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• W2016194944 abstract "The hepatitis B virus X protein induces transcriptional activation of a wide variety of viral and cellular genes. In addition to its ability to interact directly with many nuclear transcription factors, several reports indicate that the X protein stimulates different cytoplasmic kinase signal cascades. Using the yeast two-hybrid screen, we have isolated a clone designated X-associated protein 3 (XAP3) that encodes a human homolog of the rat protein kinase C-binding protein. One of the activation domains of X (amino acids 90–122) is required for binding to XAP3, while the NH2-terminal part of XAP3 is necessary for binding to X. Both X and XAP3 bound specifically to the η PKC isoenzyme synthesized in rabbit reticulocyte lysates. Overexpression of XAP3 enhanced X transactivation activity. These results support earlier findings that one of the mechanisms of transactivation by X is through involvement with the cellular protein kinase C pathway. The hepatitis B virus X protein induces transcriptional activation of a wide variety of viral and cellular genes. In addition to its ability to interact directly with many nuclear transcription factors, several reports indicate that the X protein stimulates different cytoplasmic kinase signal cascades. Using the yeast two-hybrid screen, we have isolated a clone designated X-associated protein 3 (XAP3) that encodes a human homolog of the rat protein kinase C-binding protein. One of the activation domains of X (amino acids 90–122) is required for binding to XAP3, while the NH2-terminal part of XAP3 is necessary for binding to X. Both X and XAP3 bound specifically to the η PKC isoenzyme synthesized in rabbit reticulocyte lysates. Overexpression of XAP3 enhanced X transactivation activity. These results support earlier findings that one of the mechanisms of transactivation by X is through involvement with the cellular protein kinase C pathway. Transcriptional activation is a widespread phenomenon among mammalian viral systems. Mammalian viral proteins that increase the rate of transcription can be divided into two groups based on whether they exhibit sequence-specific DNA binding. For example, the herpes simplex virus 1 (HSV-1) 1The abbreviations used are: HSV-1, herpes simplex virus 1; HBV, hepatitis B virus; CAT, chloramphenicol acetyltransferase; PKC, protein kinase C; HTLV-1, human T-cell lymphotropic virus type 1; DBD, DNA-binding domain; ONPG,o-nitrophenyl-β-d-galactoside; DAG, diacylglycerol; GST, glutathione S-transferase. Vmw175 (1Faber S.W. Wilcox K.W. Nucleic Acids Res. 1986; 14: 6067-6083Crossref PubMed Scopus (100) Google Scholar, 2Michael N. Spector D. Mavromara-Nazos P. Kristie T.M. Roizman B. Science. 1988; 239: 1531-1534Crossref PubMed Scopus (76) Google Scholar), the Epstein-Barr virus BZLF1 (3Farrell P.J. Rowe D.T. Rooney C.M. Kouzarides T. EMBO J. 1989; 8: 127-132Crossref PubMed Scopus (241) Google Scholar), the papilloma virus E2 (4Spalholz B.A. Byrne J.C. Howley P.M. J. Virol. 1988; 62: 3143-3150Crossref PubMed Google Scholar), and the simian virus 40 and polyoma virus large T antigens (5Fried M. Prives C. Cancer Cells. 1986; 4: 1-16Google Scholar) bind specific DNA sequence motifs, whereas HSV-1 VP16 (6Kristie T.M. Roizman B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4065-4069Crossref PubMed Scopus (55) Google Scholar, 7McKnight J.L.C. Kristie T.M. Roizman B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7061-7065Crossref PubMed Scopus (134) Google Scholar), the pseudorabies virus immediate early protein (8Abmayr S.M. Feldman L.D. Roeder R.G. Cell. 1985; 43: 821-829Abstract Full Text PDF PubMed Scopus (19) Google Scholar, 9Abmayr S.M. Workman J.L. Roeder R.G. Genes Dev. 1988; 2: 542-553Crossref PubMed Scopus (99) Google Scholar), and the adenovirus E1A protein (10Ferguson B. Krippl B. Andrisani O. Jones N. Westphal H. Rosenberg M. Mol. Cell. Biol. 1985; 5: 2653-2661Crossref PubMed Scopus (86) Google Scholar, 11Chatterjee P.K. Bruner M. Flint S.J. Harter M.L. EMBO J. 1988; 7: 835-841Crossref PubMed Scopus (43) Google Scholar) do not. During the last decade, many studies have shown that non-DNA binding viral transactivators