activation of the protein tyrosine kinase tyk2 by interferon α/β

Eur. J. Biochem. 223, 427-435 (1994) 0 FEBS 1994

Activation of the protein tyrosine kinase tyk2 by interferon GIIp Giovanna BARBIERI', Laura VELAZQUEZ', Marina SCROBOGNA', Marc FELLOUS' and Sandra PELLEGRINI' ' UnitC INSERM 276, Institut Pasteur, Paris, France ' Istituto San Raffaele, Milano, Italy

(Received February 28/May 5 , 1994) - EJB 94 0276/1

We previously demonstrated that the gene tyk2 rescues the phenotype of a human mutant cell line unresponsive to a (IFN) and partially responsive to IFN-P. Here, we describe functional complementation of the mutant cells with the corresponding cDNA. To characterize the putative non- receptor protein tyrosine kinase encoded by the gene tyk2 and begin to understand its functioning, we have raised polyclonal antibodies against a segment of the protein. Using these, we have identified tyk2 as a 134-kDa protein which is rapidly and transiently phosphorylated on tyrosine in response to IFN-CXIp and possesses an inducible kinase activity when tested in v i m . IFN-y has no effect on the phosphorylation state of the protein. In agreement with previous genetic evidence, these results assign a role to tyk2 in the IFN-a/p signalling pathway and not in the IFN-y pathway. Fractionation of cell lysates have helped to localize the bulk of the protein in the cytoplasm, with a minor fraction associated with the cell membrane. Both protein pools undergo activation upon short- term IFN treatment of intact cells. Through the study of the effect of pervanadate on the phosphorylation level and the activity of tyk2, we conclude that activation of tyk2 by IFN-a does not require an intermediate regulatory tyrosine phosphatase.

Interferons (IFN) constitute a family of polypeptide cytokines secreted in response to viral infection and exhibiting a wide range of biological activities [l]. IFN-a/p and IFN-y bind to distinct transmembrane glycoproteins, which lack a catalytic domain, and interact with accessory signalling components [2, 31. Rapid transcriptional activation of distinct but overlapping sets of genes occurs in response to IFN-a/p and IFN-y treatment (for review, see [4, 51). The early signalling events triggered by IFN-aIp are beginning to be clarified. IFN-aIp binding activates, within a few minutes, three proteins (p113, p91 and p84) which associate, translocate to the nucleus and bind to the IFN-stimulated response element (ISRE) in the promoter of IFN-a/p responsive genes through p48, a fourth component with DNA-binding activity (re- viewed in [6]). The whole multimeric transcriptional activator is known as IFN-stimulated gene factor 3 (ISGF3). Re- cent work has shown that, in response to IFN-a, p113, p91 and p84 undergo phosphorylation on a single tyrosine site, this event being necessary for assembly and nuclear translocation of the complex [7].

The protein tyrosine kinases (PTK) involved in these early signalling events have been recently identified through the study of cellular mutants isolated from a mutagenized human cell line (2fTGH) using a metabolic selection strategy previously described [8]. Mutants in complementation group

Correspondence to S. Pellegrini, UnitC INSERM 276, Institut Pasteur, 25, rue du Docteur Roux, F-75724, Paris Cedex 15, France

Abbreviations. JFN, interferon; ISRE, IFN-stimulated response element; ISGF3, IFN-stimulated gene factor 3 ; PTK, protein tyrosine kinase ; HAT, hypoxanthine/aminopterin/thymidine; EBV, Ep- stein Barr virus ; VSV-G, Vesicular Stomatitis virus glycoprotein ; GST, gluthatione-S-transferase; NP40, nonidet P-40.

I are unresponsive to a IFNs, partially responsive to IFN-jl and respond normally to IFN- y . Furthermore, binding of IFN-a impaired and, consequently, no ISGF3 activation occurs [8,9]. The mutant cell line 11,l (or U1) has been geneti- cally complemented and the isolation of a cosmid capable of rescuing the mutated phenotype has been reported [lo]. This cosmid was shown to encode tyk2, a member of the JAK family of non-receptor PTKs, characterized by the presence of a large non-catalytic region and two kinase domains ar- ranged in tandem. The amino-proximal domain has sequence motifs which deviate from both serinekhreonine or tyrosine kinase motifs, whereas the carboxy-terminal domain contains all the motifs conserved among tyrosine kinases [ 10 - 131. The two other known members of this family have been recently shown to complement two other IFN-response cellular mutants. JAKl [14] rescues the phenotype of mutant U4, defective in both IFN alp and IFN-y signalling [ 151, whereas JAK2 [14, 161 complements mutant ylA, defective in IFN-y signalling [ 171. Furthermore, a more general involvement of the JAKs in intracellular signalling has been suggested by the discovery of their activation by a large number of cytokines and growWdifferentiation factors [18 -231.

