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• W2087144342 abstract "tyk2 belongs to the JAK family of nonreceptor protein tyrosine kinases recently found implicated in signaling through a large number of cytokine receptors. These proteins are characterized by a large amino-terminal region and two tandemly arranged kinase domains, a kinase-like and a tyrosine kinase domain. Genetic and biochemical evidence supports the requirement for tyk2 in interferon-α/β binding and signaling. To study the role of the distinct domains of tyk2, constructs lacking one or both kinase domains were stably transfected in recipient cells lacking the endogenous protein. Removal of either or both kinase domains resulted in loss of the in vitro kinase activity. The mutant form truncated of the tyrosine kinase domain was found to reconstitute binding of interferon-α8 and partial signaling. While no contribution of this protein toward interferon-β binding was evident, increased signaling could be measured. The mutant form lacking both kinase domains did not exhibit any detectable activity. Altogether, these results show that a sequential deletion of domains engenders a sequential loss of function and that the different domains of tyk2 have distinct functions, all essential for full interferon-α and -β binding and signaling. tyk2 belongs to the JAK family of nonreceptor protein tyrosine kinases recently found implicated in signaling through a large number of cytokine receptors. These proteins are characterized by a large amino-terminal region and two tandemly arranged kinase domains, a kinase-like and a tyrosine kinase domain. Genetic and biochemical evidence supports the requirement for tyk2 in interferon-α/β binding and signaling. To study the role of the distinct domains of tyk2, constructs lacking one or both kinase domains were stably transfected in recipient cells lacking the endogenous protein. Removal of either or both kinase domains resulted in loss of the in vitro kinase activity. The mutant form truncated of the tyrosine kinase domain was found to reconstitute binding of interferon-α8 and partial signaling. While no contribution of this protein toward interferon-β binding was evident, increased signaling could be measured. The mutant form lacking both kinase domains did not exhibit any detectable activity. Altogether, these results show that a sequential deletion of domains engenders a sequential loss of function and that the different domains of tyk2 have distinct functions, all essential for full interferon-α and -β binding and signaling. The JAK family of protein tyrosine kinases presently consists of four members, tyk2, JAK1, JAK2, and JAK3(1Ziemiecki A. Harpur A.G. Wilks A.F. Trends. Cell Biol. 1994; 4: 207-212Abstract Full Text PDF PubMed Scopus (110) Google Scholar, 2Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Thierfelder W.E. Kreider B. Silvennoinen O. Trends Biochem. Sci. 1994; 19: 222-227Abstract Full Text PDF PubMed Scopus (586) Google Scholar, 3Takahashi T. Shirasawa T. FEBS Lett. 1994; 342: 124-128Crossref PubMed Scopus (73) Google Scholar, 4Johnston J.A. Kawamura M. Kirken R.A. Chen Y.Q. Blake T.B. Shibuya K. Ortaldo J.R. McVicar D.W. O'Shea J.J. Nature. 1994; 370: 151-153Crossref PubMed Scopus (490) Google Scholar, 5Witthuhn B.A. Silvennoinen O. Miura O. Lai K.S. Cwik C. Liu E.T. Ihle J.N. Nature. 1994; 370: 153-157Crossref PubMed Scopus (525) Google Scholar, 6Rane S.G. Reddy E.P. Oncogene. 1994; 9: 2415-2423PubMed Google Scholar). These 120-130-kDa proteins are characterized by the presence of (i) a carboxyl kinase domain, (ii) an adjacent kinase-like domain, and (iii) five conserved domains extending toward the amino terminus and the absence of SH2 or SH3 domains (reviewed in (1Ziemiecki A. Harpur A.G. Wilks A.F. Trends. Cell Biol. 1994; 4: 207-212Abstract Full Text PDF PubMed Scopus (110) Google Scholar) and (2Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Thierfelder W.E. Kreider B. Silvennoinen O. Trends Biochem. Sci. 1994; 19: 222-227Abstract Full Text PDF PubMed Scopus (586) Google Scholar). The COOH-terminal kinase domain (or TK ( 1The abbreviations used are: TKtyrosine kinaseKLkinase-likeIFNinterferonIFNARinterferon-α receptorSTATsignal transducer and activator of transcriptionHAThypoxanthine/aminopterine/thymidineVSV-Gvesicular stomatitis virus GlycoproteinISGF3interferon-stimulated gene factor 3kbkilobase pair(s). )domain) contains all the conserved residues associated with tyrosine kinases(7Hanks S.K. Quinn A.M. Methods Enzymol. 