achieve their task by direct interaction with different cellular sequence-specific DNA binding transcription factors. For example, VP16 interacts with the Oct-1 protein, thereby positioning the VP16 activating domain at a promoter to enhance transcription (7McKnight J.L.C. Kristie T.M. Roizman B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7061-7065Crossref PubMed Scopus (134) Google Scholar, 12Gerster T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6347-6351Crossref PubMed Scopus (234) Google Scholar, 13O'Hare P. Goding C.R. Cell. 1988; 52: 435-445Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 14O'Hare P. Goding C.R. Haigh A. EMBO J. 1988; 7: 4231-4238Crossref PubMed Scopus (95) Google Scholar, 15Preston C.M. Frame M.C. Campbell M.E.M. Cell. 1988; 52: 425-434Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 16Kristie T.M. LeBowitz J.H. Sharp P.A. EMBO J. 1989; 8: 4229-4238Crossref PubMed Scopus (164) Google Scholar, 17Stern S. Tanaka M. Herr W. Nature. 1989; 341: 624-630Crossref PubMed Scopus (293) Google Scholar). Similarly, E1A interacts with a number of cellular proteins including ATF-2 (18Liu F. Green M.R. Nature. 1994; 368: 520-525Crossref PubMed Scopus (224) Google Scholar). The hepatitis B virus (HBV) X protein is a promiscuous transcriptional transactivator (reviewed in Ref. 19Rossner M.T. J. Med. Virol. 1992; 36: 101-117Crossref PubMed Scopus (191) Google Scholar). This conclusion is derived from a large number of studies using mostly transient cotransfection of the bacterial chloramphenicol acetyltransferase (CAT) gene under control of a potential target promoter/enhancer and the X gene under the control of a heterologous promoter in mammalian cells. Induction of transcription by X usually ranges from 2- to 20-fold depending on the target promoter and cell type; whether this transactivation activity contributes to viral function, however, remains to be determined. Fusion of the X protein to the DNA-binding domain of the bacterial LexA repressor resulted in a protein that can activate transcription from a reporter plasmid bearing lexA operator sequences fused to a minimal promoter (20Seto E. Mitchell P.J. Yen T.S.B. Nature. 1990; 344: 72-74Crossref PubMed Scopus (213) Google Scholar). Similarly, fusion of the X protein to the DNA-binding domain of transcription factor C/EBP increased the ability of X to activate a reporter containing C/EBP binding sites (21Unger T. Shaul Y. EMBO J. 1990; 9: 1889-1895Crossref PubMed Scopus (77) Google Scholar). Attempts to demonstrate sequence-specific DNA binding by the X protein so far have not been successful, and, therefore, it is believed that the X protein belongs to the non-DNA binding viral transactivator family, which is brought into a transcription complex by association with cellular DNA-binding factors. So far, a number of transcription factors, including ATF-2 (22Maguire H.F. Hoeffler J.P. Siddiqui A. Science. 1991; 252: 842-844Crossref PubMed Scopus (379) Google Scholar), CREB (22Maguire H.F. Hoeffler J.P. Siddiqui A. Science. 1991; 252: 842-844Crossref PubMed Scopus (379) Google Scholar, 23Williams J.S. Andrisani O.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3819-3823Crossref PubMed Scopus (147) Google Scholar), RPB5 (24Cheong J. Yi M. Lin Y. Murakami S. EMBO J. 1995; 14: 143-150Crossref PubMed Scopus (241) Google Scholar), TATA-binding protein (25Qadri I. Maguire H.F. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1003-1007Crossref PubMed Scopus (185) Google Scholar), and p53 (26Feitelson M.A. Zhu M. Duan L.X. London W.T. Oncogene. 1993; 8: 1109-1117PubMed Google Scholar), have been shown to interact with the X protein. However, unlike most viral activators, X appears to operate through additional mechanisms. For example, X appears to function as a serine protease inhibitor and regulates the turnover of different cellular transcription factors (27Takada S. Kido H. Fukutomi A. Mori T. Koike K. Oncogene. 1994; 9: 341-348PubMed Google Scholar). Additionally, increasing evidence suggests that X may use signal transduction pathways to activate transcription. In this regard, both the protein kinase C (PKC) signaling pathway and mitogen-activated kinases have been shown to be involved in X-mediated transactivation (28Cross J.C. Wen P. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8078-8082Crossref PubMed Scopus (146) Google Scholar, 29Kekulé A.S. Lauer U. Weiss L. Luber B. Hofschneider P.H. Nature. 1993; 361: 742-745Crossref PubMed Scopus (335) Google Scholar, 30Lucito R. Schneider R.J. J. Virol. 1992; 66: 983-991Crossref PubMed Google Scholar, 31Benn J. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10350-10354Crossref PubMed Scopus (404) Google Scholar, 32Doria M. Klein N. Lucito R. Schneider R.J. EMBO J. 1995; 14: 4747-4757Crossref PubMed Scopus (275) Google Scholar, 33Benn J. Su F. Doria M. Schneider R.J. J. Virol. 1996; 70: 4978-4985Crossref PubMed Google Scholar, 34Wang H.-D. Yuh C.-H. Dang C.V. Johnson D. Mol. Cell. Biol. 1995; 15: 6720-6728Crossref PubMed Scopus (59) Google Scholar). To understand the mechanism of X transactivation, we and others have previously used the yeast two-hybrid screen to identify cellular proteins that interact with X (35Lee T.H. Elledge S.J. Butel J.S. J. Virol. 1995; 69: 1107-1114Crossref PubMed Google Scholar, 36Kuzhandaivelu N. Cong Y.-S. Inouye C. Yang W.-M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (96) Google Scholar). Among the clones analyzed were genes encoding a human homolog of a DNA repair protein (XAP1) (35Lee T.H. Elledge S.J. Butel J.S. J. Virol. 1995; 69: 1107-1114Crossref PubMed Google Scholar) and a cellular inhibitor of X (XAP2) (36Kuzhandaivelu N. Cong Y.-S. Inouye C. Yang W.-M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (96) Google Scholar). We have now sequenced and characterized an additional clone from our two-hybrid screen for encoded proteins capable of binding X. One protein, term XAP3, appears to have strong homology to a rat PKC-binding protein. PKC is a large family of phospholipid-dependent kinases involved in cell growth, differentiation, and carcinogenesis (37Housey G.M. Johnson M.D. Hsiao W.L.W. O'Brian C.A. Murphy J.P. Kirschmeier P. Weinstein I.B. Cell. 1988; 52: 343-354Abstract Full Text PDF PubMed Scopus (424) Google Scholar, 38Stabel S. Semin. Cancer Biol. 1994; 5: 277-284PubMed Google Scholar, 39Nishizuka Y. J. Am. Med. Assoc. 1989; 262: 1826-1833Crossref PubMed Scopus (355) Google Scholar, 40Jaken S. Curr. Opin. Cell Biol. 1990; 2: 192-197Crossref PubMed Scopus (53) Google Scholar, 41Glazer R.I. Kuo J.F. Protein Kinase C. Oxford University Press, New York1994: 171-198Google Scholar). The mammalian PKC enzyme family consists of at least 10 members that are divided into three groups based on enzymatic properties and common structural features (reviewed in Refs. 42Kikkawa U. Kishimoto A. Nishizuka Y. Annu. Rev. Biochem. 1989; 58: 31-44Crossref PubMed Scopus (588) Google Scholar and 43Parker P.J. Kuo J.F. Protein Kinase C. Oxford University Press, New York1994: 3-15Google Scholar). The group A (cPKC) (α, βI, βII, and γ) are calcium-dependent kinases whose activities are stimulated by diacylglycerol (DAG) or phorbol esters. The group B (nPKC) (δ, ε, η, and θ) and group C (aPKC) (ζ and λ) are different from those of group A in their regulatory domains in that a putative Ca2+-binding region is absent. The group C PKCs are different from groups A and B in that they possess a single cysteine-rich Zn2+ finger motif in the conserved C1 region. The group B PKCs are calcium-independent but can be stimulated by DAG, whereas the group C PKCs are neither calcium- nor DAG-dependent. A number of proteins have previously been identified that associate with PKC (44Chapline C. Ramsay K. Klauck T. Jaken S. J. Biol. Chem. 1993; 268: 6858-6861Abstract Full Text PDF PubMed Google Scholar, 45Dong L. Chapline C. Mousseau B. Fowler L. Ramsay K. Stevens J.L. Jaken S. J. Biol. Chem. 1995; 270: 25534-25540Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 46Mochly-Rosen D. Khaner H. Lopez J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3997-4000Crossref PubMed Scopus (442) Google Scholar, 47Mochly-Rosen D. Khaner H. Lopez J. Smith B.L. J. Biol. Chem. 1991; 266: 14866-14868Abstract Full Text PDF PubMed Google Scholar, 48Wolf M. Sayhoun N. J. Biol. Chem. 1986; 261: 13327-13332Abstract Full Text PDF PubMed Google Scholar). Some of these PKC-binding proteins serve as substrates for PKC, while interaction of the various PKC isozymes with other cellular proteins may confer the unique localization of each of the PKC enzymes. Recently, it was demonstrated that the human T-cell lymphotropic virus type 1 (HTLV-1) transactivator Tax stimulates PKC translocation to the particulate cellular membrane fraction, suggesting that Tax activates PKC in vivo(49Lindholm P.F. Tamami M. Makowski J. Brady J.N. J. Virol. 