We have now obtained a full-length tyk2 cDNA that effi- ciently rescues the altered 11,l phenotype. We demonstrate here that tyk2, a 134-kDa protein, resides in the cytosol with only a minor fraction associated with the membrane. Both protein pools are modified by tyrosyl phosphorylation in response to IFN-a/p but not to IFN-y. We have studied the effect of the tyrosine phosphatase pervanadate on the phosphorylation state and activity of tyk2 and we provide evidence for activation of tyk2 by IFN-a, even in the presence of this inhibitor.

428

MATERIALS AND METHODS Cell culture and transfections

The parental 2fTGH cell5, mutants 11,l and U1C and the DDD3-1,5 cells (derivative of 11,l) have all been previously described [8-lo]. Cells were routinely cultured in Dul- becco’s modified Eagle’s medium supplemented with 10% heat-inactivated foetal calf serum in the presence of hygro- mycin (250 pg/ml). Plasmid DNA transfections of 11,l cells were carried out using calcium phosphate as previously described [ 81 and selection for complemented transfectants was in hypoxanthine/aminopterin/thymidine (HAT) medium and 500 IU/ml Wellferon, a highly purified mixture of human IFN-a subtypes (1 Ox IU/mg protein, Wellcome Research Laboratories) [24]. The stability of the IFN-responsive phenotype of complemented clones was analyzed after culturing cells in non-selective medium for two weeks. Briefly, cells were seeded in HAT plus or minus IFN and in 6-thioguanine plus or minus IFN and examined microscopically [8]. Re- combinant human IFN-p (Asta Pharma) was a gift of Dr G. U Z ~ . Recombinant human IFN-y was kindly provided by Roussel-Uclaf. Treatment of cultured cells with the tyrosine phosphatase inhibitor was performed as follows. Stock solu- tion of H202 and sodium orthovanadate (Na,VO,) were mixed for 15 min at room temperature to generate pervanadate [25]. Freshly prepared pervanadate was added to the medium to a final concentration of 0.5 mM H,O,, 1 mM Na,VO, for the times indicated.

cDNA isolation and plasmid constructs Tyk2 cDNA was isolated from a size-selected HeLa

cDNA expression library constructed in the Epstein-Ban- virus-(EBV)-based shuttle vector, pDR2 [26] (the library was kindly provided by Dr M. Miiller). In this vector, the cDNA is driven by the Rous Sarcoma virus LTR. The EBV origin of replication and the EBNA-1 nuclear antigen allow high copy episomal replication of the plasmid in human cells. Phages (5X1O5) were plated on a lawn of Escherichia coli LE392 absorbed onto Hybond-N membranes and hybridized with a i2P-labeled 500-bp BarnHI fragment of the 3-kb cDNA clone [lo]. One positive plaque (H9) was picked and the phage DNA could be rescued as a plasmid upon infection of the bacterial strain AM1 as described [26]. Plasmid DNA was isolated by the alkaline lysis method and analyzed by restriction-enzyme mapping. The H9 insert is 4.14 kb in length, it has 5’ and 3‘ untranslated regions of 267 nucleotides and 318 nucleotides, respectively, and it contains the 18-bp stretch previously reported by us to be absent in the 3-kb cDNA molecule [ 101. Sequencing revealed that clone H9 had a deletion of 52 bp, creating a stop codon at nucleotide 2588. The deletion is flanked by a 5-bp direct repeat and might have arisen during first-strand cDNA synthesis. H9S was derived from plasmid H9 by substituting an SphI- BJsHII fragment of 1.59 kb with the corresponding fragment from the 3-kb cDNA. H9S contains an open reading frame of 3561 bp, as previously reported [12]. H9S was tagged at its 3‘ end with an oligonucleotide encoding an epitope of the Vesicular Stomatitis virus glycoprotein (VSV-G) [27]. A 492- bp sequence from the C-terminal end of tyk2 cDNA was isolated from H9S using the PCR. The 5‘ primer covered nucleotides 3336-3356, including a BssHII site at nucleotide 3344. The 3’ PCR primer covered nucleotides 3803-3828. In addition, an EcoRI site and a SmaI site were added to the end of the 5‘ and 3’ primers, respectively. The DNA was

amplified and digested with EcoRI and SmaI. The 499-bp PCR product was gel purified and ligated to an EcoRI- SmaI-digested plasmid containing the VSV-G epitope sequence (obtained from M. Arpin). The resulting construct (p499Eco/SmaVSV) contained nucleotides 3336- 3828 of tyk2 cDNA, followed by the 60-bp VSV-G sequence. To construct H9S-tag, the plasmid p499Eco/SmaVSV was digested with BssHII and XbaI and the 540-bp chimeric fragment was purified and ligated to H9S digested with BssHII and XbnI. Nucleotide sequencing was performed by the dideoxy-chain- termination method using a Sequenase kit (USB) and double- stranded plasmid DNA as template.