1991; 200: 38-62Crossref PubMed Scopus (1067) Google Scholar). The kinase-like domain (or KL domain) contains the subdomains shared by protein kinases, but lacks several residues thought to be essential for protein kinase activity, and thus it would not be predicted to exhibit kinase activity. The function of this domain has yet to be established. The remaining five blocks of homology exhibit varying degrees of conservation among the family members and their functional role is to be elucidated. It is likely that some of them might be responsible for the association of the JAKs with members of the cytokine receptor superfamily. Recent studies have in fact established the involvement of these enzymes in signaling through interferon (IFN) receptors and a large number of cytokine/growth factor receptors(1Ziemiecki A. Harpur A.G. Wilks A.F. Trends. Cell Biol. 1994; 4: 207-212Abstract Full Text PDF PubMed Scopus (110) Google Scholar, 2Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Thierfelder W.E. Kreider B. Silvennoinen O. Trends Biochem. Sci. 1994; 19: 222-227Abstract Full Text PDF PubMed Scopus (586) Google Scholar, 4Johnston J.A. Kawamura M. Kirken R.A. Chen Y.Q. Blake T.B. Shibuya K. Ortaldo J.R. McVicar D.W. O'Shea J.J. Nature. 1994; 370: 151-153Crossref PubMed Scopus (490) Google Scholar, 5Witthuhn B.A. Silvennoinen O. Miura O. Lai K.S. Cwik C. Liu E.T. Ihle J.N. Nature. 1994; 370: 153-157Crossref PubMed Scopus (525) Google Scholar). Physical interaction between JAKs and the membrane-proximal region of some of these receptor chains has also been demonstrated (reviewed in (2Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Thierfelder W.E. Kreider B. Silvennoinen O. Trends Biochem. Sci. 1994; 19: 222-227Abstract Full Text PDF PubMed Scopus (586) Google Scholar) (8Nicholson S.E. Oates A.C. Harpur A.G. Ziemiecki A. Wilks A.F. Layton J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2985-2988Crossref PubMed Scopus (175) Google Scholar, 9Narazari M. Witthuhn B.A. Yoshida K. Silvennoinen O. Yasukawa K. Ihle J.N. Kishimoto T. Taga T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2285-2289Crossref PubMed Scopus (248) Google Scholar, 10Quelle F.W. Sato N. Witthuhn B.A. Inhorn R.C. Eder M. Miyajima A. Griffin J.D. Ihle J.N. Mol. Cell. Biol. 1994; 14: 4335-4341Crossref PubMed Google Scholar, 11Colamonici O.R. Uyttendaele H. Domanski P. Yan H. Krolewski J.J. J. Biol. Chem. 1994; 269: 3518-3522Abstract Full Text PDF PubMed Google Scholar, 12Igarashi K. Garotta G. Ozmen L. Ziemiecki A. Wilks A.F. Harpur A.G. Larner A.C. Finbloom D.S. J. Biol. Chem. 1994; 269: 14333-14336Abstract Full Text PDF PubMed Google Scholar, 13Dusanter-Fourt I. Muller O. Ziemiecki A. Mayeux P. Drucker B. Djiane J. Wilks A. Harpur A.G. Fischer S. Gisselbrecht S. EMBO J. 1994; 13: 2583-2591Crossref PubMed Scopus (134) Google Scholar, 14Tanaka N. Asao H. Ohbo K. Ishii N. Takeshita T. Nakamura M. Sasaki H. Sugamura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7271-7275Crossref PubMed Scopus (65) Google Scholar). tyrosine kinase kinase-like interferon interferon-α receptor signal transducer and activator of transcription hypoxanthine/aminopterine/thymidine vesicular stomatitis virus Glycoprotein interferon-stimulated gene factor 3 kilobase pair(s). As most cytokine receptors, the IFN-α/β receptor has a multichain structure composed of the chain IFNAR1, interacting with at least another membrane component(15Uzé G. Lutfalla G. Gresser I. Cell. 1990; 60: 225-234Abstract Full Text PDF PubMed Scopus (500) Google Scholar, 16Uzé G. Lutfalla G. Bandu M.T. Proudhon D. Mogensen K.