1996; 70: 2525-2532Crossref PubMed Google Scholar). Further, Tax bound specifically to the α, δ, and η PKC isoenzymes, and the addition of Tax to in vitro kinase reaction mixtures led to the phosphorylation of Tax and an increase in the autophosphorylation of PKC. In this report, we demonstrate that X binds XAP3 in vivo and in vitro. Second, we found that X and XAP3 can both interact with PKCη. Finally, we show that an overexpression of XAP3 enhances X transactivation. These results strongly suggest the involvement of PKC as one of the mechanisms in X transactivation. The following plasmids have been described previously. pGST-X (36Kuzhandaivelu N. Cong Y.-S. Inouye C. Yang W.-M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (96) Google Scholar) contains the entire HBV-X ORF subcloned into the pGSTag vector (50Ron D. Dressler H. Biotechniques. 1992; 13: 866-869PubMed Google Scholar) in-frame with the glutathioneS-transferase (GST) polypeptide. pAS-X (36Kuzhandaivelu N. Cong Y.-S. Inouye C. Yang W.-M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (96) Google Scholar) contains the HBV (subtype adw2; Ref. 51Valenzuela P. Quiroga M. Zaldivar J. Gray W. Rutter W.J. Fields B.N. Jaenisch R. Fox C.F. Animal Virus Genetics. Academic Press, New York1980: 57-70Crossref Google Scholar) fragment from nucleotide 1375 to 1853 (NcoI-AflIII) in the Gal4 DNA-binding domain (DBD)-tagged plasmid, pAS1 (52Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1300) Google Scholar). This plasmid expresses a fusion protein containing the Gal4DBD and the full-length wild type X protein. pSP72-PKCα, pSP72-PKCη, and pSP72-PKCδ (49Lindholm P.F. Tamami M. Makowski J. Brady J.N. J. Virol. 1996; 70: 2525-2532Crossref PubMed Google Scholar) contain the different PKC isoform cDNAs under the control of T7 or SP6 phage promoters. pECE-X (53Seto E. Yen T.S.B. Peterlin B.M. Ou J.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8286-8290Crossref PubMed Scopus (150) Google Scholar) contains the HBV fragment from nucleotide 1355 to 1987 in the SV40-derived expression vector pECE (54Ellis L Clauser E. Morgan D.O. Edery M. Roth R.A. Rutter W.J. Cell. 1986; 45: 721-732Abstract Full Text PDF PubMed Scopus (697) Google Scholar). Reporter plasmids pRSVCAT (55Gorman C.M. Merlino G.T. Willingham M.C. Pastan I. Howard B.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6777-6781Crossref PubMed Scopus (881) Google Scholar) and pSV2CAT (56Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar) contain the Rous sarcoma virus and simian virus 40 promoter/enhancers upstream of the CAT gene. Plasmids pGEM-XAP3 was constructed by subcloning an XhoI fragment from the X4 clone into the XhoI site of a modified pGEM7Zf plasmid (pGEM7Zf-3X; Ref. 57Yang W.-M. Inouye C. Zeng Y. Bearss D. Seto E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12845-12850Crossref PubMed Scopus (484) Google Scholar) such that XAP3 mRNA is expressed from a T7 promoter. Different XAP3 mutants were generated by restriction enzyme digestion and religation of the pGEM-XAP3 plasmid. pGEM-TBP1 was constructed by inserting the TAT-binding protein 1 cDNA (58Nelbock P. Dillon P.J. Perkins A. Rosen C.A. Science. 