Preparation of anti-tyk2 antibodies A polyclonal antiserum was raised in rabbits against a

segment of the tyk2 protein. A 500-bp BamHI fragment from the 3-kb cDNA (nucleotides 1130-1616 in tyk2) was cloned into the bacterial expression vector pGEX-3X [28]. The recombinant plasmid encodes a 46.5-kDa gluthatione S-transferase (GST) fusion protein of which 20.5 kDa are from tyk2 (amino acids 289-451). The antisera R4 and R5 were cleared on a GST column and antibodies were immunoaffin- ity purified through a CNBr-activated Sepharose 4B (Phar- macia) column coupled to a GST-tyk2 fusion protein.

Immunoblot and immunoprecipitation analyses For immunoblotting, cells were rinsed in NaCIP,

(130 mM NaC1, 2.7 mM KCl, 10 mM Na,HP04, 1.8 mM KH2P04) and lysed in Triton X-100 lysis buffer (300mM NaC1, 50 mM Tris/HCl, pH 7.6, 0.5% Triton X-100, 10 pg/ ml leupeptin, 10 @ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). Protein concentrations were determined with the Bio-Rad Protein Assay (Bio-Rad) according to manifacturer’s instructions. For immunoprecipitation, 5 X lo6 cells were lysed in nonidet P-40 (NP40) lysis buffer (0.5% NP40, 50 mM Tris/HCl, pH 8, 10% glycerol, 0.1 mM EDTA, 150 mM NaC1, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, 3 pg/ml aprotinin, 1 pg/ml leupeptin, 0.5 pg/ml pepstatin, 1 mM sodium orthovanadate) followed by removal of insoluble material by centrifugation. Lysates were cleared with a non-specific immune antiserum previously coupled to protein-A- Sepharose (Pharmacia), then incubated for 2 h at 4°C with 1 pg affinity-purified anti-tyk, previously coupled to protein-A- Sepharose. Immunopreci- pitates were washed four times in NaClP,, 1% Triton X- 100,0.1% SDS and once in NaCVP,. The immunoprecipitates were boiled for 5 min in SDS sample buffer, resolved on a 7% SDSPAGE and electroblotted to a nitrocellulose filter (Hybond-C Super, Amersham). After blocking, filters were incubated with a monoclonal anti-phosphotyrosine (4G10; Upstate Biotechnology) or with affinity-purified anti-tyk antibodies (0.5 yg/ml). An ECL Western-blotting detection system (Amersham) was used according to the manufacturer’s instructions. When indicated, the alkaline phosphatase detection system was used. Densitometric scanning was performed with a high resolution CCD camera (Masterscan Scanalytics, Billerica).

In vitro kinase assay Immunoprecipitates were washed six times with 150 mM

NaC1, 0.1% Triton X-100 in buffer W (8 mM sodium phosphate buffer, 1 mM EDTA, 1 mM EGTA), once without Tri-

429

A B

200 -

p134 b 116 -

200 -

4 p134 116-

97 - 97-

Blt. ab : anti-tyk Ippt. ab : anti-tyk Blt. ab : anti-tyk

Fig. 1. Identification of the tyk2 protein in human cell lines. (A) Immunoblotting. Post-nuclear cell lysates (150 pg proteidlane) from subconfluent cultures were fractionated on a 7% SDSRAGE and transferred to nitrocellulose membrane. Immunostaining with the anti-tyk antibodies was revealed with an alkaline-phosphatase-coupled secondary antibody. DDD3-1,5 is a derivative of mutant 11,l complemented with genomic DNA. Mutant U1C belongs to the same complementation group as 11,l. Other human cell lines analyzed were HeLa, a human carcinoma, Daudi, a Burkitt’s lymphoma and Molt4, a T-cell line. (B) Immunoprecipitation analyzed by immunoblot. Post-nuclear extracts from 2ffGH, 11,l and H9,5 cells (5X106) were immunoprecipitated with anti-tyk, subjected to SDSRAGE and analyzed by immunoblotting with anti-tyk. Immunoreactive bands were visualized using the enhanced chemiluminescence system. The position of tyk2 (p134) is marked by an arrow.

ton X-100, once in kinase buffer (50mM Hepes, pH7.4, 10 mM MgCl,) and resuspended in a total reaction volume of 25 pl kinase buffer containing 10 pCi [y-12P]ATP (3000 Ci/ mmol, Amersham). In the experiment shown in Fig. 4B, 5 pg acid-denatured rabbit muscle enolase (Boehringer) was added to the reaction as an exogenous substrate. The kinase reaction was incubated for 10min at 30°C and terminated by adding 10 p1 50 mM EDTA. Beads were washed once in kinase buffer. Phosphorylated products were resolved on a 6% SDSPAGE which was stained with Coomassie blue, de- stained and visualized directly by autoradiography (Fig. 5B) or transferred to Immobilon-P PVDF membranes (Millipore).

