©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Jak Kinases Differentially Associate with the and (Accessory Factor) Chains of the Interferon Receptor to Form a Functional Receptor Unit Capable of Activating STAT Transcription Factors (*)

(Received for publication, February 27, 1995; and in revised form, May 11, 1995)

Minoru Sakatsume , Ken-ichi Igarashi , Karen D. Winestock , Gianni Garotta (1)(§), Andrew C. Larner , David S. Finbloom (¶)

From the Division of Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 and Pharmaceutical Research-New Technologies, Hoffmann LaRoche, Basel CH4002, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Interferon (IFN) induces the expression of early response genes by tyrosine phosphorylation of Jak kinases and transcription factors referred to as STAT proteins. The topology of the IFN receptor is partially understood and the relationship between the chain that binds the ligand and the chain that is required for signal transduction is undefined. In a cell line which expresses only the human chain, we show that these cells did not activate Jak kinases or STAT proteins with human IFN, even though Jak1 co-immunoprecipitated with the chain. In cells unexposed to IFN, Jak1 preferentially associated with the chain, while Jak2 associated with the chain. There was evidence for Jak1 kinase activity in untreated cells. For Jak2, kinase activity was IFN-dependent. Although the chain was tyrosine-phosphorylated in response to ligand, we found no evidence for tyrosine phosphorylation of the chain. These data are consistent with a model of the IFN receptor in which Jak1 associates with the chain, whereas Jak2 associates with the chain. IFN clusters at least two receptor units which results in the tyrosine phosphorylation of Jak1 and Jak2, the activation of Jak2 kinase activity, and the recruitment of STAT1 resulting in its activation by tyrosine phosphorylation.


INTRODUCTION

Cytokines such as interferons (IFN),()interleukins, and growth factors transduce signals across the cell membrane that result in the expression of early response genes. This process occurs through the activation of transcription factors, STAT proteins, (signal transducers and activators of transcription) and the tyk2/Jak family of protein tyrosine kinases(1, 2) . Whereas the STAT proteins possess SH2 and SH3 domains(3) , the Tyk/Jak kinases have two putative catalytic domains (of which only the carboxyl one is active) but lack SH2 or SH3 domains(4) . These kinases have been shown to be associated with several cytokine receptors that require dimerization by the ligand to initiate signal transduction. One such ligand is interferon (IFN), a homodimeric molecule, that bivalently binds to its receptor and activates the Jak/STAT pathway by clustering its receptor. The IFN receptor consists of a high affinity ligand binding chain () (K = 10M) that alone is incapable of transducing a signal across the membrane (5) . Another transmembrane protein (accessory factor) is required for signal transduction, and through the use of somatic cell hybrid cell lines, its gene has been determined to be located on chromosome 21(6) . The accessory factor/ chain interacts with the ligand binding component ( chain) in a species specific manner(7, 8) . The gene for the accessory factor ( chain) has been cloned and when co-expressed with the ligand binding chain reconstitutes a functional receptor unit(9, 10) . The cDNA for the chain encodes a transmembrane protein of 310 amino acids with six putative glycosylation sites and an intracytoplasmic domain of 66 amino acids.

Incubation of cells with IFN stimulates an immediate tyrosine phosphorylation of the chain(11, 12) . Although several tyrosines within the cytoplasmic domain are phosphorylated, the phosphorylation of tyrosine 440 is necessary for initiation of the signaling cascade as determined by site-directed mutagenesis(13) . Tyrosine phosphorylation of the chain occurs concomitantly with tyrosine phosphorylation of both Jak1 and Jak2(12, 14, 15, 16) . Both kinases form a multimolecular complex with the IFN receptor immediately after ligand binding(12) . STAT1 has been shown to bind to a peptide of the IFN receptor containing the phosphorylated tyrosine 440(11) . The contribution of each chain of the IFN receptor (IFNR) to the formation of this complex and the relationship of kinase activity to ligand binding still needs to be defined. We have carried out a series of experiments designed to assess the relationship between the and chains of the receptor and the Jak kinases in both cell lines, primary monocytes, and in somatic cell hybrids that express either the human chain or both the human and chains of the receptor.


