©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distinct Domains of the Protein Tyrosine Kinase tyk2 Required for Binding of Interferon-/ and for Signal Transduction (*)

(Received for publication, September 9, 1994)

Laura Velazquez (§) Knud E. Mogensen (1) Giovanna Barbieri (¶) Marc Fellous Gilles Uzé (1) Sandra Pellegrini (**)

From the Institut Pasteur, INSERM U 276, Paris 75724 Cedex 15, France Institut de Génétique Moléculaire, CNRS UMR 9942, Montpellier, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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-alpha/beta 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-alpha8 and partial signaling. While no contribution of this protein toward interferon-beta 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-alpha and -beta binding and signaling.


INTRODUCTION

The JAK family of protein tyrosine kinases presently consists of four members, tyk2, JAK1, JAK2, and JAK3(1, 2, 3, 4, 5, 6) . 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 (1) and (2) ). The COOH-terminal kinase domain (or TK (^1)domain) contains all the conserved residues associated with tyrosine kinases(7) . 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(1, 2, 4, 5) . Physical interaction between JAKs and the membrane-proximal region of some of these receptor chains has also been demonstrated (reviewed in (2) ) (8, 9, 10, 11, 12, 13, 14) .

As most cytokine receptors, the IFN-alpha/beta receptor has a multichain structure composed of the chain IFNAR1, interacting with at least another membrane component(15, 16) . Although no formal evidence exists yet, it is likely that the IFN-alpha/beta binding protein of 51 kDa recently identified by Novick et al.(17) represents a component of the receptor unit possibly interacting with IFNAR1. The signaling cascade initiated by binding of IFN-alpha/beta 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 (18, 19) . The absence of either protein prevents high affinity binding of IFN-alpha, phosphorylation of the downstream transcriptional components (p113 or STAT2 and p91/p84 or STAT1alpha/beta) and activation of inducible genes(20) . Thus, complementation of 11,1 cells with a cosmid or the cDNA encoding tyk2 was shown to restore IFN-alpha/beta responsiveness(18, 21) , whereas complementation of mutant U4A with JAK1 cDNA restored both IFN-alpha/beta and IFN- signaling(19) . In an initial study of the tyk2-deficient cell line 11,1 we found a residual sensitivity of these cells to IFN-beta, that suggested the existence of a minor IFN-beta-specific pathway and possibly a different utilization of tyk2 by the two IFN species(22, 23) .

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-alpha/beta treatment and possesses an inducible tyrosine kinase activity measurable in vitro(21) . 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-alpha and IFN-beta and the signaling activities induced in these transfectants were analyzed and compared.


MATERIALS AND METHODS

Cell Culture and Transfection

Parental 2fTGH cells, mutants 11,1, U1B, and U1C were previously described(22, 24) . 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(22) . 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-alpha subtypes (10^8 IU/mg of protein, Wellcome Research Laboratories)(25) . Recombinant human IFN-alpha8, purified to homogeneity, was a gift from CIBA-GEIGY (Basel, Switzerland). Purified recombinant human IFN-beta at 250 µg/ml was a gift from Biogen Inc. (Boston, MA).

tyk2 Deletion Constructs

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(21) . A SalI fragment, comprising nucleotides 777-4146 of tyk2 cDNA in plasmid H9S (21) 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, DeltaTK, 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 DeltaTK, was performed. The final construct generates a protein, DeltaKL, 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 DeltaKL 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.

Northern Analysis

Total RNA was extracted from subconfluent cultures with guanidinium thiocyanate(26) . 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 (26) to specific cDNA probes.

Binding Experiments with I-Labeled IFN

IFN-alpha8 and IFN-beta were labeled with I (Amersham Corp., IMS 30). IFN-alpha8 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(27) . The labeling procedure was slightly modified for IFN-beta, using a protein/iodine ratio 2.5 times higher than for IFN-alpha8, 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 times 10^5 cells/cm^2). Each point is an average from three replicate cultures, each containing 1.5 times 10^6 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 counter (Kontron, counting efficiency 75%). Conversion of radioactive counts to molecules of IFN was as described previously(28) . 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.

Immunoblotting and Immunoprecipitation

Immunoblots and immunoprecipitations were done as described elsewhere(21) . For immunoprecipitation with anti-91C antibody, cells were lysed in Nonidet P-40-containing lysis buffer (21) containing 300 mM NaCl. p91 immunoblots were blocked with 5% bovine serum albumin (fraction V, 96-99% albumin) in 1 times 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.

