In previous studies we have found that oncogenic (Val 12)-ras-p21 induces Xenopus laevis oocyte maturation that is selectively blocked by two ras-p21 peptides, 35−47, also called PNC-7, that blocks its interaction with raf, and 96−110, also called PNC-2, that blocks its interaction with jun-N-terminal kinase (JNK). Each peptide blocks activation of both JNK and MAP kinase (MAPK or ERK) suggesting interaction between the raf−MEK−ERK and JNK−jun pathways. We further found that dominant negative raf blocks JNK induction of oocyte maturation, again suggesting cross-talk between pathways. In this study, we have undertaken to determine where these points of cross-talk occur. First, we have immunoprecipitated injected Val 12-Ha-ras-p21 from oocytes and found that a complex forms between ras-p21 raf, MEK, MAPK, and JNK. Co-injection of either peptide, but not a control peptide, causes diminished binding of ras-p21, raf, and JNK. Thus, one site of interaction is cooperative binding of Val 12-ras-p21 to raf and JNK. Second, we have injected JNK, c-raf, and MEK into oocytes alone and in the presence of raf and MEK inhibitors and found that JNK activation is independent of the raf−MEK−MAPK pathway but that activated JNK activates raf, allowing for activation of ERK. Furthermore, we have found that constitutively activated MEK activates JNK. We have corroborated these findings in studies with isolated protein components from a human astrocyte (U-251) cell line; that is, JNK phosphorylates raf but not the reverse; MEK phosphorylates JNK but not the reverse. We further have found that JNK does not phosphorylate MAPK and that MAPK does not phosphorylate JNK. The stress-inducing agent, anisomycin, causes activation of JNK, raf, MEK, and ERK in this cell line; activation of JNK is not inhibitable by the MEK inhibitor, U0126, while activation of raf, MEK, and ERK are blocked by this agent. These results suggest that activated JNK can, in turn, activate not only jun but also raf that, in turn, activates MEK that can then cross-activate JNK in a positive feedback loop.
To expand our understanding of the role of Jak2 in cellular signaling, we used the yeast two-hybrid system to identify Jak2-interacting proteins. One of the clones identified represents a human homologue of the Schizosaccaromyces pombe Shk1 kinase-binding protein 1, Skb1, and the protein encoded by theSaccharomyces cerevisiae HSL7 (histone synthetic lethal 7) gene. Since no functional motifs or biochemical activities for this protein or its homologues had been reported, we sought to determine a biochemical function for this human protein. We demonstrate that this protein is a protein methyltransferase. This protein, designated JBP1 (Jak-binding protein 1), and its homologues contain motifs conserved among protein methyltransferases. JBP1 can be cross-linked to radiolabeled S-adenosylmethionine (AdoMet) and methylates histones (H2A and H4) and myelin basic protein. Mutants containing substitutions within a conserved region likely to be involved in AdoMet binding exhibit little or no activity. We mapped the JBP1 gene to chromosome 14q11.2–21. In addition, JBP1 co-immunoprecipitates with several other proteins, which serve as methyl group acceptors and which may represent physiological targets of this methyltransferase. Messenger RNA for JBP1 is widely expressed in human tissues. We have also identified and sequenced a homologue of JBP1 in Drosophila melanogaster. This report provides a clue to the biochemical function for this conserved protein and suggests that protein methyltransferases may have a role in cellular signaling.
Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.