MATERIALS AND METHODS

Cells

HeLa cells were maintained in stationary flasks or adapted for growth in spinner flasks as described previously(12) . Human peripheral blood monocytes were obtained from volunteers by leukapheresis. The monocytes were purified from the mononuclear cells by Ficoll-Hypaque (LSM, Organon Teknika, Durham, NC) sedimentation followed by countercurrent centrifugal elutriation, were >95% pure, and used without further culturing. The T41 cell line is a mouse L929 fibroblast cell line stably transfected with the human IFN receptor chain. The 3-1-12 cell line is a mouse-man somatic cell hybrid (WavR4d-F9-4a) retaining only chromosome 21 (17) and has been similarly transfected with the human IFN receptor chain. Chromosome 21 also encodes for the IFN receptor, and the responsiveness of the 3-1-12 cell line to IFN can be used to assess the ability of the cells to retain chromosome 21(18) .

Flow Cytometric Analysis

The ability of the T41 and 3-1-12 cell lines to respond to IFN or IFN was monitored by the enhancement of major histocompatibility class I antigen expression. Cells were treated with IFN for 24 h and then incubated with antibodies specific for major histocompatibility class I antigens followed by incubation with fluorescein isothiocyanate-conjugated secondary antibody. The cells were then analyzed by flow cytometric analysis on a FACScan.

Binding of IFN to Somatic Cell Hybrid Lines

Recombinant human IFN (Genentech, Inc., S. San Francisco, CA) was radiolabeled using Bolton-Hunter reagent (Amersham Corp.) to high specific activity as described(19) . Increasing concentrations of I-rIFN were incubated at 4 °C for 2 h with cells, and a 200-fold excess of unlabeled IFN was used in parallel tubes to correct for nonspecific binding. Free IFN was separated from bound IFN by centrifugation over a phthalate oil cushion as described (19) . Molecules bound per cell were calculated from the specific activity of the radiolabeled IFN.

DNA-binding Proteins

After treatment with IFN, cells were washed with cold phosphate-buffered saline and solubilized with cold whole cell extraction buffer (1 mM MgCl, 20 mM Hepes (pH 7.0), 10 mM KCl, 300 mM NaCl, 0.5 mM dithiothreitol, 0.1% Triton X-100, 200 µM phenylmethylsulfonyl fluoride, 1 mM vanadate, and 20% glycerol). DNA-binding proteins were determined by electrophoretic mobility shift assay as described previously(20) . Briefly, 10 µg of protein were incubated with the P-labeled oligonucleotide probe consisting of double-stranded GRR (5` AGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAG 3`) of the promoter of the Fcgr1 gene in binding buffer(21) . The sample was then applied to a 6% nondissociating polyacrylamide gel in order to separate free probe from probe bound to protein.

Biosynthetic Labeling of Cells

Monocytes were resuspended into medium deficient for methionine, cysteine, and glutamine for 30 min at 37 °C. This medium was replaced with fresh media deficient for the same amino acids containing 400 µCi of Translabel ([S[methionine and S-cysteine) (ICN, Costa Mesa, CA), and the cells continued in incubation for 3 h. The cells were then washed, lysed, and the extracts subjected to immunoprecipitation as described below.

Immunoprecipitations

Following treatment with IFN, cells (2 10) were solubilized in 1% Triton X-100 (Pierce) in a buffer consisting of 0.15 M NaCl, 50 mM Tris (pH 8.0), 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 mM orthovanadate. The post-nuclear lysate was then precleared with a suspension of protein G-agarose (Pharmacia Biotech Inc.) for 1 h at 4 °C. After separation of the protein G-agarose from the lysate by centrifugation, the lysate was incubated with either normal rabbit serum, rabbit anti-IFNR, rabbit anti-Jak1 (Upstate Biotechnology, Inc., Saranac Lake, NY), rabbit anti-Jak2 (Upstate Biotechnology, Inc.), or rabbit anti-STAT1 (22) for 2-18 h at 4 °C. Two different anti-IFNR antibodies were used. One is a polyclonal antibody (anti-IFNR) raised against purified chain of the receptor(23) , and the other is a polyclonal antibody (anti-AF-1) raised against a 30-amino acid peptide of the cytoplasmic domain of the accessory factor/ chain (amino acids 307-337). This antibody was affinity purified on an antigen-conjugated agarose column. Protein G-agarose was then added for 1 h at 4 °C to isolate the immune complexes. The protein G conjugates were then washed in lysis buffer and boiled in SDS sample buffer. For immunoprecipitations specifically designed to assess protein-protein interactions, cells were lysed in the same buffer as above except that digitonin was substituted for Triton X-100, and the immune complexes were washed in 0.1% digitonin containing buffer. The samples were then analyzed using 8% SDS-PAGE followed by electrophoretic transfer to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA) as described(12) .