1990; 248: 1650-1653Crossref PubMed Scopus (197) Google Scholar) into the pGEM7Zf vector (Promega). Different X deletion mutants in pAS1 vector were constructed by first subcloning the X coding sequence (a BglII fragment from pECE-X) into theBamHI site of pGEM7Zf-3X, then using different restriction enzymes to subdivide the X coding region, and finally subcloning each individual X mutant into the NcoI/BamHI site of pAS1. pGST-XAP3 was constructed by subcloning an XAP3 cDNA fragment from pGEM-XAP3 into pGSTag. pCMV-XAP3 was constructed by cloning anXhoI fragment from clone X4 into the SalI site of pcDNAI/Amp (Invitrogen). All plasmid constructions were verified by dideoxy sequencing (59Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52749) Google Scholar). To generate 35S-labeled XAP3, TBP-1, PKCα, PKCη, or PKCδ proteins; pGEM-XAP3, pGEM-TBP1, pSP72PKCα, pSP72PKCη, or pSP72PKCδ were transcribed and translated with T7 or SP6 RNA polymerase and [35S]methionine in a transcription/translation coupled system (Promega). Bacterially expressed GST, GST-X, or GST-XAP3 fusion proteins were purified according to Frangioni et al. (60Frangioni J.V. Neel B.G. Anal. Biochem. 1993; 210 (2nd Ed.): 179-187Crossref PubMed Scopus (833) Google Scholar). Briefly, DH5α cells harboring either the pGSTag, pGST-X, or pGST-XAP3 plasmids were grown to log phase and induced with isopropyl-1-thio-β-d-galactopyranoside for 4 h. After sonication in STE buffer (10 mm Tris-HCl (pH 8), 150 mm NaCl, 1 mm EDTA, and 5 mmdithiothreitol) containing 1% sarcosyl (w/v, final concentration), solubilized proteins were recovered by centrifugation and incubated with glutathione-agarose beads in the presence of 3% Triton X-100 (final concentration) for 30 min at 4 °C, and washed several times with ice-cold phosphate-buffered saline. For binding assays, beads were mixed with in vitrotranslated, 35S-labeled proteins for 1 h at room temperature. Unbounded proteins were washed extensively with STE buffer containing 0.1% Nonidet P-40, and bound proteins were eluted from the beads by boiling in SDS loading buffer (50 mm Tris-HCl (pH 6.8), 100 mm dithiothreitol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol). Final products were analyzed on a 12.5% SDS-polyacrylamide gel and detected by autoradiography. To determine XAP3 interacting domains within X, library-derived X4 plasmid was transformed into Y153 alone or Y153 containing either pAS-X or pAS-X mutants. Transformants were assayed for the presence of β-galactosidase activity. X4, a positive clone from a two-hybrid screen that showed specific interaction with X but not to other unrelated proteins were subcloned into pGEM7Zf-3X, and nucleotide sequences of the subcloned cDNA were obtained using dideoxy sequencing. To obtain a full-length XAP3 cDNA, a HeLa λgt11 cDNA library (CLONTECH) was screened with a32P-labeled, random-primed 368-base pair probe (XhoI/BglII fragment from clone X4) using standard protocols (61Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Inserts from positive clones were subcloned into pGEM7Zf-3X, and sequences were determined. The final sequence of the XAP3 cDNA was determined from both DNA strands. Homology searches for DNA and deduced amino acid sequences were performed at the National Center for Biotechnology Information with the BLAST network service. Filter lift assays were performed essentially as described (62Breeden L. Nasmyth K. Cold Spring Harbor Symp. Quant. Biol. 1985; 50: 643-650Crossref PubMed Scopus (470) Google Scholar). Briefly, transformants were allowed to grow at 30 °C for 2–4 days, transferred onto nitrocellulose filters, and frozen under liquid nitrogen. Filters were then placed on Whatman no. 3MM paper presoaked with 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside solution, incubated at 30 °C, and checked periodically for production of blue color. For quantitation of β-galactosidase activity in yeast, liquid culture assays were done usingo-nitrophenyl-β-d-galactoside (ONPG) as described (63Guarente L. Methods Enzymol. 1983; 101: 181-191Crossref PubMed Scopus (874) Google Scholar). Individual transformants were inoculated into the appropriate medium and incubated at 30 °C until the cultures reached mid-log phase (A 600 of 1.0). For each culture, 0.1 ml of culture was mixed with 0.9 ml of Z buffer (60 mmNa2HPO4, 40 mmNaH2PO4, 10 mm KCl, 1 mm MgSO4, and 50 mmβ-mercaptoethanol). The cells were permeabilized with 0.05 ml of CHCl3 and 0.05 ml of 0.1% sodium dodecyl sulfate (SDS). After addition of 0.2 ml of ONPG solution (4 mg/ml ONPG in 0.1m phosphate buffer, pH 7.0), reactions were incubated at 30 °C for 1 h and quenched by the addition of 0.5 ml of 1m Na2CO3. β-Galactosidase activities were determined by measuring absorbances at 420 nm. Standard protocols