Interleukin-10 (IL-10)-related T cell-derived inducible factor (IL-TIF; provisionally designated IL-22) is a cytokine with limited homology to IL-10. We report here the identification of a functional IL-TIF receptor complex that consists of two receptor chains, the orphan CRF2-9 and IL-10R2, the second chain of the IL-10 receptor complex. Expression of the CRF2-9 chain in monkey COS cells renders them sensitive to IL-TIF. However, in hamster cells both chains, CRF2-9 and IL-10R2, must be expressed to assemble the functional IL-TIF receptor complex. The CRF2-9 chain (or the IL-TIF-R1 chain) is responsible for Stat recruitment. Substitution of the CRF2-9 intracellular domain with the IFN-γR1 intracellular domain changes the pattern of IL-TIF-induced Stat activation. The CRF2-9 gene is expressed in normal liver and kidney, suggesting a possible role for IL-TIF in regulating gene expression in these tissues. Each chain, CRF2-9 and IL-10R2, is capable of binding IL-TIF independently and can be cross-linked to the radiolabeled IL-TIF. However, binding of IL-TIF to the receptor complex is greater than binding to either receptor chain alone. Sharing of the common IL-10R2 chain between the IL-10 and IL-TIF receptor complexes is the first such case for receptor complexes with chains belonging to the class II cytokine receptor family, establishing a novel paradigm for IL-10-related ligands similar to the shared use of the gamma common chain (γc) by several cytokines, including IL-2, IL-4, IL-7, IL-9, and IL-15. Interleukin-10 (IL-10)-related T cell-derived inducible factor (IL-TIF; provisionally designated IL-22) is a cytokine with limited homology to IL-10. We report here the identification of a functional IL-TIF receptor complex that consists of two receptor chains, the orphan CRF2-9 and IL-10R2, the second chain of the IL-10 receptor complex. Expression of the CRF2-9 chain in monkey COS cells renders them sensitive to IL-TIF. However, in hamster cells both chains, CRF2-9 and IL-10R2, must be expressed to assemble the functional IL-TIF receptor complex. The CRF2-9 chain (or the IL-TIF-R1 chain) is responsible for Stat recruitment. Substitution of the CRF2-9 intracellular domain with the IFN-γR1 intracellular domain changes the pattern of IL-TIF-induced Stat activation. The CRF2-9 gene is expressed in normal liver and kidney, suggesting a possible role for IL-TIF in regulating gene expression in these tissues. Each chain, CRF2-9 and IL-10R2, is capable of binding IL-TIF independently and can be cross-linked to the radiolabeled IL-TIF. However, binding of IL-TIF to the receptor complex is greater than binding to either receptor chain alone. Sharing of the common IL-10R2 chain between the IL-10 and IL-TIF receptor complexes is the first such case for receptor complexes with chains belonging to the class II cytokine receptor family, establishing a novel paradigm for IL-10-related ligands similar to the shared use of the gamma common chain (γc) by several cytokines, including IL-2, IL-4, IL-7, IL-9, and IL-15. interleukin-10 signal transducers and activators of transcription peripheral blood mononuclear cells lipopolysaccharide IL-10-related T cell-derived inducible factor interferon polymerase chain reaction major histocompatibility complex electrophoretic mobility shift assay polyacrylamide gel electrophoresis peptide:N-glycosidase F cytomegalovirus-encoded IL-10 Six new ligands with limited sequence homology (19–27% identity) to IL-101 have been recently identified (1Dumoutier L. Louahed J. Renauld J.C. J. Immunol. 2000; 164: 1814-1819Crossref PubMed Scopus (443) Google Scholar, 2Gallagher G. Dickensheets H. Eskdale J. Izotova L.S. Mirochnitchenko O.V. Peat J.D. Pestka S. Vazquez N. Donnelly R.P. Kotenko S.V. Genes Immun. 2000; 1: 442-450Crossref PubMed Scopus (261) Google Scholar, 3Jiang H. Lin J.J. Su Z.Z. Goldstein N.I. Fisher P.B. Oncogene. 1995; 11: 2477-2486PubMed Google Scholar, 4Knappe A. Hor S. Wittmann S. Fickenscher H. J. Virol. 2000; 74: 3881-3887Crossref PubMed Scopus (194) Google Scholar, 5Kotenko S.V. Saccani S. Izotova L.S. Mirochnitchenko O.V. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1695-1700Crossref PubMed Scopus (408) Google Scholar). One of these IL-10 homologs is a viral protein, whereas others are encoded in the genome. Cytomegalovirus-encoded IL-10, designated cmvIL-10 (5Kotenko S.V. Saccani S. Izotova L.S. Mirochnitchenko O.V. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1695-1700Crossref PubMed Scopus (408) Google Scholar), demonstrates only 27% identity to human IL-10. Despite this limited homology, cmvIL-10 binds to and signals through the canonical IL-10 receptor complex (5Kotenko S.V. Saccani S. Izotova L.S. Mirochnitchenko O.V. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1695-1700Crossref PubMed Scopus (408) Google Scholar, 6Kotenko S.V. Krause C.D. Izotova L.S. Pollack B.P. Wu W. Pestka S. EMBO J. 1997; 16: 5894-5903Crossref PubMed Scopus (333) Google Scholar). cmvIL-10 is produced by cytomegalovirus-infected cells and is likely to play a role in immune evasion helping virus to avoid clearance by the host immune system (5Kotenko S.V. Saccani S. Izotova L.S. Mirochnitchenko O.V. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1695-1700Crossref PubMed Scopus (408) Google Scholar, 7Lockridge K.M. Zhou S.S. Kravitz R.H. Johnson J.L. Sawai E.T. Blewett E.L. Barry P.A. Virology. 2000; 268: 272-280Crossref PubMed Scopus (140) Google Scholar). Another IL-10 homolog was cloned as a protein whose expression is elevated in terminally differentiated human melanoma cells and was designated mda-7, for melanoma differentiation-associated gene 7 (3Jiang H. Lin J.J. Su Z.Z. Goldstein N.I. Fisher P.B. Oncogene. 1995; 11: 2477-2486PubMed Google Scholar). The expression of the rat mda-7 analog was linked to wound healing (8Soo C. Shaw W.W. Freymiller E. Longaker M.T. Bertolami C.N. Chiu R. Tieu A. Ting K. J. Cell. Biochem. 1999; 74: 1-10Crossref PubMed Scopus (67) Google Scholar) (the protein was designated c49a) and to ras transformation (9Zhang R. Tan Z. Liang P. J. Biol. Chem. 2000; 275: 24436-24443Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) (the protein was designated mob-5). The expression of rat mda-7 (c49a) was localized primarily to fibroblast-like cells at the wound edge and base. During wound healing the level of c49a mRNA was transiently elevated 9- to 12-fold above unwounded controls (8Soo C. Shaw W.W. Freymiller E. Longaker M.T. Bertolami C.N. Chiu R. Tieu A. Ting K. J. Cell. Biochem. 1999; 74: 1-10Crossref PubMed Scopus (67) Google Scholar). In addition, expression of rat mda-7 (mob-5) was demonstrated to be induced by expression of oncogenicras. Moreover, mob-5 and its putative receptor are oncogenicras-specific targets; mob-5 binds to the cell surface ofras-transformed cells but not of parental untransformed cells (9Zhang R. Tan Z. Liang P. J. Biol. Chem. 2000; 275: 24436-24443Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Another IL-10 homolog, designated ak155, was cloned as a protein expressed by Herpesvirus saimiri-transformed T lymphocytes (4Knappe A. Hor S. Wittmann S. Fickenscher H. J. Virol. 2000; 74: 3881-3887Crossref PubMed Scopus (194) Google Scholar). Transcription of the gene of a fourth IL-10 homolog, designated IL-19, was demonstrated to be induced in monocytes by LPS treatment. The appearance of IL-19 mRNA in LPS-stimulated monocytes coincided with the expression of IL-10 mRNA (2Gallagher G. Dickensheets H. Eskdale J. Izotova L.S. Mirochnitchenko O.V. Peat J.D. Pestka S. Vazquez N. Donnelly R.P. Kotenko S.V. Genes Immun. 2000; 1: 442-450Crossref PubMed Scopus (261) Google Scholar). An additional protein with homology to IL-10 was designated Zcyto10 (GenBank™ accession number AF224266), but there is no published information available about its activities or expression. Finally, an IL-10 homolog, designated IL-TIF (IL-10-related T cell-derived inducible factor), is expressed by IL-9-treated murine T cells (1Dumoutier L. Louahed J. Renauld J.C. J. Immunol. 2000; 164: 1814-1819Crossref PubMed Scopus (443) Google Scholar). Its human analog (human IL-TIF or, provisionally, IL-22) was recently reported (10Dumoutier L. Van Roost E. Colau D. Renauld J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10144-10149Crossref PubMed Scopus (324) Google Scholar, 11Xie M.H. Aggarwal S. Ho W.H. Foster J. Zhang Z. Stinson J. Wood W.I. Goddard A.D. Gurney A.L. J. Biol. Chem. 2000; 275: 31335-31339Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Murine IL-TIF expression can be induced by IL-9 in thymic lymphomas, T cells, and mast cells in vitroand by LPS in various organs in vivo. It was also demonstrated that IL-TIF injection induced production of acute-phase reactants in mouse liver, suggesting involvement of IL-TIF in the inflammatory response (10Dumoutier L. Van Roost E. Colau D. Renauld J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10144-10149Crossref PubMed Scopus (324) Google Scholar). IL-TIF can induce activation of Stat proteins (Stat1, Stat3, and Stat5) in several cell lines, including mesangial MES13, neuronal PC12, and hepatoma HepG2 cell lines (1Dumoutier L. Louahed J. Renauld J.C. J. Immunol. 