In Vitro Kinase Assays

Cells, untreated or treated with IFN, were washed, solubilized, and the extracts subjected to immunoprecipitation as described above. The immunoprecipitates were then resuspended in kinase assay buffer (100 mM NaCl, 50 mM Hepes (pH 7.3), 0.1% Triton X-100, 6.25 mM MnCl, 0.5 mM dithiothreitol). To this buffer was added either 25 µM ATP and 5 µCi of [-P]ATP when autoradiography was performed or 2.5 mM ATP when anti-phosphotyrosine immunoblotting was performed. The reaction mixture was incubated for 15 min at 30 °C and the immunoprecipitates washed several times with lysis buffer prior to the addition of SDS sample buffer. The samples were then subjected to SDS-PAGE analysis followed by either autoradiography or immunoblotting with anti-phosphotyrosine antibodies (PY20) and enhanced chemiluminescent imaging.


RESULTS

IFN Signaling in the Somatic Cell Hybrid Cell Lines

The ability of the somatic cell hybrid 3-1-12 cell line to retain chromosome 21 was assessed using IFN. For the T41 cells there was no change in human IFN-induced major histocompatibility class I expression as measured by peak channel fluorescence (134 in untreated cells versus 132 for IFN treatment). This finding was consistent with the fact that the L929 cells are murine in origin and do not respond to human IFN (these cells do respond to murine IFN). In contrast, the 3-1-12 cell line responded to human IFN by enhanced peak channel fluorescence from 124 to 141. These data confirmed that chromosome 21 was present within this cell line.

In order to ensure that the transfected IFNR chain was adequately expressed, both the T41 and the 3-1-12 cell line were examined for the surface expression of IFNR by measuring the binding of I-rIFN (Fig. 1). The chain alone is sufficient to impart an high affinity binding state. Both cell lines expressed approximately similar numbers of binding sites: the T41 cells bound about 6000 molecules/cell, whereas the 3-1-12 cells bound about 5000 molecules/cell. The concentration at half-maximal binding for both cell lines was about 10M, which was consistent with a high affinity binding site. The affinity and number of molecules per cell for these cell lines was similar to previously reported values for other cells(19) .


Figure 1: Saturation binding of I-IFN to T41 and 3-1-12 cells. Cells were incubated at 4 °C with increasing concentrations of I-IFN with or without a 200-fold molar excess of unlabeled IFN. The cells were then centrifuged through phthalate oils and the radioactivity in the pellet (bound) and supernatant (free) were measured. Data are represented as molecules (mol) of IFN bound per cell. The curves represent specific binding of I-IFN to the cells. Open circles, T41 cells; closed circles, 3-1-12 cells.



The ability of the two cell lines to signal was examined next by measuring the formation of the IFN-activated GRR binding complex, FcRF (Fig. 2). In T41 cells only murine IFN stimulated the formation of FcRF (Fig. 2, lane 5). In contrast both murine (Fig. 2, lane 2) and human IFN (Fig. 2, lane 3) resulted in assembly of the FcRF complex in 3-1-12 cells. That the intensity of the signal was considerably less for human IFN compared with murine IFN in the 3-1-12 cell line was probably the result of limited expression and/or association of the component with the component of the IFNR. Since both the Jak1 and Jak2 tyrosine kinases have been shown to be involved in the signal transduction pathway for IFN, we next examined whether the component alone was sufficient for the ligand-induced tyrosine phosphorylation of these tyrosine kinases. Incubation of the 3-1-12 cell line with IFN resulted in the tyrosine phosphorylation of both Jak1 (Fig. 3A, lane 2) and Jak2 (Fig. 3B, lane 2). In contrast, in those cells expressing only the component of the receptor, no tyrosine phosphorylation of Jak1 (Fig. 3A, lane 4) or Jak2 (Fig. 3B, lane 4) was observed. This implied that the chain alone in spite of being capable of binding ligand with high affinity could not facilitate ligand induced tyrosine phosphorylation of the Jak kinases. When these same cells were examined for the tyrosine phosphorylation of STAT1 in response to IFN, the results were similar to that observed for the Jak kinases (Fig. 3C). Only in 3-1-12 cells where a functional / receptor unit existed was there human IFN-induced tyrosine phosphorylation of the STAT1 (Fig. 3C, lane 3 versus 6). T41 cells that were treated with murine IFN exhibited ligand-induced tyrosine phosphorylation of Jak1 and Jak2 (data not shown) and STAT1 (Fig. 3C, lane 5). There was no STAT1 tyrosine phosphorylation in response to human IFN in the T41 cell line (Fig. 3C, lane 6). Equal amounts of immunoprecipitated protein (Jak1, Jak2, and STAT1) were loaded onto each lane (Fig. 3, A-C, lower panel).