In Vitro Kinase Assay

The in vitro kinase assay was performed as previously reported(21) . Gel was transferred to Hybond-C Super membrane (Amersham Corp.), and phosphorylated proteins were visualized by autoradiography.


RESULTS

Biological Activity of tyk2 Deletion Mutants

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 DeltaTK is a truncated tyk2 form lacking the tyrosine kinase domain. The DeltaKL protein lacks the kinase-like domain but retains an intact tyrosine kinase domain, whereas the protein designated N corresponds to the NH(2)-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 neo^r 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 (21) . 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.


Figure 1: Structure and expression of the deleted tyk2 proteins. A, Schematic representation of the tyk2 deleted forms. Names of the proteins are on the left and their predicted molecular masses in kilodaltons (kDa) are on the right. w.t., wild-type; KL, kinase-like domain; TK, tyrosine kinase domain; N, NH(2)-terminal region. The striped box at the C termini corresponds to the VSV-G epitope. B, anti-tyk2 immunoblot to compare the level of protein in 2fTGH cells and in clones transfected with the indicated cDNAs. Fifteen micrograms of total protein obtained from crude cell extracts were loaded on a SDS-7% polyacrylamide gel. Minor forms of higher mobility were routinely detected in cells overexpressing tyk2.



We tested the ability of the four neo^r transfectants to grow in the selective medium (HAT) in the presence of different concentrations of IFN-alpha. This medium allows survival of 11,1 derivatives which have reverted to IFN sensitivity (22) 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-alpha (Wellferon, a mixture of purified human alpha subtypes). DeltaTK-expressing cells survived in HAT medium only when a higher concentration of IFN-alpha was used. In contrast, 11,1 cells and DeltaKL- and N-expressing cells failed to grow at any given concentration of IFN-alpha tested (Fig. 2A). To rule out the possibility that this behavior was unique to 11,1 derivatives, we generated DeltaTK and DeltaKL neo^r transfectants from cell lines U1B and U1C, two independent mutants of the same complementation group as 11,1(24) . The IFN responsiveness of each transfectant gave comparable results (data not shown).


Figure 2: Biological assay of the activity of wild-type and deleted tyk2 forms. A, The sensitivity to IFN-alpha of the indicated cell lines was tested by seeding 3 times 10^4 cells/well in a 24-well plate in HAT medium without or with the indicated concentrations of IFN-alpha (Wellferon). After 6-7 days, cells were fixed and stained. B, comparison of the dose-response range for IFN-alpha8 and IFN-beta of mutant 11,1 cells and its derivative transfectants expressing wild-type tyk2 or the deleted DeltaTK form. The IFN response was assayed as described in A. After fixation, staining intensity was visually compared for the three cell lines. Boxes are closed on the left side at approx1% growth response, and on the right side at approx100% growth response. A half-log difference is regarded as significant. 1 nM corresponds approximately to 4000 IU/ml. The same profile of sensitivity was obtained for the 11,1 cell line and the clones expressing DeltaTK and N.



We next tested the sensitivities of the 11,1 transfectants to recombinant IFN-alpha8 and -beta. These results are summarized in Fig. 2B. The specific activity of IFN-beta was higher than that of IFN-alpha8 on all clones tested. There was a 30-100-fold difference in the sensitivities of DeltaTK-expressing cells and 11,1 to either IFN. Interestingly, the sensitivity of DeltaTK-expressing cells to IFN-alpha8 attained the level of sensitivity of 11,1 cells to IFN-beta. The analysis of the DeltaKL- 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 DeltaTK form can, however, partially reconstitute sensitivity to both IFNs.

As a measure of the response of the transfectants to IFN-alpha, 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-alpha (Fig. 3). A comparable level of transcript accumulated in DeltaTK-expressing cells treated with 5000 IU/ml IFN-alpha. In contrast, DeltaKL-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-alpha (data not shown).


Figure 3: Induction of the 6-16 mRNA by IFN-alpha in cells expressing wild-type or deleted tyk2 forms. Northern transfers of 10 µg of total RNA from untreated cells(-) or cells treated with the indicated amounts of IFN-alpha (Wellferon) for 4 h were hybridized to a 6-16-specific cDNA probe. Comparable signals in each lane were obtained with a beta-actin probe (data not shown).