Subcellular fractionation 2X107 cells were washed twice in ice-cold NaCI/P, and

scraped in ice-cold hypotonic buffer (5 mM KC1, 1 mM MgC12, 20 mM Hepes, 2 mM phenylmethylsulfonyl fluoride, 1 pg/ml leupeptin, 3 pg/ml aprotinin, 1 mM dithiothreitol and 1 mM sodium orthovanadate, pH 7.5) [29] for 15 min on ice. Cells were disrupted with a tight-fitting pestle of a Potter homogenizer (=50 strokes). The extent of cell breakage was monitored microscopically. Nuclei were separated by centrifugation at 8OOXg for 10min. The supernatant was centrifuged at 50000Xg for 1 h to pellet the particulate fraction and yield the cytosolic fraction. The crude nuclear pellet was washed twice in hypotonic buffer containing 0.5% NP-40 and 0.25% sodium deoxycolate and lysed by sonication in 50 mM Tris, pH 7.5, 150 mM NaCI, 0.5% NP-40, 0.25% sodium deoxycolate, 0.1% SDS, l mM sodium orthovanadate and protease inhibitors. The nuclear fraction was centrifuged at 1OOOOXg for 30min to pellet debris, then analyzed directly by immunoblotting (Fig. 6A). In Fig. 6B, the membrane fractions were extracted in buffer A (25 mM Tris,

pH7.5, 250mM sucrose, 2.5mM MgC12, 5 mM EGTA, 5 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 1 mM Na,VO, and protease inhibitors) for 30min in ice, centrifuged at 1OOOOXg for 30 min to pellet debris and immunoprecipitated.

RESULTS ‘Qk2 cDNA reverts the 11,l phenotype

We have previously reported the identification of tyk2 as the gene capable of restoring IFN sensitivity in mutant 11,l [lo]. A full-length cDNA (plasmid H9S) was isolated from a HeLa library made in an EBV-based expression vector that allows episomal replication and maintenance of the transfected cDNA in human cells (see Materials and Methods) [26]. To demonstrate the biological activity of this clone, we tested its ability to complement the mutant phenotype. For this, 11,l cells were transfected with H9S and selected in HAT medium plus IFN. This medium selects transfectants that have reverted to IFN-a/P sensitivity [lo]. H9S reverted the phenotype of transfected cells at a frequency 10-fold higher than the tyk2-encoding cosmid [lo]. A similar result was obtained when mutant UlC, of the same complementation group as 11,l [9], was used as recipient for transfection. We, therefore, conclude that the plasmid H9S encodes the protein capable of reverting the phenotype of mutants be- longing to complementation group I.

Complemented cDNA transfectants expressed variable levels of two tyk-specific transcripts (4.1 kb and 4.4 kb), the largest transcript most likely representing a product utilizing the simian virus 40 poly-adenylation signal provided by the vector. Clone H9,5 was further studied as it expressed high tyk2 mRNA levels (data not shown) and displayed a stable

430

A B IFN a: - 2' 5' 15' 30' 60' - 2' 5' 15'30' 60'

- 200 -

p134 b 4 p134 - 116 -

- 97 -

Blt. ab : anti-P-Tyr Blt. ab : anti-tyk

Fig.2. Time course of IFN-a-induced tyrosyl phosphorylation of tyk2. Lysates of 2ffGH cells (5X106), untreated or treated for the indicated times with IFN-rx (1 000 IU/ml), were immunoprecipitated with anti-tyk. Immunocomplexes were fractionated on SDSPAGE and immunoblotted fint with anti-phosphotyrosine (A), then with anti-tyk (B). The position of tyk2 (p134) is marked by an arrow.

2fTGH 1 1 , l H9,5

I 1 2 3 4 5 1 I1 2 3 4 5 1 I1 2 3 4 5 1

200 -

p134 b 116-

97 -

200 -

p134 b 116-

97 -

Blt. ab:

anti-P-Tyr

anti-tyk

Fig. 3. Tyrosyl phosphorylation of tyk2 in response to IFNs. Anti-tyk immunoprecipitates from parental 2fTGH, mutant 11,l and complemented H9,5 Iysates (5 X 10' cells) were immunoblotted with either anti-phosphotyrosine (upper panels) or anti-tyk (lower panels). IFN treatments (1000 IU/ml) were as follows: lanes 1, no treatment; lanes 2, 16 h with IFN-y; lanes 3, 15 min with IFN-a; lanes 4, 15 min with IFN-/I; lanes 5 , 15 min with IFN-y. The position of tyk2 (p134) is marked by an arrow.

complemented phenotype due to maintenance of the epi- soma1 DNA during propagation in non-selective medium.