Figure 2: Activation of GRR-binding proteins in 3-1-12 and T41 cells. Cells were exposed to either human IFN (lanes 3 and 6) or murine IFN (Genentech) (lanes 2 and 5) for 10 min at 37 °C and then solubilized in whole cell extract buffer. Extracts were incubated with P-GRR and DNA bound proteins were separated on 6% nondissociating gels. The figure is an autoradiogram. FcRF denotes the GRR binding complexes induced by IFN. CTL, untreated control cells. m-IFN, murine IFN. h-IFN, human IFN.




Figure 3: Activation of Jak kinases and STAT1 in response to IFN in 3-1-12 and T41 cells. Cells were exposed to either murine or human IFN for 10 min at 37 °C and then solubilized in 1% Triton X-100 buffer. A, the extracts were incubated with anti-Jak1 and analyzed by immunoblotting. The upper panel represents the membrane probed with anti-phosphotyrosine (PY20) and then developed using ECL. The lower panel represents the same membrane reprobed for Jak1 protein and developed using colorimetric analysis. B, the extracts were incubated with anti-Jak2 and analyzed as described in A. C, the extracts were exposed to anti-STAT1 and analyzed as described in A. CTL, untreated control cells. m-IFN, murine IFN. h-IFN, human IFN.



We have previously shown that the Jak1 kinase constitutively associated with the IFNR and that ligand binding then resulted in its tyrosine phosphorylation(12) . We examined whether the chain or the chain was required for this association. Both the T41 and 3-1-12 cell lines were solubilized and subjected to immunoprecipitation with anti-IFNR chain antibodies. The immunoprecipitates were probed with anti-Jak1 antibodies (Fig. 4). Both the T41 (Fig. 4, lane 3, upper panel) and the 3-1-12 cells (Fig. 4, lane 4, upper panel) had Jak1 constitutively associated with the IFNR. The IFNR protein was immunoprecipitated equally in both lanes (Fig. 4, lanes 3 and 4, lower panel). Since in T41 cells only the chain is present, these data suggested that Jak1 preferentially associated with the component. When a similar experiment was attempted and probed with Jak2, no association was seen in the T41 or the 3-1-12 cells even when these cells were treated with IFN (data not shown). Presumably, if Jak2 preferentially associates with the chain, the poor expression of this gene may account for our inability to detect the association in the 3-1-12 cell line. The lack of association in the T41 cells suggests that Jak2 does not associate with the chain.


Figure 4: Co-immunoprecipitation of Jak1 with the IFNR in T41 and 3-1-12 cells. Cells (untreated) were solubilized in 1% digitonin buffer and the extracts incubated with anti-IFNR (lanes 3 and 4) or normal rabbit serum (NS) (lanes 1 and 2). The immunoprecipitates were then split between two polyacrylamide gels and analyzed by immunoblotting with anti-Jak1 antibodies (upper panel) or with the immunoprecipitating antibody (anti-IFNR) (lower panel). The immunoblots were developed using ECL.



Jak Kinase Activity in Untreated and IFN-treated Cells

Since Jak1 associated with the IFN receptor in a untreated cells, we assessed whether this kinase demonstrated catalytic activity as measured by an in vitro kinase assay. Immunoprecipitates prepared with anti-Jak1 and anti-Jak2 antisera were assayed for kinase activity using either [-P]ATP (Fig. 5) or unlabeled ATP followed by phosphotyrosine detection by immunoblotting (Fig. 6). In cells untreated with IFN there was a substantial amount of Jak1 kinase activity as judged by autophosphorylation of Jak1 (Fig. 5, lane 3, upper panel). There was no increased activity after treatment with IFN (Fig. 5, lane 4, upper panel). In contrast there was no observed Jak2 kinase activity in cells untreated with IFN. Only with IFN treatment was Jak2 kinase activity observed (Fig. 5, lane 6, upper panel). When the kinased immunoprecipitated Jak1 was subjected to phosphoamino acid analysis, essentially all of the incorporated radioactivity was on tyrosine moieties (data not shown). Equal amounts of the immunoprecipitated Jak kinases were loaded in each lane (Fig. 5, lanes 3-6, lower panel). When the experiment was performed using unlabeled ATP in conjugation with phosphotyrosine analysis by immunoblot, similar results were obtained (Fig. 6). Jak1 demonstrated constitutive tyrosine kinase activity (autophosphorylation) (Fig. 6A, lane 3) with no further increased kinase activity after IFN treatment (Fig. 6A, lane 4). On the other hand, Jak2 (Fig. 6A, lane 5 and 6) demonstrated tyrosine kinase activity only after cells had been stimulated with IFN. With no ATP present in the assay buffer only the endogenous tyrosine phosphorylation is observed in response to IFN (Fig. 6B, lanes 4 and 6).