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4774-4778Crossref PubMed Scopus (85) Google Scholar). Although no formal evidence exists yet, it is likely that the IFN-α/β binding protein of 51 kDa recently identified by Novick et al.(17Novick D. Cohen B. Rubinstein M. Cell. 1994; 77: 391-400Abstract Full Text PDF PubMed Scopus (564) Google Scholar) represents a component of the receptor unit possibly interacting with IFNAR1. The signaling cascade initiated by binding of IFN-α/β to its receptor relies on the activity of tyk2 and JAK1, as was first demonstrated through the study of the IFN-resistant cell lines 11,1 (or U1A) and U4A, deficient in one or the other of these protein tyrosine kinases (18Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (684) Google Scholar, 19Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Crossref PubMed Scopus (624) Google Scholar). The absence of either protein prevents high affinity binding of IFN-α, phosphorylation of the downstream transcriptional components (p113 or STAT2 and p91/p84 or STAT1α/β) and activation of inducible genes(20Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4764) Google Scholar). Thus, complementation of 11,1 cells with a cosmid or the cDNA encoding tyk2 was shown to restore IFN-α/β responsiveness(18Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (684) Google Scholar, 21Barbieri G. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (55) Google Scholar), whereas complementation of mutant U4A with JAK1 cDNA restored both IFN-α/β and IFN-γ signaling(19Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Crossref PubMed Scopus (624) Google Scholar). In an initial study of the tyk2-deficient cell line 11,1 we found a residual sensitivity of these cells to IFN-β, that suggested the existence of a minor IFN-β-specific pathway and possibly a different utilization of tyk2 by the two IFN species(22Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (312) Google Scholar, 23John J. McKendry R. Pellegrini S. Flavell D. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1991; 11: 4189-4195Crossref PubMed Scopus (117) Google Scholar). We have recently reported on the biochemical characterization of tyk2 as a 134-kDa protein mostly cytosolic, with a minor membrane-associated fraction. The protein is transiently phosphorylated on tyrosine in response to IFN-α/β treatment and possesses an inducible tyrosine kinase activity measurable in vitro(21Barbieri G. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (55) Google Scholar). To understand the role of the kinase domains of tyk2, here we have constructed deleted forms of the protein lacking either or both kinase domains and stably expressed them in the tyk2-deficient cell line. The binding of IFN-α and IFN-β and the signaling activities induced in these transfectants were analyzed and compared. Parental 2fTGH cells, mutants 11,1, U1B, and U1C were previously described(22Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (312) Google Scholar, 24McKendry R. John J. Flavell D. Müller M. Kerr I.M. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11455-11459Crossref PubMed Scopus (227) Google Scholar). Cells were propagated in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum and hygromycin (250 μg/ml). Plasmid DNA transfections of 11,1 cells were carried out using calcium phosphate as described previously(22Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (312) Google Scholar). Two days after transfection, cells were seeded 1:10 in medium containing 450 μg/ml G418 (Sigma). Resistant colonies were ring-cloned 2 weeks later and expanded. Survival in hypoxanthine/aminopterine/thymidine (HAT) medium was assayed in the presence of different concentrations of IFN. Wellferon is a highly purified mixture of human IFN-α subtypes (108 IU/mg of protein, Wellcome Research Laboratories)(25Allen G. Fantes K.H. Burke D.C. Morser J. J. Gen. Virol. 