DeltaTK Contributes to the Affinity of Binding of IFN-alpha

Previous work had suggested that tyk2 exerts an effect on receptor-mediated uptake of IFN-alpha, i.e. mutant 11,1 cells lacking tyk2 showed reduced uptake of labeled IFN-alpha2(22) , whereas complementing the defect by tyk2 transfection restored binding as well as signaling(18) . Furthermore, 11,1 cells are known to be partially responsive to IFN-beta (Fig. 2B)(22, 23) . To complete these observations, we studied the kinetics of uptake of radiolabeled IFN-beta and IFN-alpha8 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 DeltaKL- 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-beta seen in parental 2fTGH cells (Fig. 4A). While a similar level of uptake was attained in both 11,1 cells and DeltaTK-expressing cells, there was no down phase as in parental cells. Similar results were obtained with IFN-alpha8 (Fig. 4B) except that 11,1 cells showed a much reduced uptake than DeltaTK-expressing cells.


Figure 4: Binding of human IFN-alpha8 and IFN-beta to cells expressing wild-type or deleted tyk2 forms. A, Kinetics of uptake of I-labeled human IFN-beta (280 pM) at 37 °C by , 2fTGH cells; ; 11,1 cells; up triangle; wild-type tyk2-expressing cells; and box, DeltaTK-expressing cells. Values are femtomoles of IFN taken up per million cells. B, kinetics of uptake of I-labeled human IFN-alpha8 (350 pM) at 37 °C by bullet 11,1 cells; , wild-type tyk2-expressing cells; circle, DeltaTK-expressing cells. Values are femtomoles of IFN taken up per million cells. C, Scatchard plots of direct binding of I-labeled human IFN-alpha8 (bullet, circle) and IFN-beta (, box) for 6 h at 4 °C to circle, box, DeltaTK-expressing cells; bullet, , DeltaKL-expressing cells. Values are for femtomoles of IFN bound per million cells.



To investigate this further and to eliminate the dynamic aspects of cellular uptake, we compared the binding of IFN-alpha8 and IFN-beta at 4 °C on the partially sensitive DeltaTK-expressing cells and on the IFN-insensitive DeltaKL-expressing cells. The results, in the form of Scatchard graphs, are shown in Fig. 4C. The binding of IFN-beta was nearly the same on the two cell lines, showing that, for IFN-beta, receptor expression appears to be unaffected by the status of tyk2. The binding of IFN-alpha8 on the DeltaTK-expressing cells was greater and shows a Scatchard plot with a slope 2.4 times greater than that obtained on the DeltaKL-expressing cells. This suggests that the mutant protein DeltaTK contributes to the affinity of binding of IFN-alpha over the concentration range used. The Scatchard coefficient gave K(d) equivalent to 0.54 nM and 0.62 nM for IFN-beta, 0.87 nM and >2.1 nM for IFN-alpha8, on DeltaTK- and DeltaKL-expressing cells, respectively (the K(d) 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-alpha8 on DeltaKL cells is a lower binding affinity.

Deletion of Either Kinase Domain Affects Phosphorylation of tyk2

It has been previously reported that tyrosine phosphorylation of tyk2 occurs within min of IFN-alpha/beta treatment(11, 19, 21) . We therefore investigated the in vivo phosphorylation state of deleted tyk2 forms in the transfected clones. Cells were left untreated or treated with IFN-alpha 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-alpha, 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. 3in (21) ). The N and DeltaTK mutant forms were not phosphorylated (Fig. 5A, lanes 4 and 8). Conversely, the DeltaKL 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).


Figure 5: In vivo and in vitro tyrosine phosphorylation of tyk2 in response to IFN-alpha in wild-type and deleted tyk2-expressing cells. A, Anti-tyk2 immunoprecipitates from wild-type, deleted tyk2-expressing, and mutant 11,1 cells (5 times 10^6 cells) were immuno-blotted with either anti-phosphotyrosine 4G10 (upper panel) or anti-tyk2 (lower panel) antibody. IFN treatment (1000 IU/ml of Wellferon) was as follows: lanes 1, 3, 5, 7, and 9, no treatment; lanes 2, 4, 6, 8, and 10, 5 min with IFN-alpha. The position of wild-type and mutant tyk2 forms is marked by an arrow. Faster migrating bands are routinely detected in cells overexpressing tyk2. B, anti-tyk2 immunoprecipitates from post-nuclear lysates (600 µg) of untreated or IFN-alpha treated (1000 IU/ml Wellferon) wild-type, 11,1, DeltaKL, and DeltaTK-expressing cells were assayed in an in vitro kinase assay. Acid-denatured enolase was added to the reaction as an exogenous substrate. Kinase reaction products were fractionated on a 7% SDS-polyacrylamide gel electrophoresis and transferred to Hybond-C super membrane. Following autoradiography, the filter was probed with anti-tyk2 (bottom panel) antibodies. The expected positions of tyk2, enolase (En), and mutant DeltaKL and DeltaTK forms are marked.