were followed (64Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). For each sample, yeast transformants were grown at 30 °C in 1 ml of selective SC medium containing 2% dextrose to anA 600 of 1–2. Cells were collected by centrifugation, and lysates were prepared according to a standard protocol (61Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Briefly, cells were washed in ice-cold 50 mmTris-HCl (pH 8.0) and resuspended in lysis buffer (50 mmTris-HCl (pH 8.0), 0.1% Triton X-100, and 0.5% SDS) containing 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, and 1 μg/ml leupeptin. After addition of acid-washed glass beads (Sigma), samples were vortexed and cell extract recovered. One-tenth of each sample was resolved on a 12.5% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membrane. After blocking with nonfat dried milk, the membrane was treated with 1:1000 diluted Gal4-DNA-binding domain polyclonal antiserum (Santa Cruz Biotechnology) followed by 1:7500 diluted alkaline phosphatase-conjugated rabbit anti-mouse IgG. Subsequently, the blot was developed by 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. Total RNA was purified using the acid guanidinium thiocyanate-phenol-chloroform extraction method (65Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63228) Google Scholar), separated on a formaldehyde agarose gel, and transferred onto a Hybond membrane (Amersham). Multiple human tissue Northern blot was obtained from CLONTECH. Prehybridization, hybridization, and high stringency washes were performed as described (61Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). To control for the relative amount of RNA in each lane, after hybridization with XAP3, the blots were stripped by incubation in 0.5% SDS at 95 °C, and reprobed with the human β-actin cDNA. HepG2 cells were grown in minimal essential medium supplemented with 10% fetal bovine serum. Transfections were done using the calcium phosphate method (66Graham F. van der Eb A. Virology. 1973; 52: 456-457Crossref PubMed Scopus (6498) Google Scholar), which included a 1-min glycerol shock 4 h after the addition of DNA precipitate. Forty-eight h after transfection, cells were harvested and CAT activity was determined (56Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar) in 30-min reactions. All transfections were normalized to equal amounts of DNA with pECE or pcDNAI/Amp plasmids. Previously, we have used the yeast two-hybrid screen to detect human cDNAs coding for products that interact with the X protein (36Kuzhandaivelu N. Cong Y.-S. Inouye C. Yang W.-M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (96) Google Scholar). Of 5.6 × 106 colonies screened, 12 of the clones were active only when cotransformed with plasmid encoding Gal4DBD fused to X, but not to Rb, SNF1, or several unrelated transcription factors (36Kuzhandaivelu N. Cong Y.-S. Inouye C. Yang W.-M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (96) Google Scholar). Preliminary DNA sequence analysis revealed seven novel sequences and five that were highly homologous to known sequences. Of the seven novel sequences, six of them were overlapping clones of cDNA encoding XAP2 (36Kuzhandaivelu N. Cong Y.-S. Inouye C. Yang W.-M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (96) Google Scholar). Of the five known sequences, we have now completely sequenced one cDNA insert designated X4 (we will refer to the product encoded by X4 as X-associated protein-3 (XAP3)). To determine whether XAP3 binds X in vitro, we expressed the X protein as a fusion to GST and used it to test for its ability to bind specifically toin vitro 35S-labeled XAP3. As shown in Fig.1, XAP3 binds to GST-X but not GST (lanes 4and 6). An unrelated protein, TBP-1, was used as an additional negative control; and, as expected, TBP-1 did not bind GST-X in this assay (lane 7). Taken together, this suggests that X and XAP3 interact both in vivo and in vitro and the interaction is specific. The X protein sequences required for binding XAP3 were examined to determine whether they coincided with previously defined activation or regulatory domains. A series of carboxyl-terminal X deletion mutants, as well as internal deletion mutants previously