Identification of p134 as the tyk2 protein A polyclonal antiserum was raised against a segment of

the protein produced in bacteria and used in immunoblotting and immunoprecipitation experiments (see Materials and Methods). Fig. 1 A shows that a protein of 134 kDa (p134) was recognized by anti-tyk in immunoblots of post-nuclear lysates from various human cell lines. This protein was absent in mutant 11 , I and present in complemented transfectants, such as DDD3-1,5 [lo] and H9,5. The level of p134 in H9,5 cells was higher than in 2ffGH cells, as expected from

a more abundant transcript level. Two minor immunoreactive bands were detected in Western blots as well as in immunoprecipitation experiments in all cDNA transfectants analyzed and could represent aberrant spliced products (H9,5 in Fig. 1 ; see also T2 in Fig. 4B). In mutant UlC, which expresses very low levels of tyk mRNA [lo], p134 was not present. p134 was not detected when pre-immune serum or a non- specific antiserum were used in immunoblots (data not shown) .

The ability of the anti-tyk to immunoprecipitate pl34 was tested in parental, mutant and complemented H9,5 cells. Im- munoprecipitates were subjected to immunoblot using anti- tyk; p134 was detected in parental cells and, in higher amount, in H9,5 (Fig. 1B). No trace of p134 was detected in immunoprecipitates of 11,l lysates.

431

A

2fTGH 1 1 , l I - 1 ' 5' 15' 30' 60' 1 - 5' I

- 200

+- P I 3 4

-116

- 97

- 66

B T2 1 1 , l

-1 - 200

f- P I 3 4

- 116

- 97

- 66

- IgG - En

- p134 - p134

C

+

P-peptides 0 , i i i j

o r i 0 I + Fig. 4. IFN-a stimulation activates tyk2 in vitro kinase activity. (A) Anti-tyk immunoprecipitates from post-nuclear lysates (800 pg) of untreated or IFN-a-treated 2ffGH and 11,1 cells were assayed in an in vitro kinase assay. Kinase reaction products were fractionated on a 6% SDSPAGE and transferred to a membrane. Following autoradiography, the filter was probed with anti-tyk (bottom panel). (B) Anti- (VSV-G tag) immunoprecipitates from lysates (700 pg) of T2 and 11,1 cells were assayed as described above, except that enolase (En) was added to the reaction as an exogenous substrate. The positions of tyk2 (p134), enolase (En) and immunoglobulins (IgG) are marked. The filter was subsequently probed with anti-tyk (bottom panel). (C) Tyk2 was immunoprecipitated from 2ffGH cells treated for 5 min with IFN-a and subjected to an in vitro kinase assay. The 134-kDa phosphoprotein product was hydrolyzed from the poly(viny1idine difluoride) membrane in 6 M HC1 (60 min at 110°C) and the phosphoamino acids were analyzed by two-dimensional thin-layer electrophoresis [39]. The positions of the origin and of the phosphoamino acid standards are indicated.

IFN-a and IFN-j?, but not IFN-y, induce tyrosyl phosphorylation of tyk2

Protein tyrosine kinases have been shown to autophosphorylate at tyrosine residues in vitro and in vivo in response to the proper stimuli and this event is commonly associated with enhanced kinase activity. Thus, we first investigated whether treatment of cells with IFN-a had an effect on the in vivo tyrosine phosphorylation content of the protein. For this, tyk2 immunoprecipitates of 2ff GH lysates were analyzed by immunoblotting with either anti-tyk or anti-phosphotyrosine antibodies. Both antibodies recognized pl34. In the experiment shown in Fig. 2, the phosphorylated p134

band was barely detectable in untreated cells. However, we have observed some variability among experiments in the intensity of this signal. Within 2 min of addition of IFN-a, we observed an increase in the tyrosine phosphorylation of p134, which reached a maximum at 15 min and returned to the basal level after 60 min.

The phosphotyrosine content of tyk2 was also studied in parental, mutant and complemented H9,5 cells, before and after 15-min treatment with either IFN-a or IFN-P. These treatments increased pl34 phosphorylation in parental and complemented cell lines (Fig. 3, upper panels, lanes 3 and 4). The basal and the induced levels of phosphorylated p134

432

A were higher in H9.5 cells than in 2fTGH. From these results, we conclude that the interaction of IFN-a/P with its cognate receptor triggers rapid phosphorylation of tyk2 on tyrosine residues. Conversely, 15 min or 16 h treatment with IFN-y (Fig. 3, lanes 2 and 5) had no detectable effect on the abun-

fore not modified in response to IFN-y.

