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 ()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-/ 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-/ 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-/ 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-, phosphorylation of the downstream transcriptional components (p113 or STAT2 and p91/p84 or STAT1/) 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-/ responsiveness(18, 21) , whereas complementation of mutant U4A with JAK1 cDNA restored both IFN-/ 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-, that suggested the existence of a minor IFN--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-/ 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- and IFN- and the signaling activities induced in these transfectants were analyzed and compared.

MATERIALS AND METHODS

Cell Culture and Transfection

tyk2 Deletion Constructs

Northern Analysis

Binding Experiments with I-Labeled IFN

Immunoblotting and Immunoprecipitation

In Vitro Kinase Assay

RESULTS

Biological Activity of tyk2 Deletion Mutants

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-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 transfectants to grow in the selective medium (HAT) in the presence of different concentrations of IFN-. 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- (Wellferon, a mixture of purified human subtypes). TK-expressing cells survived in HAT medium only when a higher concentration of IFN- was used. In contrast, 11,1 cells and KL- and N-expressing cells failed to grow at any given concentration of IFN- tested (Fig. 2A). To rule out the possibility that this behavior was unique to 11,1 derivatives, we generated TK and KL neo 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- of the indicated cell lines was tested by seeding 3 10 cells/well in a 24-well plate in HAT medium without or with the indicated concentrations of IFN- (Wellferon). After 6-7 days, cells were fixed and stained. B, comparison of the dose-response range for IFN-8 and IFN- of mutant 11,1 cells and its derivative transfectants expressing wild-type tyk2 or the deleted TK 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 1% growth response, and on the right side at 100% 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 TK and N.

We next tested the sensitivities of the 11,1 transfectants to recombinant IFN-8 and -. These results are summarized in Fig. 2B. The specific activity of IFN- was higher than that of IFN-8 on all clones tested. There was a 30-100-fold difference in the sensitivities of TK-expressing cells and 11,1 to either IFN. Interestingly, the sensitivity of TK-expressing cells to IFN-8 attained the level of sensitivity of 11,1 cells to IFN-. The analysis of the KL- 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 TK form can, however, partially reconstitute sensitivity to both IFNs.

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

Figure 3: Induction of the 6-16 mRNA by IFN- 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- (Wellferon) for 4 h were hybridized to a 6-16-specific cDNA probe. Comparable signals in each lane were obtained with a -actin probe (data not shown).

TK Contributes to the Affinity of Binding of IFN-

Figure 4: Binding of human IFN-8 and IFN- to cells expressing wild-type or deleted tyk2 forms. A, Kinetics of uptake of I-labeled human IFN- (280 pM) at 37 °C by , 2fTGH cells; ; 11,1 cells; ; wild-type tyk2-expressing cells; and , TK-expressing cells. Values are femtomoles of IFN taken up per million cells. B, kinetics of uptake of I-labeled human IFN-8 (350 pM) at 37 °C by 11,1 cells; , wild-type tyk2-expressing cells; , TK-expressing cells. Values are femtomoles of IFN taken up per million cells. C, Scatchard plots of direct binding of I-labeled human IFN-8 (, ) and IFN- (, ) for 6 h at 4 °C to , , TK-expressing cells; , , KL-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-8 and IFN- at 4 °C on the partially sensitive TK-expressing cells and on the IFN-insensitive KL-expressing cells. The results, in the form of Scatchard graphs, are shown in Fig. 4C. The binding of IFN- was nearly the same on the two cell lines, showing that, for IFN-, receptor expression appears to be unaffected by the status of tyk2. The binding of IFN-8 on the TK-expressing cells was greater and shows a Scatchard plot with a slope 2.4 times greater than that obtained on the KL-expressing cells. This suggests that the mutant protein TK contributes to the affinity of binding of IFN- over the concentration range used. The Scatchard coefficient gave K equivalent to 0.54 nM and 0.62 nM for IFN-, 0.87 nM and >2.1 nM for IFN-8, on TK- and KL-expressing cells, respectively (the K 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-8 on KL cells is a lower binding affinity.

Deletion of Either Kinase Domain Affects Phosphorylation of tyk2

Figure 5: In vivo and in vitro tyrosine phosphorylation of tyk2 in response to IFN- in wild-type and deleted tyk2-expressing cells. A, Anti-tyk2 immunoprecipitates from wild-type, deleted tyk2-expressing, and mutant 11,1 cells (5 10 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-. 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- treated (1000 IU/ml Wellferon) wild-type, 11,1, KL, and TK-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 KL and TK forms are marked.

IFN- 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 TK and KL mutant forms. As shown in Fig. 5B, wild-type tyk2 exhibited increased autophosphorylating activity upon IFN- treatment. In contrast, TK and KL 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- treatment, enolase was markedly phosphorylated by wild-type tyk2 (Fig. 5B, lane 2). In contrast, the exogenous substrate was not phosphorylated in KL and TK-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

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- (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- (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- treatment of cells expressing the tyk2 deletion mutants. Phosphorylated p113 was detected in wild-type tyk2-expressing cells after 20 min of IFN- 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 p113p91 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 TK-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 KL 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. ()Thus, it appears that, when juxtaposed to the NH-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 KL 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 KL 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 KL. Tyk2 deleted of its TK domain (TK) 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 KL and N behaved essentially as cells lacking tyk2. Conversely, expression of the TK protein resulted in a partial reconstitution of sensitivity toward IFN- and in an increased sensitivity to IFN- (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 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- uptake is partially restored by the TK mutant protein, probably through an increase in binding affinity (Fig. 4). The inability of the N and the KL 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) . ()Despite an increase in the affinity of binding of IFN- on TK-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 TK protein toward ligand binding is much less evident for IFN- than for IFN-. While there is a partial reconstitution of sensitivity towards IFN- with the TK 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-/ response, with a more direct structural relationship with IFN- than with IFN-, 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- can utilize a minor or tyk2-independent binding pathway(s), an option not available to IFN-. Further work will be needed to identify elements unique to the IFN- response and the recent report of a protein that can be coprecipitated with one ligand binding protein and phosphorylated only following IFN- treatment goes into that direction(40) . If the apparently greater flexibility of IFN- is a reflection of its evolutionary seniority (41) , then the close dependence of IFN- and tyk2 may have its origin in the diversification of the mammalian IFNs.

Figure 7: Tyk2 in the receptor-mediated pathways induced by IFN- and -. 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-, whereas IFN- 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 TK-expressing cells in response to IFN-, possibly due to the low number of molecules activated and the limited sensitivity of this assay. Recently, it has been shown that IFN-/ induce tyrosine phosphorylation of the p95 proto-oncogene in hematopoietic cell lines, suggesting that vav may be involved in an alternative IFN-/ 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-/ 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 TK form contribute to receptor function. In parallel, the study of the JAK1-deficient U4A mutant cell line, whose IFN- 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-/-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.

()

The abbreviations used are: TK, tyrosine kinase; KL, kinase-like; IFN, interferon; IFNAR, interferon- 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).

()

L. Velazquez, R. McKendry, S. Pellegrini, unpublished observation.

()

G. Barbieri, K. Cheung, L. Ling, S. Pellegrini, unpublished observation.

ACKNOWLEDGEMENTS

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