IFN-alpha induces tyk2 kinase activity and this can be measured in vitro in an anti-tyk2 immunocomplex(21) . 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 DeltaTK and DeltaKL mutant forms. As shown in Fig. 5B, wild-type tyk2 exhibited increased autophosphorylating activity upon IFN-alpha treatment. In contrast, DeltaTK and DeltaKL 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-alpha treatment, enolase was markedly phosphorylated by wild-type tyk2 (Fig. 5B, lane 2). In contrast, the exogenous substrate was not phosphorylated in DeltaKL and DeltaTK-expressing cells treated with IFN (Fig. 5B, lanes 4 and 6). The abundance of the immunoprecipitated proteins was visualized by blotting the filter with anti-tyk2 antibodies (Fig. 5B, lower panel). These results indicate that both kinase domains appear necessary for the enzymatic activity of the protein since deletion of either domain generates a kinase-inactive protein.

No Detectable Activated JAK1, p91, and p113 in Cells Expressing tyk2 Deletion Mutants

To try to uncover the mechanism underlying the low IFN-alpha signaling observed in DeltaTK-expressing cells, we analyzed the phosphorylation state of components known to be involved in the IFN-alpha/beta signaling pathway. In wild-type cells, JAK1 is phosphorylated on tyrosine after IFN-alpha treatment, whereas no phosphorylation is detected in cells lacking tyk2(19, 29, 30) . We therefore examined the phosphorylation of JAK1 in cells expressing deleted tyk2 forms (Fig. 6A). No phosphorylated JAK1 was detected in cells expressing the deleted tyk2 forms even after long treatment with IFN (Fig. 6A, lanes 4 and 6). The protein levels were comparable in all cell lines tested (Fig. 6A, bottom panel).


Figure 6: Tyrosine phosphorylation of JAK1, p113, and p91 in cells expressing the wild-type and the deleted tyk2 forms. A, cells were left untreated (-, lanes 1, 3, 5, and 7) or were treated (+, lanes 2, 4, 6, and 8) for the indicated times (minutes) with IFN-alpha (1000 IU/ml Wellferon). Cell lysates were immunoprecipitated with anti-JAK1 and immunoblotted with anti-phosphotyrosine (upper panel) or anti-JAK1 (lower panel) antibodies. B and C, cells were left untreated (-, lanes 1, 3, 5, and 7) or were treated (+, lanes 2, 4, 6, and 8) for 20 min with IFN-alpha (1000 IU/ml of Wellferon). Cell lysates were immunoprecipitated with anti-p113 (B) or anti-p91C (C) antibodies, followed by immunoblotting with anti-phosphotyrosine 4G10 (B, upper panel) or a mixture of anti-phosphotyrosine 4G10 and PY20 antibodies (C, upper panel). The blots were stripped and reprobed with anti-p113 or/and anti-p91 as indicated (lower panels). The positions of JAK1, p113, and p91 are marked.



We next investigated whether p113 and p91, components of the transcriptional complex ISGF3, were phosphorylated upon IFN-alpha treatment of cells expressing the tyk2 deletion mutants. Phosphorylated p113 was detected in wild-type tyk2-expressing cells after 20 min of IFN-alpha treatment (Fig. 6B, lanes 2), whereas no phosphorylated p113 was detected in cells expressing the deleted tyk2 forms (Fig. 6B, lanes 4 and 6). Similarly, neither phosphorylated p91 nor p113bulletp91 complex (Fig. 6C, lane 2) were observed in cells expressing mutant tyk2 forms (Fig. 6C). Comparable levels of proteins were present in all extracts (Fig. 6, B and C, bottom panels). We were not successful at detecting phosphorylated JAK1, p91, or p113 in the DeltaTK-expressing cells even when different experimental conditions, such as increased dose and time of IFN treatment or increased amount of cell extract immunoprecipitated, were assayed.