1982; 63: 207-212Crossref PubMed Scopus (36) Google Scholar). Recombinant human IFN-α8, purified to homogeneity, was a gift from CIBA-GEIGY (Basel, Switzerland). Purified recombinant human IFN-β at 250 μg/ml was a gift from Biogen Inc. (Boston, MA). All constructs were made in the pRc/CMV expression vector (InVitrogen). In this vector, the cDNA of interest is under the control of the cytomegalovirus immediate-early promoter. All plasmids were tagged at their 3′ end with an oligonucleotide encoding an epitope of the vesicular stomatitis virus (VSV-G) as previously described(21Barbieri G. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (55) Google Scholar). A SalI fragment, comprising nucleotides 777-4146 of tyk2 cDNA in plasmid H9S (21Barbieri G. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (55) Google Scholar) was subcloned into the phagemid vector pBluescript KS+ (Stratagene) to facilitate subsequent deletion constructs (b.s.3.3 B-Sal). To delete the tyrosine kinase domain, a 2.1-kb EcoRI-Eco47III fragment (nucleotides 777-2950) from b.s.3.3 B-Sal was cloned into the EcoRI-SmaI site of a plasmid containing the VSV-G epitope sequence. The new plasmid was digested with SalI and XbaI, the 2.2-kb fragment was purified, and, together with an 850-base pair fragment comprising the 5′-untranslated sequence and nucleotides 1-777 of tyk2 cDNA, it was ligated to the HindIII-XbaI-digested vector. The final construct, ΔTK, encodes a protein containing amino acids 1-895 of tyk2. To delete the kinase-like domain, b.s.3.3 B-Sal was digested with AatII and EcoRV, treated with DNA polymerase to form blunt ends and religated. The resulting plasmid was digested with EcoRI, blunted as before and then digested with SpeI. The SpeI site is in the vector portion of the plasmid. The 3.9-kb band was isolated and ligated to a 1.3-kb SpeI-PvuII fragment (nucleotides 777-2040) of b.s. 3.3 B-Sal. A 2.2-kb SalI-XbaI fragment was prepared from this plasmid, and a three-part ligation, with the 850-base pair HindIII-SalI fragment and the HindIII-XbaI vector band as described for ΔTK, was performed. The final construct generates a protein, ΔKL, in which amino acids 594-877 have been deleted, and an additional Tyr has been inserted after amino acid 593. To generate a protein deleted of both kinase domains, a 2.1-kb HindIII-Eco47III fragment from plasmid ΔKL was purified and ligated to the VSV-G plasmid digested with HindIII-SmaI. The resulting plasmid was digested with HindIII and XbaI, and the subsequent 2.2-kb fragment was ligated to the HindIII-XbaI-digested vector. This construct encodes a protein, N, that contains amino acids 1-591, followed by a three-amino acid insertion of Ile-Glu-Phe and finally amino acids 878-895 of tyk2. All final plasmids were sequenced across the junctions of the deletions to confirm that the coding region remained in frame. Sequence analysis was performed by the dideoxy chain termination method using a Sequenase kit (U. S. Biochemical Corp.) and double-stranded plasmid DNA as template. Total RNA was extracted from subconfluent cultures with guanidinium thiocyanate(26Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). Ten micrograms of RNA per sample were fractionated in 1.2% agarose gels containing 2.2 M formaldehyde, transferred to Hybond-N membrane (Amersham Corp.), and hybridized under standard conditions (26Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar) to specific cDNA probes. IFN-α8 and IFN-β were labeled with 125I (Amersham Corp., IMS 30). IFN-α8 was labeled to a specific radioactivity of 25 Bq/fmol of monomeric IFN and stored at −80°C at a concentration of 20 nM, as described previously(27Mogensen K.E. Uzé G. Methods Enzymol. 1986; 119: 267-276Crossref PubMed Scopus (25) Google Scholar). The labeling procedure was slightly modified for IFN-β, using a protein/iodine ratio 2.5 times higher than for IFN-α8, to give a specific radioactivity of 10 Bq/fmol of monomeric IFN at 50 nM. Cell binding was carried out on subconfluent monolayer cultures (1.5 × 105 cells/cm2). Each point is an average from three replicate cultures, each containing 1.5 × 106 cells. Binding measured at 37°C was terminated by placing the cultures on ice and aspirating the supernatant. The cultures were then washed three times with equivalent volumes of ice-cold medium containing 1% of fetal calf serum, detached in trypsin and EDTA for transfer to a γ counter (Kontron, counting efficiency 75%). Conversion of radioactive counts to molecules of IFN was as described previously(28Mogensen K.E. Bandu M.