2000; 164: 1814-1819Crossref PubMed Scopus (443) Google Scholar, 10Dumoutier L. Van Roost E. Colau D. Renauld J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10144-10149Crossref PubMed Scopus (324) Google Scholar). In addition, there are data linking IL-TIF to allergy and asthma. IL-TIF is induced by IL-9, a Th2 cytokine active on T and B lymphocytes, mast cells, and eosinophils, and potentially involved in allergy and asthma (12Temann U.A. Geba G.P. Rankin J.A. Flavell R.A. J. Exp. Med. 1998; 188: 1307-1320Crossref PubMed Scopus (408) Google Scholar, 13Levitt R.C. McLane M.P. MacDonald D. Ferrante V. Weiss C. Zhou T. Holroyd K.J. Nicolaides N.C. J. Allergy Clin. Immunol. 1999; 103: S485-S491Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 14McLane M.P. Haczku A. van de Rijn M. Weiss C. Ferrante V. MacDonald D. Renauld J.C. Nicolaides N.C. Holroyd K.J. Levitt R.C. Am. J. Respir. Cell Mol. Biol. 1998; 19: 713-720Crossref PubMed Scopus (168) Google Scholar). The IL-TIF gene (and also the ak155 gene) is located on human chromosome 12q, where several loci potentially linked to asthma and atopy have been identified by genetic studies, particularly in the 12q13.12-q23.3 region (for review see Ref. 15Cookson W. Nature. 2000; 402s (11.): B5-9BGoogle Scholar). The strongest evidence for linkage is in a region near the gene encoding IFN-γ (16Barnes K.C. Freidhoff L.R. Nickel R. Chiu Y.F. Juo S.H. Hizawa N. Naidu R.P. Ehrlich E. Duffy D.L. Schou C. Levett P.N. Marsh D.G. Beaty T.H. J. Allergy Clin. Immunol. 1999; 104: 485-491Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 17Barnes K.C. Neely J.D. Duffy D.L. Freidhoff L.R. Breazeale D.R. Schou C. Naidu R.P. Levett P.N. Renault B. Kucherlapati R. Iozzino S. Ehrlich E. Beaty T.H. Marsh D.G. Genomics. 1996; 37: 41-50Crossref PubMed Scopus (215) Google Scholar, 18Nickel R. Wahn U. Hizawa N. Maestri N. Duffy D.L. Barnes K.C. Beyer K. Forster J. Bergmann R. Zepp F. Wahn V. Marsh D.G. Genomics. 1997; 46: 159-162Crossref PubMed Scopus (108) Google Scholar, 19Wilkinson J. Thomas N.S. Morton N. Holgate S.T. Int. Arch. Allergy Immunol. 1999; 118: 265-267Crossref PubMed Scopus (14) Google Scholar). However, the gene for IFN-γ appears to be highly conserved (no sequence variations were detected in 265 individuals), suggesting that mutations of the IFN-γ gene are unlikely to be a significant cause of inherited asthma (20Hayden C. Pereira E. Rye P. Palmer L. Gibson N. Palenque M. Hagel I. Lynch N. Goldblatt J. Lesouëf P. Clin. Exp. Allergy. 1997; 27: 1412-1416PubMed Google Scholar). The IL-TIF and ak155 genes are positioned next to the IFN-γ gene on the bacterial artificial chromosome BAC RPCI11–444B24 (GenBank™ accession numberAC007458) and, thus, are possible candidates for linkage to asthma. Cytokines exert their actions by binding to specific cell surface receptors that leads to the activation of cytokine-specific signal transduction pathways. The functional IL-10 receptor complex consists of two chains (6Kotenko S.V. Krause C.D. Izotova L.S. Pollack B.P. Wu W. Pestka S. EMBO J. 1997; 16: 5894-5903Crossref PubMed Scopus (333) Google Scholar), the ligand binding IL-10R1 subunit (21Liu Y. Wei S.H. Ho A.S. de Waal M. Moore K.W. J. Immunol. 1994; 152: 1821-1829PubMed Google Scholar) and the second IL-10R2 subunit that supports signaling through the IL-10R1 chain (6Kotenko S.V. Krause C.D. Izotova L.S. Pollack B.P. Wu W. Pestka S. EMBO J. 1997; 16: 5894-5903Crossref PubMed Scopus (333) Google Scholar). Both chains belong to the class II cytokine receptor family (22Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1879) Google Scholar, 23Thoreau E. Petridou B. Kelly P.A. Djiane J. Mornon J.P. FEBS Lett. 1991; 282: 26-31Crossref PubMed Scopus (129) Google Scholar), which also includes two receptor chains for type I interferons (IFNs), two receptor chains for type II IFN, and the tissue factor that binds coagulation factor VIIa (for review see Ref. 24Kotenko S.V. Pestka S. Oncogene. 