Figure 5: In vitro kinase assay of Jak1 and Jak2 in IFN-treated HeLa cells. Cells were either exposed to IFN (lanes 2, 4, and 6) or untreated (lanes 1, 3, and 5) and solubilized in 1% Triton X-100. The extracts were then incubated with either normal rabbit serum (lanes 1 and 2), anti-Jak1 (lanes 3 and 4), or anti-Jak2 (lanes 5 and 6). The immunoprecipitates were washed and subjected to an in vitro kinase assay using [-P]ATP and analyzed by SDS-PAGE followed by electrophoretic transfer to a polyvinylidene difluoride membrane. The upper panel is an autoradiogram of the membrane. The membrane was then reprobed with the immunoprecipitating antibodies for specific protein loaded onto each lane (lower panel). The membrane was developed using colorimetric chemistry. CTL, control untreated cells. NS, normal rabbit serum.




Figure 6: In vitro kinase assay of Jak1 and Jak2 in IFN-treated HeLa cells using anti-phosphotyrosine immunoblotting. Cells were treated as described in the legend to Fig. 5. Immunoprecipitates were subjected to an in vitro kinase assay in the absence (B) or presence (A) of ATP. A, in vitro kinase assay in the presence of ATP. The upper panel represents the immunoblot probed with anti-phosphotyrosine antibody (PY20). The lower panel represents the same membrane reprobed with the immunoprecipitating antibody and developed using colorimetric chemistry. B, in vitro kinase assay in the absence of ATP. The upper and lower panel are as described in A. The presence of tyrosine-phosphorylated Jak1 and Jak2 in the upper panel represent endogenous tyrosine phosphorylation in IFN-treated cells. NS, normal rabbit serum. CTL, untreated control cells. P-Tyr, phosphotyrosine.



Since the IFNR chain was rapidly tyrosine-phosphorylated following ligand binding, we next determined whether the kinase that was responsible for this was pre-associated with the IFN receptor. Cells were treated with or without IFN and then solubilized in digitonin to maintain any protein-protein interactions. The extracts were then immunoprecipitated with antibodies directed against the IFNR chain. The immunoprecipitate was then analyzed by an in vitro kinase reaction. In untreated cells (Fig. 7A, lanes 1 and 2) there was tyrosine phosphorylation of the IFNR chain (arrow) in the presence of ATP (lane 2). For comparison, the endogenous tyrosine phosphorylation of the IFNR (arrow) and Jak kinases (asterisk) was measured in response to IFN (Fig. 7A, lane 3). When the immunoprecipitates from untreated cells were washed under more stringent conditions with 1% Triton X-100 prior to performing the in vitro kinase assay, the ability to tyrosine-phosphorylate the IFNR was lost (Fig. 7A, lane 8 versus 6). This suggested that the tyrosine kinase that was responsible for tyrosine phosphorylation of the IFNR chain was pre-associated with the IFNR in a receptor-kinase complex. Since Jak1 was shown to pre-associate with the chain and be active in untreated cells by an in vitro kinase assay, Jak1 was a likely candidate responsible for the tyrosine phosphorylation of the chain of receptor. When the anti-IFNR chain immunoprecipitates were probed for the presence of Jak1, there was a loss of Jak1 in those cells washed with 1% Triton X-100 (Fig. 7B, lanes 5 and 6 versus 7 and 8) even though total IFNR protein was the same (Fig. 7C, lanes 5-8).


Figure 7: Association of tyrosine kinase activity with anti-IFNR immunoprecipitations. A, HeLa cells were exposed to IFN (lanes 3 and 4) or untreated (lanes 1, 2, 5-8) and solubilized in 1% digitonin buffer. The extracts were then incubated with anti-IFNR antibody. The immunoprecipitates were then either washed in 0.1% digitonin buffer (lanes 1-6) or 1% Triton X-100 buffer (lanes 7 and 8). The immunoprecipitates were then subjected to an in vitro kinase assay with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) ATP. The immunoprecipitates were then analyzed by immunoblotting with anti-phosphotyrosine antibodies. Lanes 1 and 2 represent a kinase activity co-immunoprecipitating with the IFNR in untreated cells. Lane 3 represents endogenous tyrosine phosphorylation of the IFNR chain (arrowhead) and Jak kinases (asterisk) in IFN-treated cells. Lanes 5 and 6 are as described for lanes 1 and 2. Lanes 7 and 8 represent the loss of kinase activity co-immunoprecipitating with the IFNR. B and C, the immunoblot of the membrane represented in A (lanes 5-8) was divided in two portions. The upper potion (B) was reprobed with anti-Jak1 and developed using colorimetric chemistry. The lower portion (C) was reprobed with anti-IFNR and developed using colorimetric chemistry. CTL, untreated control cells. P-Tyr, phosphotyrosine. IgH, immunoglobulin heavy chain.