-T. Eur. J. Biochem. 1983; 134: 355-364Crossref PubMed Scopus (43) Google Scholar). Binding at 4°C was carried out by equilibrating the cultures on ice for 30 min before adding labeled IFN and terminated as above by aspiration and washing. As incubation times were necessarily longer at 4°C than at 37°C, the culture medium was buffered by addition of Hepes to 50 mM. After several hours at 4°C, cells detach easily, and extreme care was exercised during the washing. Binding results are presented as receptor-specific with background (estimated from cultures treated with labeled IFN in the presence of a 200-fold molar excess of unlabeled IFN) substracted. Backgrounds were typically at 0.05% of the input radioactivity. Immunoblots and immunoprecipitations were done as described elsewhere(21Barbieri G. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (55) Google Scholar). For immunoprecipitation with anti-91C antibody, cells were lysed in Nonidet P-40-containing lysis buffer (21Barbieri G. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (55) Google Scholar) containing 300 mM NaCl. p91 immunoblots were blocked with 5% bovine serum albumin (fraction V, 96-99% albumin) in 1 × TBST (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20) buffer. Tyrosine phosphorylation of p91 was detected with a mixture of PY20 (ICN) and 4G10 (Upstate Biotechnology, Inc.) antibodies. Antisera to p113 and p91 (a gift of C. Schindler) were used at a 1:1,000 and 1:500 dilution respectively for immunoprecipitation and at a 1:10,000 dilution for immunoblotting. Affinity-purified anti-JAK1 antibodies (a gift of A. Ziemiecki) were used at a 1:250 dilution for immunoprecipitation and at a 1:2,000 dilution for immunoblotting. An ECL Western blotting detection system (Amersham Corp.) was used according to the manufacturer's instructions. The in vitro kinase assay was performed as previously reported(21Barbieri G. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (55) Google Scholar). Gel was transferred to Hybond-C Super membrane (Amersham Corp.), and phosphorylated proteins were visualized by autoradiography. To carry out a structure-function analysis of tyk2, we generated three deleted cDNA forms lacking one or both kinase domains. The proteins encoded by these constructs are schematically depicted in Fig. 1A. The mutant protein designated ΔTK is a truncated tyk2 form lacking the tyrosine kinase domain. The ΔKL protein lacks the kinase-like domain but retains an intact tyrosine kinase domain, whereas the protein designated N corresponds to the NH2-terminal region. The wild-type and the deleted cDNAs were cloned in the pRc/CMV eukaryotic expression vector, which contains the neomycin-resistance marker. Mutant 11,1 cells were transfected with the four constructs, selected in G418, and independent neor clones arising from each transfection were analyzed for the presence of tyk2 by immunoblot. All transfectants analyzed expressed tyk2 forms of the predicted size, at levels ranging from 0.5- to 10-fold the endogenous tyk2 level present in parental 2fTGH cells (21Barbieri G. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (55) Google Scholar). Minor tyk2 forms of higher mobility were routinely detected in tyk2-overexpressing cells (Fig. 1B) and might result from in vivo degradation. Four representative clones (Fig. 1B) expressing almost comparable levels of protein (approximately 5-fold the endogenous tyk2 level in 2fTGH cells) were chosen for further studies. We tested the ability of the four neor transfectants to grow in the selective medium (HAT) in the presence of different concentrations of IFN-α. This medium allows survival of 11,1 derivatives which have reverted to