DISCUSSION

In this study we have examined the effects of deletions on the activity of tyk2 expressed in an IFN-resistant cell line lacking the endogenous protein. Deletions of one or both kinase domains were found to abrogate tyk2 catalytic activity when measured in vitro for autophosphorylation and phosphorylation of an exogenous substrate. The absence of autophosphorylation could simply be consequent to the removal of major phosphorylation sites. However, the mutant DeltaKL protein retains two contiguous tyrosine residues, recently identified as targets of in vitro IFN-induced autophosphorylation in the wild-type tyk2. These sites (Tyr-Tyr) map in the TK domain, between subdomains VII and VIII, and, as shown for other protein tyrosine kinases, their phosphorylation parallels activation of the enzyme. (^2)Thus, it appears that, when juxtaposed to the NH(2)-terminal region, the TK domain of tyk2 is not active. This could either reflect a conformational alteration of the protein caused by the deletion, or be a consequence of the excision of a domain playing an essential role in the activity of the enzyme. Despite the fact that the DeltaKL mutant protein is catalytically inactive, it is phosphorylated in vivo, independently of the presence of IFN (Fig. 5A). When expressed in IFN-treated cells containing endogenous tyk2, the mutant form DeltaKL showed no evidence of phosphorylation above the basal level (data not shown), suggesting that IFN-induced trans-phosphorylation between the wild-type and the mutant protein cannot take place. Although at this time the site and the functional significance of this IFN-independent phosphorylation is unknown, these observations argue for the existence of another cellular protein tyrosine kinase capable of phosphorylating DeltaKL. Tyk2 deleted of its TK domain (DeltaTK) is catalytically inactive, indicating that the KL domain lacks intrinsic kinase activity. Indeed, no activity of the bacterially expressed JAK1 kinase-like domain could be detected for a number of exogenous substrates(31) .

One fundamental question regarding tyk2 and the other JAK family members concerns their mode of regulation which might differ from that of cytoplasmic protein tyrosine kinases possessing an SH2 domain and a single kinase domain(32) , resembling more that of other bifunctional proteins bearing a kinase-related domain(33, 34, 35) . One such protein is the cell surface receptor for atrial natriuretic peptide containing in its intracellular region a kinase-like domain next to a guanylyl cyclase catalytic domain. Deletion of the kinase domain constitutively activates the cyclase(33, 36) . Thus, a negative regulatory role has been proposed for this kinase-like domain, repressing the guanylyl cyclase in absence of atrial natriuretic peptide. This is not the case for tyk2, since deletion of its KL domain does not create a constitutively active enzyme. It is likely that a more complex mode of regulation involving both kinase domains exists for the JAK family.

To study the biological activity of tyk2 mutant forms, we have taken advantage of a unique property of 11,1 cells in which the expression of the marker guanine phosphoribosyl transferase and the consequent resistance to HAT is dependent on IFN and tyk2(18) . Thus, we have found that the function of tyk2 is completely abrogated by removal of the KL domain or of both kinase domains: cells expressing the mutant tyk2 forms DeltaKL and N behaved essentially as cells lacking tyk2. Conversely, expression of the DeltaTK protein resulted in a partial reconstitution of sensitivity toward IFN-alpha and in an increased sensitivity to IFN-beta (Fig. 2). This effect could be appreciated only when transfectants were expanded in G418, prior to testing their IFN responsiveness. When transfected cells were directly seeded in HAT, no survivors could be isolated in up to 10^4 IU/ml IFN, probably due to the poor colony-forming ability of these cells.

Our binding studies showed that the capacity for receptor-mediated IFN-alpha uptake is partially restored by the DeltaTK mutant protein, probably through an increase in binding affinity (Fig. 4). The inability of the N and the DeltaKL proteins to perform this function suggests that the KL domain in its proper conformation contributes toward the binding affinity of the receptor unit and indeed may be in contact with some component of that unit. In relation to this, tyk2 has been recently found to physically associate with the chain IFNAR of the receptor complex, independently of the presence of the ligand (11) . (^3)Despite an increase in the affinity of binding of IFN-alpha on DeltaTK-expressing cells, there are dynamic aspects of the interaction that have not been reconstituted, and that must require the TK domain. One hypothesis awaiting further investigation is that ligand-induced tyk2 kinase activity could play a role in post-binding events such as the internalization rate of the receptor unit, as proposed for the intrinsic kinase activity of epidermal growth factor and insulin receptors(37, 38, 39) .