HrOz : 30' - 5' 15' 30' Pervanad.:

dance of pl34 or on its phosphorylation state. Tyk2 is there- ~ ' I F N - c x : - + - + - + - + - +

In vitro kinase activity of tyk2 Blt. ab : anti-P-Tyr

Blt. ab : anti-tyk2

To determine if tyk2 possessed kinase activity, the protein was immunoprecipitated from 2fTGH cells untreated or treated for various times with IFN-a, and tested for autophosphorylation in an in vitro kinase reaction (see Materials and Methods). 11,l cells were assayed in parallel. As shown in Fig. 4A, a phosphorylated 134-Da band was detected only in the 2ffGH immunoprecipitates and its intensity increased 5-10-fold upon 5 min of IFN-a treatment. The other phosphorylated bands are not tyk2 co-immunoprecipitating proteins since they are also present in the 11,l reactions. Simi-

sent a protein structurally related to tyk2. To eliminate the possibility of precipitating cross-reactive kinases, a monoclonal antibody to a VSV-G epitope 1271 was used to immuno- Pervanad.: 5' 15' 30' precipitate a tagged version of the tyk2 protein expressed into an 11,l complemented derivative named T2. Enolase was added to this in vitro reaction as an exogenous substrate. As shown in Fig. 4B, only tyk2 and enolase were phosphory-

profile of enolase was obtained with 2fTGH cells (data not shown).

To verify that tyk2 incorporates phosphate into tyrosyl

larly, the slow migrating band of over 200 kDa could repre- B

5' 1FN-a : -+I[ - + + ' - + + ' I - + + ' lated following IFN-rx treatment. A similar phosphorylation +

residues, phosphoamino acid analysis was performed on the - c 7

in vitrt, "P-labeled 134-kDa band (Fig. 4A). The radio- .- c 7

.- c 7

activity was incorporated almost exclusively into tyrosyl residues (Fig. 4C). Fig. 5. Effect of pervanadate on the tyrosine phosphorylation

state of tyk2 and on its in vitro kinase activity. 2ffGH cells were treated for the indicated times with pervanadate (orthovanadate

IFN-dependent activation of tyk2 does not require the action of a phosphatase

We next investigated whether the activity of a phosphatase is required for the IFN-dependent in vivo and in vitro activation of tyk2. For this, 2fTGH cells were cultured for increasing lengths of time in the presence of the tyrosine phosphatase inhibitor sodium pervanadate. Cells were then treated for 5 min with IFN-a. Anti-tyk immunoprecipitates were analyzed by immunoblotting to measure the in vivo phosphotyrosjne content of the protein. As shown in Fig. 5A, pervanadate progressively increased the amount of the phosphorylated tyk2 band in untreated cells. IFN-induced phosphorylation of the protein was still observed above this basal level. The in vitro kinase activity from parallel cultures was also assayed. As shown in Fig. SB, no difference in the basal or in the induced tyk2 activity was detected between control and pervanadate-treated cells. A similar phosphorylation profile of the exogenous substrate enolase was observed (data not shown). Altogether. these results suggest that the activation of tyk2 by IFN-u does not rely on the action of a tyrosine phosphatase.

Subcellular localization of the tyk2 protein

mixed with H,O,) or with H,O, alone and stimulated for 5 min with IFN-a. In (A), tyk2 immunoprecipitates were analyzed by western blotting to anti-phosphotyrosine and to anti-tyk2. In (B), 2ffGH cultures (and 11,l when indicated) were treated as described above. Anti-tyk immunoprecipitates were subjected to in vifro kinase as- says, fractionated on a 6% SDSBAGE and the products were visualized by autoradiography. The arrow indicates the position of tyk2.

spectively, in Fig. 6A) were assayed by SDS/PAGE and immunoblotting. The purity of the fractions was confirmed by analyzing the distribution of enolase, an abundant protein which is solely cytosolic, and of human brm, a nuclear transcriptional activator [30]. Tyk2 immunoblotting revealed that the bulk of the protein (-80%) is cytosolic and -15% is membrane associated. A weak but reproducible signal was present in the nuclear fraction. IFN-a treatment did not cause a detectable change in the distribution of the protein (data not shown). When a similar experiment was performed on T2 cells, the two minor tyk2 forms appeared localized solely in the cytosol (data not shown), probably due to the inability of these shorter proteins to associate with membrane components.