The IFNR Chain and Signal Transduction

In order to fully assess the role of the IFNR chain, we raised polyclonal antibodies against a peptide of the chain consisting of amino acids 307-337 of the intracytoplasmic domain. The reactivity of this affinity-purified antibody was assessed on biosynthetically labeled human peripheral blood monocytes. The IFNR chain and other associated chains have been extensively studied in these cells by both radiolabeling and biosynthetic labeling followed by immunoprecipitation (24) . When monocytes were biosynthetically labeled and the digitonin lysates subjected to immunoprecipitation with the anti-IFNR chain (anti-AF-1), there was a prominent band at about 38 kDa (Fig. 8A, lane 2, arrow) that was not observed with control antibodies (Fig. 8A, lane 1) or antibodies directed against the IFNR chain (Fig. 8A, lane 3). A similar band was noted in cells solubilized in 1% Triton X-100 (data not shown). The migration of the immunoprecipitated IFNR chain as a 38-kDa protein is consistent with the predicted amino acid sequence of 310 amino acids with some degree of post-translational modification such as glycosylation. Under the conditions utilized the antibodies against the chain co-immunoprecipitate the chain, but antibodies against the chain only precipitate the chain and associated Jak kinases (Fig. 8A, lane 3 and Fig. 9, lane 6). The ability of the anti-IFNR antisera to co-immunoprecipitate the chain of the receptor was directly measured by incubating extracts prepared with digitonin with anti-IFNR and probing the membrane with anti-IFNR (Fig. 8B). Under these condition the IFNR chain co-immunoprecipitates with the chain in cells untreated (Fig. 8B, lane 3) or treated (Fig. 8B, lane 4) with IFN.


Figure 8: A, biosynthetic labeling of the IFNR chain. Monocytes were incubated with Translabel for 3 h, washed, and solubilized. The extracts were then incubated with either normal rabbit IgG (nIgG) (lane 1), affinity purified anti-IFNR chain (anti-AF-1) (lane 2), or anti-IFNR chain (lane 3). The arrow represents the 38-kDa IFNR chain. The figure is an autoradiogram. B, the and chains of the IFNR are associated in untreated cells. HeLa cells were solubilized with 1% digitonin buffer and the extract incubated with normal rabbit IgG (nIgG) (lanes 1 and 2) or anti-IFNR (anti-AF-1) (lanes 3 and 4). The immunoprecipitates were then analyzed by immunoblotting with anti-IFNR chain and developed using ECL. nIgG, normal rabbit IgG. anti-AF-1, anti-IFNR chain. anti-IFNR, anti-IFNR chain.




Figure 9: The IFNR chain is not tyrosine-phosphorylated in response to IFN. HeLa cells were either exposed to IFN (lanes 2, 4, and 6) or untreated (lanes 1, 3, and 5) and solubilized in 1% digitonin buffer. The extracts were then incubated with either normal rabbit IgG (nIgG) (lanes 1 and 2), anti-IFNR chain (anti-AF-1) (lanes 3 and 4), or anti-IFNR chain (anti-IFNR) (lanes 5 and 6). The immunoprecipitates were then analyzed by immunoblotting with anti-phosphotyrosine antibodies (PY20). NS, represents nonspecific bands.



In order to examine whether or not the IFNR chain like the chain undergoes tyrosine phosphorylation in response to the binding of IFN to the cells, we treated cells with IFN and then solubilized them in digitonin (Fig. 9). Using anti-IFNR chain antibody it was evident that the chain of the receptor was clearly tyrosine-phosphorylated in response to IFN (Fig. 9, lane 5 versus 6). In addition, a band migrating slightly slower than the IFNR chain at about 130 kDa represented the tyrosine phosphorylated Jak1/2 kinases. This has been confirmed by reprobing the blots with anti-Jak kinase antibodies (data not shown). In contrast, when the anti-IFNR chain antibodies (anti-AF-1) were utilized, there was no observable tyrosine phosphorylation of the chain (Fig. 9, lane 3 versus 4). However, it was evident that the antibody was indeed immunoprecipitating the receptor complex since the tyrosine-phosphorylated chain at 90 kDa and the Jak kinases at 130 kDa were present in the immunoprecipitate.