The contribution of the DeltaTK protein toward ligand binding is much less evident for IFN-beta than for IFN-alpha. While there is a partial reconstitution of sensitivity towards IFN-beta with the DeltaTK protein, the early receptor binding events appear to be unaffected. Full reconstitution requires the tyrosine kinase domain. These data suggest that tyk2 has both structural and enzymatic functions in the IFN-alpha/beta response, with a more direct structural relationship with IFN-alpha than with IFN-beta, and this has been schematized in Fig. 7. In wild-type cells, the different elements are obviously well integrated. From the study of mutant cell lines, we would suggest that IFN-beta can utilize a minor or tyk2-independent binding pathway(s), an option not available to IFN-alpha. Further work will be needed to identify elements unique to the IFN-beta response and the recent report of a protein that can be coprecipitated with one ligand binding protein and phosphorylated only following IFN-beta treatment goes into that direction(40) . If the apparently greater flexibility of IFN-beta is a reflection of its evolutionary seniority (41) , then the close dependence of IFN-alpha and tyk2 may have its origin in the diversification of the mammalian IFNs.


Figure 7: Tyk2 in the receptor-mediated pathways induced by IFN-alpha and -beta. The central box contains all known and unknown components, other than tyk2, involved in signal transduction. Ifnar1 and Ifnar2 are ligand binding proteins(43) . Both binding pathways are deemed to be open to IFN-beta, whereas IFN-alpha is restricted to the major binding pathway.



We have not yet identified the signaling components involved in the minor pathway(s). Phosphorylated JAK1 or STATs could not be detected in DeltaTK-expressing cells in response to IFN-alpha, possibly due to the low number of molecules activated and the limited sensitivity of this assay. Recently, it has been shown that IFN-alpha/beta induce tyrosine phosphorylation of the p95 proto-oncogene in hematopoietic cell lines, suggesting that vav may be involved in an alternative IFN-alpha/beta signaling pathway in addition to the ISGF3 pathway(42) . It will be interesting to determine if signaling proteins similar to vav are activated in response to IFN-alpha/beta in cells other than hematopoietic cell lines, as it may provide a hint of a possible alternative IFN signaling pathway.

Certainly, more work will be needed to understand how tyk2 and its deleted DeltaTK form contribute to receptor function. In parallel, the study of the JAK1-deficient U4A mutant cell line, whose IFN-alpha binding activity is impaired(19, 20) , should help to clarify the role of JAK1 in receptor function. In relation to this, recently Novick et al.(17) have shown that the IFN-alpha/beta-binding chain that they have identified can be co-precipitated with JAK1. Altogether, our studies support a model in which heterodimerization of receptor chains and consequent juxtaposition and activation of associated JAK1 and tyk2 all contribute to the generation or stabilization of ligand-induced, conformationally correct, and productive receptor units.


FOOTNOTES

*
This project was supported by grants from the Association Française Contre les Myopathies (AFM) and the Ligue Nationale Contre le Cancer. The work at the Institut de Génétique Moléculaire, Montpellier, was supported in part by grants from the Direction des Recherches, Etudes et Techniques (94/2513A), Association pour la Recherche sur le Cancer, INSERM(920102), Fondation pour la Recherche Médicale, and Biogen Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from the Universidad Nacional Autonoma de Mexico (UNAM).

Recipient of an EEC Human Capital and Mobility fellowship.

**
To whom correspondence should be addressed: INSERM U 276, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France. Tel.: 33-1-40-61-32-16; Fax: 33-1-40-61-31-53.

(^1)
The abbreviations used are: TK, tyrosine kinase; KL, kinase-like; IFN, interferon; IFNAR, interferon-alpha receptor; STAT, signal transducer and activator of transcription; HAT, hypoxanthine/aminopterine/thymidine; VSV-G, vesicular stomatitis virus Glycoprotein; ISGF3, interferon-stimulated gene factor 3; kb, kilobase pair(s).

(^2)
L. Velazquez, R. McKendry, S. Pellegrini, unpublished observation.

(^3)
G. Barbieri, K. Cheung, L. Ling, S. Pellegrini, unpublished observation.


ACKNOWLEDGEMENTS

We thank A. Ziemiecki for providing the anti-JAK 1 antibodies, C. Schindler for the anti-p91 and p113 antibodies, and A. Alcover, O. Acuto, and C. Alcaide for critical reading of the manuscript.


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