We next compared the tyrosyl phosphorylation state of the membrane-associated and of the cytosolic tyk2, in un-

To determine the subcellular localization of tyk2, 2ffGH cells were fractionated (see Materials and Methods) and the particulate, cytosolic and nuclear fractions (M, C and N, re-

treated versus IFN-treated 2fTGH cells. For this; each fraction was immunoprecipitated with anti-tyk and immunoblotted. While the amount of tyk2 protein in each fraction re-

433

lppt ab : anti-tyk 2

- 5'15'30'60' IFN a - 5' 15'30'60'

- 200

p134 - 116

- 97

En - 45

-p134-

IFN a - 5' 15'30'60' - 5'15'30'60'

8 - 0

- 200 anti-P-tyr

A

pl34 -

anti-tyk 2

Fig. 6. Subcellular localization of tyk2 by fractionation of 2ff GH cells. (A) Immunoblotting of subcellular fractions. 2ffGH cells were fractionated into membrane (M), cytoplasmic (C) and nuclear (N) fractions. Aliquots from each fraction, equivalent to 1.8X lo6 cells, were tested for the presence of tyk2 protein by immunoblotting with anti-tyk2. In the cytoplasmic and nuclear fractions, bands cross-reacting with the polyclonal serum are visible. After stripping, the blot was probed with anti-enolase using alkaline phosphatase detection (bottom panel). Aliquots from each fractions were reacted with purified antibodies specific for the nuclear brm protein of 180 kDa [30]. (B) Phosphorylation of tyk2 in membrane and cytosolic fractions. Membrane and cytosolic fractions from 2ffGH cells untreated or IFN-a treated for the indicated times were immunoprecipitated with anti-tyk. The amount of tyk2 imunoprecipitated from the cytosolic fractions was adjusted to approximate the protein level in the membrane fractions. Immunoblots were analyzed with either anti-phosphotyrosine (left panel) or anti-tyk2 (right panel).

mained constant, increased phosphorylation of the protein was detected in both pools following addition of IFN-a (Fig. 6B). Treatments as short as 1 min yielded comparable results.

DISCUSSION Our understanding of the mechanism of signalling by

IFNs has recently progressed through the study of mutant cell lines and, in parallel, through the biochemical characterization of signalling components [6]. As in other pathways, regulated protein phosphorylation appears to play a central role in the propagation of the signal. We have started to characterize tyk2, a PTK identified by genetic means on the basis of its ability to complement the defect of a mutant cell line unresponsive to IFN-a. The gene tyk2 restores responsive- ness to IFN-a as it allows survival of the mutant in a defined selective medium [lo]. Here we extend our previous finding and demonstrate complementation of the mutant phenotype with the full-length cDNA. The generation of antibodies has allowed us to identify the product of the tyk2 gene as a 134- kDa protein constitutively expressed in human cell lines of fibroblastic and lymphoid origins. We have observed a low, though variable, basal level of tyrosyl phosphorylation of tyk2. One possible interpretation of this constitutive phosphorylation is that at any one time a small fraction of tyk2 might be in an activated state. It is conceivable that environ-

mental changes (concentration of serum constituents, cell density) could induce the production of small amounts of endogenous IFN (or other cytolunes) which in turn would activate tyk2. The study of a kinase-inactive mutant form of the protein should help to distinguish between a low level of tyk2 autophosphorylation and the action of another kinase. We have shown that treatment of cells with either IFN-a or IFN-/3 leads, within 1 min, to phosphorylation of tyk2. In addition, we have demonstrated an IFN-a stimulated tyk2 tyrosine kinase activity.

Three of the four components of the primary transcriptional activator of IFN-a//3 sensitive genes (ISGF3) have been shown to undergo phosphorylation on a tyrosine residue in response to IFN-a. The time course of IFN-induced tyk2 activation parallels the phosphorylation of ISGF3, suggesting that this kinase could directly phosphorylate one or more ISGF3 components in vivo.

We have tested whether a tyrosine phosphatase could be involved in tyk2 activation by IFN using the inhibitor pervanadate. Our data show that the IFN-induced phosphorylation of tyk2 in vivo as well as its in v i m activation occur in the presence of the inhibitor. This result, whose significance needs to be restricted to such phosphatases as inhibited under the experimental conditions used here, contrasts with the ob- servation made by David et al. [31]. Using isolated membranes, this group has provided indirect evidence for an IFN- a-dependent tyrosine phosphatase activity, preceding the ac-

434

tion of a tyrosine kinase and necessary for reconstitution of ISGF3 DNA-binding activity in a cell-free system [31]. In- terestingly, both activities appear to be membrane associated. One way to reconcile the discrepancy is to hypothesize a pervanadate-sensitive step downstream of tyk2 activation and yet necessary for ISGF3 formation. For instance, if recmite- ment of the ISGF3 components as substrates for phosphorylation is mediated by association between their src homology 2 (SH2) domains 132, 331 and specific phosphotyrosine-containing sequences, one could envisage the need for a phosphatase to dissociate the complex and release the modified ISGF3. Pervanadate-mediated inactivation of such phosphatase would abolish the release and thus the ISGF3 DNA- binding activity measured by David et al. without, however, affecting tyk2 activation by IFN.