In order to ascertain whether Jak2 preferentially associated with the IFNR chain, HeLa cells were either untreated or treated with IFN and then solubilized in digitonin. Immunoprecipitates with anti-IFNR were resolved by SDS-PAGE and then immunoblotted with anti-Jak2 (Fig. 10). Jak2 associated with the chain in a constitutive fashion (Fig. 10, lane 3). However, upon IFN signaling there was a consistent increase in the amount of Jak2 that could be co-immunoprecipitated with the chain, suggesting that recruitment of Jak2 occurred during signal transduction or that the affinity of Jak2 increased for the clustered IFNR (Fig. 10, lane 4). When antibodies against Jak1 were utilized to probe anti-IFNR immunoprecipitates, no co-immunoprecipitation of Jak1 was detectable (data not shown), suggesting either that there was no direct interaction between Jak1 and the IFNR chain or that the anti-IFNR antibody itself disrupted any weak interaction that may have occurred.


Figure 10: Jak2 co-immunoprecipitates with the anti-IFNR chain. HeLa cells were incubated with IFN (lanes 2 and 4) or untreated (lanes 1 and 3) and solubilized in 1% digitonin buffer. The extracts were then incubated with either normal rabbit serum (nIgG) (lanes 1 and 2) or anti-IFNR chain (anti-AF-1) (lanes 3 and 4) and analyzed by immunoblotting with anti-Jak2 and developed using ECL. IgH, immunoglobulin heavy chain. nIgG, normal rabbit serum. anti-AF-1, anti-IFNR chain.



Upon activation by IFN there is rapid tyrosine phosphorylation of STAT1. In order to determine if STAT1 could be directly detected within the receptor-Jak complex, we exposed cells to IFN at 4 °C and then solubilized them in digitonin. The extracts were incubated with anti-STAT1 antibodies and the membranes probed with anti-phosphotyrosine. In cells treated with IFN (Fig. 11, lane 4) there were two additional co-immunoprecipitating tyrosine-phosphorylated bands that correspond to the IFNR chain and the Jak1/2 kinases both of which can also be shown to be immunoprecipitated with the anti-IFNR (lane 6).


Figure 11: STAT1 is part of the IFNRJak complex. HeLa cells were either untreated (lanes 1, 3, and 5) or treated with IFN (lanes 2, 4, and 6) for 10 min at 4 °C, solubilized in digitonin buffer, and subjected to immunoprecipitation with either normal rabbit serum (NRS) (lanes 1 and 2), anti-STAT1 (lanes 3 and 4), or anti-IFNR (anti-IFNR) (lanes 5 and 6). The immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting with anti-phosphotyrosine (P-Tyr) antibodies (PY20) and developed using ECL.




DISCUSSION

Interferon activation of STAT1 requires both Jak1 and Jak2 as well as tyrosine phosphorylation of the chain of the IFN receptor. Although it has been known that another component of the receptor is necessary for signal transduction to occur, only recently has the gene for this accessory factor ( chain) been cloned and shown to encode for a transmembrane protein of 310 amino acids with a 66-amino acid intracytoplasmic domain(9, 10) . Nothing is known regarding the role of this protein in the formation of the ligand induced multi-molecular complex. In this report we show that both the and components are required to activate STAT proteins. In cells expressing only the chain, we show that Jak1 constitutively associates with this component but does not become tyrosine-phosphorylated after treatment with IFN in spite of high affinity binding of the ligand to the chain. Only in cells expressing both the and component are Jak1 and Jak2 tyrosine-phosphorylated in response to IFN treatment. Jak1 pre-associates with the component and shows no increased association with the receptor after IFN treatment. In contrast, Jak2 is pre-associated with the component, but also is either recruited to the receptor after ligand binding or its affinity for the chain increases after ligand binding. The data derived from the somatic cell hybrid cells suggest that the association of Jak2 with the receptor chain is an absolute requirement for subsequent activation of STAT1 by tyrosine phosphorylation. Although the chain is tyrosine-phosphorylated in IFN-treated cells, we have been unable to observe any ligand-dependent tyrosine phosphorylation of the chain. Therefore, it is likely that this component may not directly interact with the STAT proteins as has been proposed to occur for the chain of the IFN receptor (11) and the ligand binding component of the IL-4 receptor(25) . We have also shown that in digitonin-solubilized extracts the and components can be co-immunoprecipitated in untreated cells, and therefore, it is likely that dimerization of at least two complexes initiates signal transduction.