IFN sensitivity (22Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (312) Google Scholar) and constitutes our biological assay for tyk2 activity. Wild-type tyk2-expressing cells showed IFN-dependent growth with as little as 10 IU/ml IFN-α (Wellferon, a mixture of purified human α subtypes). ΔTK-expressing cells survived in HAT medium only when a higher concentration of IFN-α was used. In contrast, 11,1 cells and ΔKL- and N-expressing cells failed to grow at any given concentration of IFN-α tested (Fig. 2A). To rule out the possibility that this behavior was unique to 11,1 derivatives, we generated ΔTK and ΔKL neor transfectants from cell lines U1B and U1C, two independent mutants of the same complementation group as 11,1(24McKendry R. John J. Flavell D. Müller M. Kerr I.M. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11455-11459Crossref PubMed Scopus (227) Google Scholar). The IFN responsiveness of each transfectant gave comparable results (data not shown). We next tested the sensitivities of the 11,1 transfectants to recombinant IFN-α8 and -β. These results are summarized in Fig. 2B. The specific activity of IFN-β was higher than that of IFN-α8 on all clones tested. There was a 30-100-fold difference in the sensitivities of ΔTK-expressing cells and 11,1 to either IFN. Interestingly, the sensitivity of ΔTK-expressing cells to IFN-α8 attained the level of sensitivity of 11,1 cells to IFN-β. The analysis of the ΔKL- and N-expressing clones gave results essentially identical to 11,1. These data indicate that both tyk2 kinase domains are required to restore a wild-type response to both IFN species. The truncated ΔTK form can, however, partially reconstitute sensitivity to both IFNs. As a measure of the response of the transfectants to IFN-α, we studied the transcriptional induction of the endogenous IFN-responsive 6-16 gene by Northern blot. Analysis of total RNA from wild-type tyk2-expressing cells treated for 4 h showed accumulation of the 6-16 transcript with as little as 10 IU/ml IFN-α (Fig. 3). A comparable level of transcript accumulated in ΔTK-expressing cells treated with 5000 IU/ml IFN-α. In contrast, ΔKL-expressing cells failed to induce the 6-16 message, behaving as mutant 11,1. These results confirm the fine correlation between the transcriptional activation of the 6-16 gene promoter by IFN and the ability of the cells to grow in HAT medium. The steady-state mRNA level of three other IFN-responsive genes (ISG-54, GBP, and IRF-1) was analyzed in the various cell lines. None of these transcripts accumulated in cells expressing the deleted tyk2 forms, treated with up to 5000 IU/ml IFN-α (data not shown). Previous work had suggested that tyk2 exerts an effect on receptor-mediated uptake of IFN-α, i.e. mutant 11,1 cells lacking tyk2 showed reduced uptake of labeled IFN-α2(22Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (312) Google Scholar), whereas complementing the defect by tyk2 transfection restored binding as well as signaling(18Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (684) Google Scholar). Furthermore, 11,1 cells are known to be partially responsive to IFN-β (Fig. 2B)(22Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (312) Google Scholar, 23John J. McKendry R. Pellegrini S. Flavell D. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1991; 11: 4189-4195Crossref PubMed Scopus (117) Google Scholar). To complete these observations, we studied the kinetics of uptake of radiolabeled IFN-β and IFN-α8 in the mutant cell line and in its derivatives (Fig. 4). The kinetic results shown here are for a single concentration of IFN (∼300 pM), but the relative form of the curves was similar across the dose range 50-500 pM. Since essentially identical curves were obtained from ΔKL- and N-expressing cells and 11,1 cells, only data from 11,1 were represented in Fig. 4, A and B. Cells expressing wild-type tyk2 showed the characteristic binding dynamics for IFN-β seen in parental 2fTGH cells (Fig. 4A). While a similar level of uptake was attained in both 11,1 cells and