Treatment of intact cells with pervanadate has an effect on the tyrosine phosphorylation of tyk2. However, under this condition tyk2 kinase activity is not augmented (Fig. 5B). Two alternative interpretations, as described, could explain this result: (a) pervanadate could lead to phosphorylation of tyrosyl residues which do not derepress the enzyme and which differ from the positive regulatory sites modified by IFN ; (b) pervanadate-induced tyrosine phosphorylation would not be sufficient to activate the enzyme and a second signal, triggered by IFN, would be needed. Mapping of the residues phosphorylated in response to IFN and pervanadate should help to distinguish between these two alternatives.

IFN-./ interacts with a receptor distinct from the IFN-a/b receptor and yet stimulates tyrosine phosphorylation and nuclear translocation of p91, one of the ISGF3 components [34, 351. The reaction occurs on the same site after IFN-a and IFN- ^J treatment. IFN-y activated p91 binds to the y- activation site in the promoter of 1FN-y-inducible genes and stimulates transcription. There is now clear genetic evidence for the involvement of p91 in both IFN-dP and IFN-y signalling [36]. Our finding that IFN-./ does not induce phosphorylation of tyk2 and yet activation of IFN-y-inducible genes occurs, even in tyk2-deficient cells, strongly suggest that this kinase is not the one responsible for IFN-y-dependent p91 phosphorylation.

A number of non-receptor PTKs are physically associated to cell surface proteins in haemopoietic cells 1371. Although it is sometimes difficult to demonstrate the biological significance of these associations, at least in some cases a direct role in early signalling has been demonstrated [38]. In this work, we have found that a minor fraction of tyk2 in the cell is associated with the membrane. No detectable relocaliza- tion of the protein was observed upon IFN-a treatment, suggesting that the association of tyk2 with membrane components is not ligand dependent. The inability to dissociate the activation event occurring at the membrane from the event in the cytosol (Fig. 6B) is most likely due to the rapidity of the phosphorylation event and the high turnover of the phosphorylated protein. We do not know what type of interaction retains tyk2 at the membrane, since the protein is not mirystoylated 1121. The finding that 11,l cells lack high affinity binding sites for IFN-a and that tyk2 complements this defect [8, 101 assigns a critical role to tyk2 in the formation of functional binding sites. In the simplest model, tyk2 would interact with the cytoplasmic domain of one or more receptor components and would autophosphorylate upon ligand binding.

It has recently been shown that in mutant U4, defective in both IF"-a/P and IFN-.,I signalling and complemented by the tyrosine kinase JAK1, neither tyk2 nor JAK2 can be acti-

vated by IFN-a or IFN-y, respectively [15]. These results point to a double role of JAKl in tyk2 activation by IFN-a/ p and in JAK2 activation by IFN-y and suggest a possible trans-phosphorylation event occurring between two juxta- posed members of the JAK family [15]. In order to further demonstrate the requirement of functional tyk2 for JAKl activation, we are studying JAKl activation in 11,l cells ex- pressing a kinase-inactive form of tyk2 which is still capable of restoring IFN-a binding.

It is now possible to address questions regarding the interaction of tyk2 with receptor components, its mode of regulation and the role of the kinase-related domain. A major goal is to understand the interdependence between JAKl and tyk2 activities, their precise role in receptor-complex formation and their direct substrate specificity. Preliminary data indicate that both kinase domains of tyk2 are necessary for full functional complementation of the mutant cell line (Vel- azquez, L. and Pellegrini, S., unpublished results), suggesting that the kinase-related domain is critical for the IFN-induced activity of the enzyme. The existence of the tyk2-deficient and JAKl-deficient cell lines will facilitate in vivo structure/ function analyses of mutated and chimaeric forms of these proteins.

We thank M. Arpin, T. Kreis, A. Giallongo, M. Muller and C. Muchardt for providing reagents, M. Arpin, P. Duplay and A. Smith for useful and technical suggestions during this work, M. Mac- Kichan for help with phosphoamino acid mapping, P. Duplay, G. Cossu and 0. Acuto for critical reading of the manuscript. G. B. was a recipient of a Poste Vert INSERM; L. Velazquez was supported by a fellowship from the Universidad Nacionul Autonoma de Mexico (UNAM) and M. Scrobogna was a student in the EEC ERASMUS project. This project was supported by grants from the Association Frangaise contre les Myopathies (AFM) and the Ligue Nationale contre le Cancer.

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activation of the protein tyrosine kinase tyk2 by interferon α/β

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