2000; 19: 2557-2565Crossref PubMed Scopus (186) Google Scholar). In addition, there are currently at least five orphan receptors CRF2-8, CRF2-9, CRF2-10, CRF2-11, and CRF2-12 (cytokinereceptor family class II members) and the extracellular domains of CRF2-8, CRF2-9, and CRF2-10 are mostly homologous to the IL-10R1 extracellular domain (24Kotenko S.V. Pestka S. Oncogene. 2000; 19: 2557-2565Crossref PubMed Scopus (186) Google Scholar). 2S. V. Kotenko and S. Pestka, unpublished data. In this study we demonstrate that the functional IL-TIF receptor complex consists of two receptor chains, the orphan CRF2-9 chain and the IL-10R2 chain, which we demonstrate to be a common shared chain between the IL-TIF and the IL-10 receptor complexes. Primers 5′-CCGGTACCAATGGCCGCCCTGCAGAAATCTG-3′ and 5′-GCGAATTCAAATGCAGGCATTTCTCAG-3′ (tif1) and total RNA isolated from PBMCs obtained from a healthy donor were used for reverse transcription-PCR to clone the human IL-TIF cDNA into plasmid pcDEF3 (25Goldman L.A. Cutrone E.C. Kotenko S.V. Krause C.D. Langer J.A. BioTechniques. 1996; 21: 1013-1015Crossref PubMed Scopus (152) Google Scholar) with the use of KpnI andEcoRI restriction endonucleases, resulting in plasmid pEF-IL-TIF. The PCR product obtained with primers 5′-CCGGATCCACAGGGAGGAGCAGCTGCGCCC-3′ (tif2) and tif1 and plasmid pEF-IL-TIF as a template was digested with BamHI andEcoRI restriction endonucleases and cloned into corresponding sites of the pEF-SPFL vector (5Kotenko S.V. Saccani S. Izotova L.S. Mirochnitchenko O.V. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1695-1700Crossref PubMed Scopus (408) Google Scholar), resulting in plasmid pEF-SPFL-IL-TIF. This plasmid encodes IL-TIF tagged at its N terminus with the FLAG epitope (FL-IL-TIF). The PCR product obtained with primers tif2 and 5′-CCGAATTCATGCGACTGACGCTCGTCGAATGCAGGCATTTCTCAGAGAC-3′ and plasmid pEF-IL-TIF as a template was digested with BamHI andEcoRI restriction endonucleases and cloned into corresponding sites of the pEF-SPFL vector, resulting in plasmid pEF-SPFL-IL-TIF-P. This plasmid encodes FL-IL-TIF tagged at its C terminus with the Arg-Arg-Ala-Ser-Val-Ala sequence (FL-IL-TIF-P), which contains the consensus amino acid sequence recognizable by the catalytic subunit of the cAMP-dependent protein kinase (26Kemp B.E. Graves D.J. Benjamini E. Krebs E.G. J. Biol. Chem. 1977; 252: 4888-4894Abstract Full Text PDF PubMed Google Scholar, 27Edelman A.M. Blumenthal D.K. Krebs E.G. Annu. Rev. Biochem. 1987; 56: 567-613Crossref PubMed Scopus (1018) Google Scholar, 28Li B.L. Langer J.A. Schwartz B. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 558-562Crossref PubMed Scopus (62) Google Scholar, 29Pestka S. Lin L. Wu W. Izotova L. Protein Expr. Purif. 1999; 17: 203-214Crossref PubMed Scopus (10) Google Scholar). Primers 5′-CCGGTACCGATGAGGACGCTGCTGACCATC-3′ and 5′-GGCGCTAGCAAGGTCCATGTCCGGTCTGGCAGTG-3′ and a library containing cDNA isolated from human fetal liver (CLONTECH, catalog no. HL4029AH) were used for PCR to clone the extracellular domain of the CRF2-9 protein (24Kotenko S.V. Pestka S. Oncogene. 2000; 19: 2557-2565Crossref PubMed Scopus (186) Google Scholar) into plasmid pEF3-IL-10R1/γR1 with the use of KpnI and NheI restriction endonucleases, resulting in plasmid pEF-CRF2-9/γR1. Primers 5′-GGCGCTAGCCTCCGGAGCCTTCCTGTTCTCCATG-3′ and 5′-CCGAATTCAGGACTCCCACTGCCAGTCAG-3′ and the same library were used for PCR to clone the CRF2-9 intracellular domain into plasmid pEF-CRF2-9/γR1 with the use of NheI and EcoRI restriction endonucleases, resulting in plasmid pEF-CRF2-9. A “tandem vector” encoding two receptors, the CRF2-9/γR1 and the IL-10R2 chains, in which the expression of each receptor is controlled by separate promoters and polyadenylation signals was created as follows. The fragment containing the EF-1α promoter, the IL-10R2 coding sequence, and the bovine