generated and subcloned into pAS1 were tested for their abilities to interact with XAP3 in the two-hybrid system. As shown in Fig. 2 A, deletion of the X protein carboxyl-terminal from amino acids 122–154 had no effect on β-galactosidase activities, as measured by filter lift or liquid assays. However, deletion of amino acids 109–154 eliminated β-galactosidase activities. Further amino-terminal and internal deletions revealed that amino acids 90–122 of X were important for binding XAP3. Western blot analysis indicated that mutants which did not bind XAP3 were expressed efficiently in yeast cells (Fig. 2 B), indicating that the loss of binding property of these mutants is not a reflection of loss in protein expressions. The predicted amino acid sequence of XAP3 was determined by theoretical translation of the cDNA clone open reading frame. Since the predicted open reading frame of the X4 cDNA clone isolated from the two-hybrid screen remains open at the 5′ side (beginning at nucleotide 559, Fig. 3 A), we initially suspected that this clone represented only a partial XAP3 coding sequence. To obtain a full-length XAP3 cDNA, a λgt11 HeLa cDNA library was screened with a radiolabeled probe corresponding to the 5′ end of clone X4. The complete DNA sequence of the newly isolated clone is illustrated in Fig. 3 A. Analysis of the predicted amino acid sequence of XAP3 revealed an open reading frame of 468 amino acids (1.4 kilobases) with an in-frame stop codon upstream of the first methionine at nucleotide 540 (Fig. 3 A). This indicated that the original X4 clone did, in fact, contain a full-length XAP3 coding region. Sequence motif searches indicate that XAP3 contains a C3HC4 zinc finger (amino acids 240–284) and three PKC substrate recognition sites (amino acids 32–34, amino acids 41–43, and amino acids 116–119). As shown in Fig. 3 B, sequence homology searches revealed that XAP3 shares 76% identity and 85% similarity to a rat PKC-binding protein. 2S. Kuroda, C. Tokunaga, Y. Kiyohara, H. Konishi, and U. Kikkawa, unpublished results (NCBI accession numberU48248). In attempt to uncover the functional significance of X and XAP3 interaction, Northern blot analysis was performed to determine the expression pattern of XAP3. We found that a message of approximately 2.5–3.0 kilobases was present in high levels in human brain, placenta, and pancreas; intermediate levels in the heart and skeletal muscle; and low levels in the lung, liver, and kidney (Fig. 4). To localize a domain within XAP3 that binds the X protein, we in vitro synthesized different35S-labeled XAP3 mutants and used them to test for binding to GST-X. As shown in Fig. 5, carboxyl-terminal deletion of amino acids 212–468 or internal deletions of amino acids 97–262 or 123–347 had little or no effect on the binding of XAP3 to the X protein (lanes 3–10, 12, and 13). However, deletion of amino acids 13–74 completely eliminated XAP3's ability to bind X (lane 11). Taken together, these data suggest that the amino-terminal part of the XAP3 protein (amino acids 13–74 in particular) is essential for binding to the X protein. This finding is consistent with the observation that amino acids 13 to 74 is included in the original X4 clone derived from the initial two-hybrid screen. GST binding assays were performed to analyze X protein binding to 35S-labeled PKC isoenzymes translated in vitro with rabbit reticulocyte lysates. Equal quantities of bacterial expressed GST or GST-X fusion protein coupled to glutathione-Sepharose beads were incubated with PKCα, PKCη, or PKCδ. Interestingly, PKCη but not PKCα or PKCδ bound specifically to GST-X but not to GST (Fig.6, lanes 4–9). To determine which PKC isoenzyme XAP3 binds, similar experiments were performed using GST-XAP3 fusion protein. Intriguingly, like the X protein, XAP3 also" @default.
• W2016194944 created "2016-06-24" @default.
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• W2016194944 title "The Hepatitis B Virus X-associated Protein, XAP3, Is a Protein Kinase C-binding Protein" @default.
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• W2016194944 doi "https://doi.org/10.1074/jbc.272.26.16482" @default.
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