In untreated cells Jak1 is pre-associated with the chain and, by in vitro kinase assay, is enzymatically active as measured by autophosphorylation. When the IFNR chain is immunoprecipitated and subjected to in vitro kinase assay, the chain along with the co-immunoprecipitating Jak kinases are tyrosine-phosphorylated in cells unexposed to IFN. This suggests that the kinase responsible for the tyrosine phosphorylation of the chain in vivo is pre-associated with the receptor and may be Jak1. Washing of the anti-IFNR immunoprecipitate in 1% Triton X-100 results in loss of the activity and loss of co-immunoprecipitating Jak1. We have shown previously that under similar conditions in untreated cells no detectable Jak2 is immunoprecipitated with the anti-IFNR chain (12) . This finding is similar to that observed with the leukemia inhibitory factor receptor chain or gp130 of the IL-6 receptor. Both Jak1 and Jak2 are pre-associated with the components of the IL-6 and leukemia inhibitory factor receptor, and upon immunoprecipitation of the receptors in Brij96 (a detergent with properties similar to digitonin), in vitro tyrosine phosphorylation of the component is lost after a Nonidet P-40 wash (26, 27) .

The degree of activation of the Jak kinase itself in cells unexposed to ligand varies depending upon the cells utilized. In the IL-6/leukemia inhibitory factor studies there was slight but perceptible in vitro kinase activity for Jak1 but not Jak2 in untreated cells (26) . In PHA-activated T cells and factor-starved FWT-2 cells, there was kinase activity of Jak1 in untreated cells that was enhanced after IL-2 treatment(28, 29) . In untreated MOLT cells, there was in vitro tyrosine autophosphorylation of Jak1 that did not increase after IL-2 exposure(29) . Therefore, varying degrees of in vitro kinase activity for Jak1 exist in untreated cells that may or may not be enhanced after ligand stimulation. For receptors utilizing Jak1 and Jak2 or Jak3, it appears that Jak1 rather than Jak2/3 tends to demonstrate more in vitro kinase activity in cells untreated with ligand. These data are consistent with the concept that the initial reaction after clustering of receptors by ligand may allow Jak1 access to substrate. Jak2 activation quickly follows and in addition is recruited to the receptor or binds more tightly to the clustered complex(12) . It is at this point that STAT1 can be shown to become part of the receptor-Jak complex.

Most of the Jak/Tyk kinases have been shown to interact with membrane proximal domains (Box1/Box 2 regions) of the cytoplasmic portion of receptors that are present in many cytokine receptors(30, 31) . However, it is clear that there is differential binding of Jak/Tyk kinases to these regions. Jak1 primarily associates with the chain and Jak2 with the chain of the IFN receptor. For the IL-2 receptor, Jak1 associates with the serine-rich membrane proximal region of the IL-2R, whereas Jak3 associated with the COOH-terminal region of the component, a region mutated in X-linked severe combined immunodeficiency patients who have altered T cell function(28, 29) . In cell lines expressing all three Jak kinases, Jak2 did not associate with the IL-2 receptor. The kinase-specific association to distinct receptor components may be a mechanism for specificity of signal transduction through the Jak/Tyk pathway. The interaction of Jak2 with the chain of the IFNR appears to be a requirement for IFN signaling. This was also seen for the IL-2Rc/Jak3 association(28, 29) . From our previous studies and the data in this report, it appears that Jak2 is required for the tyrosine phosphorylation of STAT1, whereas Jak1 is more closely affiliated with the tyrosine phosphorylation of the IFNR chain. Therefore, in receptor systems that utilize paired Tyk/Jak kinases, one of the kinases pre-associated with the receptor may facilitate the tyrosine phosphorylation of one or more chains of the receptor, whereas the other kinase may be more responsible for the activation of STAT proteins.


FOOTNOTES

*
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.

§
Current address: Human Genome Sciences, 9410 Key West Ave., Rockville, MD 20850.

To whom all correspondence should be addressed: Division of Cytokine Biology, Center for Biologics Research and Evaluation, 29 Lincoln Dr., MSC 4555, Bethesda, MD 20892-4555. Tel.: 301-496-0894; Fax: 301-402-1659.

The abbreviations used are: IFN, interferon ; PAGE, polyacrylamide gel electrophoresis; IL, interleukin.


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