growth hormone polyadenylation signal was released from the pEF-CRF (or pEF-IL-10R2) vector (6Kotenko S.V. Krause C.D. Izotova L.S. Pollack B.P. Wu W. Pestka S. EMBO J. 1997; 16: 5894-5903Crossref PubMed Scopus (333) Google Scholar) by digestion with BsaI and BssHII restriction endonucleases and ligated into the BsaI and MluI sites of the pEF-CRF2-9/γR1 plasmid. The resulting plasmid was designated pEF-CRF2-9/γR1+IL-10R2. The nucleotide sequences of the modified regions of all constructs were verified in their entirety by DNA sequencing. The 16-9 hamster x human somatic cell hybrid line is the Chinese hamster ovary cell (CHO-K1) hybrid containing a translocation of the long arm of human chromosome 6 encoding the human IFNGR1 (Hu-IFN-γR1) gene and a transfected human HLA-B7 gene (30Soh J. Donnelly R.J. Mariano T.M. Cook J.R. Schwartz B. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8737-8741Crossref PubMed Scopus (53) Google Scholar). The cells were maintained in F-12 (Ham) medium (Sigma) containing 5% heat-inactivated fetal bovine serum (Sigma). COS-1 cells, an SV40-transformed fibroblast-like simian CV-1 cell line, were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% heat-inactivated fetal bovine serum. Cells were transfected as described previously (5Kotenko S.V. Saccani S. Izotova L.S. Mirochnitchenko O.V. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1695-1700Crossref PubMed Scopus (408) Google Scholar, 6Kotenko S.V. Krause C.D. Izotova L.S. Pollack B.P. Wu W. Pestka S. EMBO J. 1997; 16: 5894-5903Crossref PubMed Scopus (333) Google Scholar) except that stable COS cell transfectants were selected with 350 μg/ml G418. COS cell supernatants were collected at 72 h as a source of the expressed proteins. Leukocytes were obtained from a normal donor by leukapheresis. Peripheral blood mononuclear cells (PBMCs) were then isolated by density centrifugation with polysucrose and sodium diatrizoate according to the manufacturer's suggested protocol (Sigma, HISTOPAQUE-1077). To detect cytokine-induced MHC class I antigen (HLA-B7) expression, cells were treated with COS cell supernatants or purified recombinant proteins as indicated in the text for 72 h and analyzed by flow cytometry. Cell surface expression of the HLA-B7 antigen was detected by treatment with mouse anti-HLA (W6/32) (31Barnstable C.J. Bodmer W.F. Brown G. Galfre G. Milstein C. Williams A.F. Ziegler A. Cell. 1978; 14: 9-20Abstract Full Text PDF PubMed Scopus (1598) Google Scholar) monoclonal antibody followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology Inc., catalog no. SC-2010). The cells then were analyzed by cytofluorography as described previously (32Kotenko S.V. Izotova L.S. Mirochnitchenko O.V. Lee C. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5007-5012Crossref PubMed Scopus (44) Google Scholar). Cells were starved overnight in serum-free media and then treated with IL-10 or IL-TIF as indicated in the text for 15 min at 37 °C and used for EMSA experiments to detect activation of Stat1, Stat3, and Stat5 as described (6Kotenko S.V. Krause C.D. Izotova L.S. Pollack B.P. Wu W. Pestka S. EMBO J. 1997; 16: 5894-5903Crossref PubMed Scopus (333) Google Scholar). EMSAs were performed with a 22-base pair sequence containing a Stat1α-binding site corresponding to the GAS (IFN-γ activation sequence) element in the promoter region of the human IRF-1 gene (5′-GATCGATTTCCCCGAAATCATG-3′) as described (33Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 34Kotenko S.V. Izotova L.S. Pollack B.P. Muthukumaran G. Paukku K. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1996; 271: 17174-17182Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Three days after transfection, conditioned media from COS-1 cells transiently transfected with expression plasmids was collected and subjected to Western blotting with anti-FLAG epitope-specific M2 monoclonal antibody (Sigma) as described (5Kotenko S.V. Saccani S. Izotova L.S. Mirochnitchenko O.V. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1695-1700Crossref PubMed Scopus (408) Google Scholar). Northern blotting was performed with two blots (CLONTECH, catalog nos. 7757-1 and 7780-1) and a CRF2-9 probe corresponding to the coding region of the CRF2-9 cDNA as described (2Gallagher G. Dickensheets H. Eskdale J. Izotova L.S. Mirochnitchenko O.V. Peat J.D. Pestka S. Vazquez N. Donnelly R.P. Kotenko S.V. Genes Immun. 2000; 1: 442-450Crossref PubMed Scopus (261) Google Scholar). The RNA loading was adjusted by the manufacturer with a β-actin signal. The FL-IL-TIF-P protein was transiently expressed in COS cells and purified from conditioned media by immunoaffinity chromatography with the anti-FLAG M1 gel (Sigma) according to the manufacturer's suggested protocols. FL-IL-TIF-P was labeled with [32P]ATP and used for cross-linking as described (28Li B.L. Langer J.A. Schwartz B. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 558-562Crossref PubMed Scopus (62) Google Scholar, 29Pestka S. Lin L. Wu W. Izotova L. Protein Expr. Purif. 1999; 17: 203-214Crossref PubMed Scopus (10) Google Scholar, 33Kotenko S.V. Izotova L.S. Pollack B.P. Mariano T.M. Donnelly R.J. Muthukumaran G. Cook J.R. Garotta G. Silvennoinen O. Ihle J.N. Pestka S. J. Biol. Chem. 1995; 270: 20915-20921Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The following ligands and receptors and their derivatives were created and used in this study. Human IL-TIF (IL-21) (GenBank™ accession no.AJ277247) is a cytokine with limited homology to IL-10 (10Dumoutier L. Van Roost E. Colau D. Renauld J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10144-10149Crossref PubMed Scopus (324) Google Scholar, 11Xie M.H. Aggarwal S. Ho W.H. Foster J. Zhang Z. Stinson J. Wood W.I. Goddard A.D. Gurney A.L. J. Biol. Chem. 2000; 275: 31335-31339Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Three expression vectors were created (Fig.1 A) encoding intact human IL-TIF, N-terminal FLAG-tagged IL-TIF (FL-IL-TIF), or FL-IL-TIF with the consensus amino acid sequence Arg-Arg-Ala-Ser-Val-Ala (phosphorylatable site, P), recognizable by the catalytic subunit of the cAMP-dependent protein kinase (26Kemp B.E. Graves D.J. Benjamini E. Krebs E.G. J. Biol. Chem. 1977; 252: 4888-4894Abstract Full Text PDF PubMed Google Scholar, 27Edelman A.M. Blumenthal D.K. Krebs E.G. Annu. Rev. Biochem. 1987; 56: 567-613Crossref PubMed Scopus (1018) Google Scholar, 28Li B.L. Langer J.A. Schwartz B. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 558-562Crossref PubMed Scopus (62) Google Scholar, 29Pestka S. Lin L. Wu W. Izotova L. Protein Expr. Purif. 1999; 17: 203-214Crossref PubMed Scopus (10) Google Scholar) fused to its C terminus (FL-IL-TIF-P). COS cells were transiently transfected with the expression vectors, and 3 days later conditioned media containing FL-IL-TIF or FL-IL-TIF-P were tested by Western blotting with anti-FLAG antibody for protein expression (Fig. 1 B, lanes 3and 4). Western blotting revealed that FL-IL-TIF was secreted from COS cells and migrated on the SDS-PAGE gel as several bands in the region of about 25–40 kDa, suggesting possible glycosylation of the protein. Indeed, there are three potential sites for N-linked glycosylation (Asn-Xaa-Thr/Ser) in human IL-TIF. Treatment of the conditioned media with peptide:N-glycosidase F (PNGase F) resulted in the disappearance of the higher bands and enhancement of a band in the region of 21 kDa (Fig. 1 B, lane 4), consistent with glycosylation of the 25–40 kDa proteins. FL-IL-TIF-P, purified by affinity column chromatography, was also analyzed by Western blotting with anti-FLAG antibody (Fig. 1 B, lane 5). The32P-labeled FL-IL-TIF-P ([32P]FL-IL-TIF-P) was also resolved on the gel and autoradiographed (Fig. 1 B,lane 6). Human IL-10 tagged with the FLAG epitope at the N terminus and with the phosphorylation site at the C terminus was used as a control (Fig. 1 B, lane 2). CRF2-9 is an orphan human receptor from the class II cytokine receptor family as shown in Fig. 1 D (24Kotenko S.V. Pestka S. Oncogene. 