ΔTK-expressing cells, there was no down phase as in parental cells. Similar results were obtained with IFN-α8 (Fig. 4B) except that 11,1 cells showed a much reduced uptake than ΔTK-expressing cells. To investigate this further and to eliminate the dynamic aspects of cellular uptake, we compared the binding of IFN-α8 and IFN-β at 4°C on the partially sensitive ΔTK-expressing cells and on the IFN-insensitive ΔKL-expressing cells. The results, in the form of Scatchard graphs, are shown in Fig. 4C. The binding of IFN-β was nearly the same on the two cell lines, showing that, for IFN-β, receptor expression appears to be unaffected by the status of tyk2. The binding of IFN-α8 on the ΔTK-expressing cells was greater and shows a Scatchard plot with a slope 2.4 times greater than that obtained on the ΔKL-expressing cells. This suggests that the mutant protein ΔTK contributes to the affinity of binding of IFN-α over the concentration range used. The Scatchard coefficient gave Kd equivalent to 0.54 nM and 0.62 nM for IFN-β, 0.87 nM and >2.1 nM for IFN-α8, on ΔTK- and ΔKL-expressing cells, respectively (the Kd of 2.1 nM falls outside the range of experimental points). The linear regressions extrapolated to infinite ligand concentration meet the intercept at values close enough to suggest that the main reason for the reduced uptake of IFN-α8 on ΔKL cells is a lower binding affinity. It has been previously reported that tyrosine phosphorylation of tyk2 occurs within min of IFN-α/β treatment(11Colamonici O.R. Uyttendaele H. Domanski P. Yan H. Krolewski J.J. J. Biol. Chem. 1994; 269: 3518-3522Abstract Full Text PDF PubMed Google Scholar, 19Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Crossref PubMed Scopus (624) Google Scholar, 21Barbieri G. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (55) Google Scholar). We therefore investigated the in vivo phosphorylation state of deleted tyk2 forms in the transfected clones. Cells were left untreated or treated with IFN-α for 5 min and tyk2 immunoprecipitates were analyzed by blotting with antibodies against phosphotyrosine and, after stripping, with anti-tyk2 antibodies. In response to IFN-α, wild-type tyk2 was rapidly phosphorylated on tyrosine above a basal level of phosphorylation (Fig. 5A, lanes 1 and 2, upper panel; see also Fig. 3 in (21Barbieri G. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (55) Google Scholar). The N and ΔTK mutant forms were not phosphorylated (Fig. 5A, lanes 4 and 8). Conversely, the ΔKL mutant protein showed an IFN-independent phosphorylation (Fig. 5A, lanes 5 and 6). Comparable levels of protein were present in all extracts analyzed (Fig. 5A, lower panel). IFN-α induces tyk2 kinase activity and this can be measured in vitro in an anti-tyk2 immunocomplex(21Barbieri G. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (55) Google Scholar). To determine whether the deletions had an effect on the kinase activity of the protein, in vitro kinase assays were performed on the wild-type tyk2 and the ΔTK and ΔKL mutant forms. As shown in Fig. 5B, wild-type tyk2 exhibited increased autophosphorylating activity upon IFN-α treatment. In contrast, ΔTK and ΔKL did not exhibit autophosphorylating activity. A similar result was obtained when the two deletion constructs were co-expressed in 11,1 cells (data not shown). To investigate the ability of these proteins to phosphorylate an exogenous substrate, the in vitro kinase assay was performed in the presence of acid-denatured enolase. Upon IFN-α treatment, enolase was markedly phosphorylated by wild-type tyk2 (Fig. 5B, lane 2). In contrast, the exogenous substrate was not phosphorylated in ΔKL and ΔTK-expressing cells treated with IFN (Fig. 5B, lanes 4 and 6). The abundance of the immunoprecipitated proteins was visualized by blotting" @default.
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• W2087144342 title "Distinct Domains of the Protein Tyrosine Kinase tyk2 Required for Binding of Interferon-α/β and for Signal Transduction" @default.
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