB-mediated transcription induced by either TNFα or RelA in CPAP siRNA-treated cells was decreased to <50% of that in cells transfected with control siRNA (Fig. 2C, upper panel). In contrast, reporter activity from the plasmid containing mutated NF-κB-binding sites was not affected by knocking down CPAP (Fig. 2C, lower panel). These findings indicate that endogenous CPAP is required for full activation of NF-κB-dependent reporter gene expression. Next, we examined whether CPAP affects expression of endogenous target genes. After treatment with TNFα, total RNA was isolated from MCF-7 cells transfected with either control or CPAP siRNA and analyzed by reverse transcription-PCR to detect the mRNA levels of interferon-β and TNF receptor-associated factor-1, which are known to be induced by NF-κB. As shown in Fig. 2D, transfection with CPAP siRNA, but not control siRNA, down-regulated TNFα-induced expression of interferon-β and TNF receptor-associated factor-1 mRNAs. These results indicate that CPAP plays an important role in NF-κB-mediated transcriptional activation in cells. Translocation of CPAP into the Nucleus upon TNFα Treatment—RelA is translocated from the cytoplasm into the nucleus upon stimulation by specific cytokines. To determine whether the localization of CPAP is similarly altered by activation of the NF-κB pathway, we examined the subcellular localization of CPAP in MCF-7 cells by indirect immunofluorescence analysis with or without TNFα treatment. As reported previously (23.Peng B. Sutherland K.D. Sum E.Y. Olayioye M. Wittlin S. Tang T.K. Lindeman G.J. Visvader J.E. Mol. Endocrinol. 2002; 16: 2019-2033Crossref PubMed Scopus (42) Google Scholar), CPAP was found to localize primarily in the cytoplasm, although some protein was also detectable in the nucleus without stimulation (Fig. 3a). As CPAP was immunoprecipitated with RelA from the cytoplasmic fraction of such cells (data not shown), it seemed likely that a cytoplasmic complex is present before TNFα treatment. However, following TNFα treatment for 20 min, a portion of CPAP was observed to accumulate in the nucleus (Fig. 3e), similar to RelA (b and f). These results suggest that at least a portion of cytoplasmic CPAP enters the nucleus in a TNFα-dependent manner. Recruitment of CPAP to the NF-κB-binding Motif—The increase in NF-κB-dependent transcriptional activation by CPAP, the nuclear accumulation of CPAP in response to TNFα stimulation, and the physical interaction of CPAP with RelA all suggested the possibility that CPAP, together with RelA, is recruited to the transcriptional promoters of NF-κB target genes. To examine this possibility, we performed a DNA-protein complex immunoprecipitation assay. As shown in Fig. 4 (upper panel, lanes 1–3), a DNA fragment containing an NF-κB-binding motif was detected by PCR in complexes specifically immunoprecipitated by either anti-FLAG or RelA antibodies from lysates of 293T cells transfected with pNF-κB-luc, FLAG-tagged CPAP, and RelA expression plasmids. In contrast, no DNA fragment was amplified from cells transfected with pNF-κB-mt-luc instead of pNF-κB-luc (Fig. 4, lower panel, lanes 2 and 3). These data suggest that CPAP is recruited to the transcriptional promoter region containing an NF-κB-binding motif via association with RelA. CPAP Can Activate Gene Expression When Tethered to a Transcriptional Promoter—We showed above that CPAP interacted with RelA, up-regulated NF-κB-mediated transcription, and formed part of the complex binding to a transcriptional promoter containing NF-κB-binding motifs. These data suggest that CPAP acts as a transcriptional coactivator of NF-κB. We examined this possibility using a one-hybrid assay system with fusion proteins consisting of the Gal4 DNA-binding domain (DBD) and full-length CPAP or its C-terminal region in mammalian cells. As demonstrated in Fig. 5 (second bar), Gal4 DBD-fused CPAP up-regulated luciferase expression from pFR-luc, a reporter plasmid containing a Gal4-responsive transcriptional promoter. In contrast, CPAP by itself had no effect on the same promoter (Fig. 5, third bar). No difference in luciferase levels was observed among the exogenous Gal4 DBD-containing constructs (data not shown). This suggests that CPAP has a transactivation capacity when tethered to the transcriptional promoter. This activity is lik
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