2000; 19: 2557-2565Crossref PubMed Scopus (186) Google Scholar). We constructed expression vectors encoding intact CRF2-9 and a chimeric CRF2-9/γR1 receptor that has the CRF2-9 extracellular domain fused to the transmembrane and intracellular domains of the human IFN-γR1 chain (Fig.1 C). The previously constructed pEF-CRF (or pEF-IL-10R2) vector (6Kotenko S.V. Krause C.D. Izotova L.S. Pollack B.P. Wu W. Pestka S. EMBO J. 1997; 16: 5894-5903Crossref PubMed Scopus (333) Google Scholar) was also utilized in this study. In addition, to express both receptor chains in a single transfected cell the tandem vector encoding two receptors, the CRF2-9/γR1 and the IL-10R2 chains, in which expression of each receptor is controlled by separate set of promoter and polyadenylation signal was constructed. COS cells were transfected with the expression plasmid encoding CRF2-9, and transfectants were selected by growth in 350 μg/ml G418 for 3 weeks and pooled. To test for responsiveness to IL-TIF, pooled cells were treated with conditioned media from COS cells expressing FL-IL-TIF or left untreated as control, and the detergent-free total cellular lysates were prepared for electrophoretic mobility shift assays (EMSAs). The formation of Stat DNA-binding complexes was detected in FL-IL-TIF-treated COS cells transfected with the plasmid expressing the CRF2-9 chain and not in untreated cells or in FL-IL-TIF-treated control COS cells transfected with the blank expression vector (Fig.2). The DNA-binding complexes (Fig. 2) were shown to consist mainly of two Stats with anti-Stat1 and anti-Stat3 antibodies: Stat1α and Stat3. Thus, the pattern of IL-TIF-induced Stat DNA-binding complexes observed in COS cells expressing CRF2-9 correlates with the pattern of Stat activation demonstrated for IL-TIF signaling in PC-12 or MES-13 cells (1Dumoutier L. Louahed J. Renauld J.C. J. Immunol. 2000; 164: 1814-1819Crossref PubMed Scopus (443) Google Scholar). COS cells were also stably transfected with an expression vector encoding the chimeric CRF2-9/γR1 receptor with the CRF2-9 extracellular domain fused to the transmembrane and the intracellular domains of the IFN-γR1 chain (Fig. 1 C). This chimeric receptor was made to enable us to detect IFN-γ-like biological activities induced by IL-TIF. Because IL-TIF-specific biological activities are not well characterized and may be restricted to specific cell types, and because we expect that the CRF2-9 receptor complex structurally mimics the IL-10 receptor complex, we followed the same approach that was used to create the chimeric IL-10-IFN-γ receptor complex (6Kotenko S.V. Krause C.D. Izotova L.S. Pollack B.P. Wu W. Pestka S. EMBO J. 1997; 16: 5894-5903Crossref PubMed Scopus (333) Google Scholar). We predicted that, in cells expressing the chimeric CRF2-9/γR1 receptor, IL-TIF would induce IFN-γ-specific biological activities. As expected, in COS cells expressing the chimeric CRF2-9/γR1 chain, IL-TIF treatment induced activation of Stat1 DNA-binding complexes as demonstrated by EMSA with anti-Stat1 antibody (Fig. 2). We hypothesized that the IL-TIF receptor complex might be structurally homologous to the IL-10 receptor complex, and to consist of two receptor chains with one common chain shared between these two receptor complexes. It has been demonstrated that, in hamster cells, unlike COS cells, hamster IL-10R2 failed to support signaling by the human IL-10R1 chain (6Kotenko S.V. Krause C.D. Izotova L.S. Pollack B.P. Wu W. Pestka S. EMBO J. 1997; 16: 5894-5903Crossref PubMed Scopus (333) Google Scholar). To determine whether a similar situation holds for the human IL-TIF receptor complex expressed in hamster cells, hamster cells were transfected with the chimeric CRF2-9/γR1 receptor. In these cells IL-TIF treatment failed to induce Stat activation (Fig. 2) or MHC class I antigen expression (Fig. 3 B). In contrast, hamster cells transfected with the tandem vector encoding both the CRF2-9/γR1 and the IL-10R2 chains responded to IL-TIF treatment by activation of Stat1 DNA-binding complexes (Fig. 2) and up-regulation of MHC class I antigen expression (Fig. 3 D). Parental hamster cells and cells expressing the human IL-10R2 chain (6Kotenko S.V. Krause C.D. Izotova L.S. Pollack B.P. Wu W. Pestka S. EMBO J. 1997; 16: 5894-5903Crossref PubMed Scopus (333) Google Scholar) did not respond to the IL-TIF treatment (Figs. 2, 3 A, and 3 C). F
