The Proximal Tyrosines of the Cytoplasmic Domain of the beta  Chain of the Type I Interferon Receptor Are Essential for Signal Transducer and Activator of Transcription (Stat) 2 Activation
EVIDENCE THAT TWO Stat2 SITES ARE REQUIRED TO REACH A THRESHOLD OF INTERFERON alpha -INDUCED Stat2 TYROSINE PHOSPHORYLATION THAT ALLOWS NORMAL FORMATION OF INTERFERON-STIMULATED GENE FACTOR 3*

Owen W. Nadeauab, Paul Domanskiab, Anna Usachevaac, Shahab Uddind, Leonidas C. Plataniasd, Paula Pithae, Regina Razf, David Levyf, Beata Majchrzakg, Eleanor Fishg, and Oscar R. Colamoniciah

From the a Department of Pathology, University of Tennessee, Memphis, Tennessee 38163, the c Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, Russia, d Section of Hematology/Oncology, University of Illinois, Chicago, Illinois 60606, g Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 3E2, Canada, f Department of Pathology, New York University School of Medicine, New York, New York 10016, and the e Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231

    ABSTRACT
Top
Abstract
Introduction
References

The precise role of the different subunits (alpha /IFNAR1 and beta L/IFNAR2) of the type I interferon receptor (IFN-R) in the activation of signal transducer and activator of transcription (Stat) 1, Stat2, and Stat3 has not yet been established. In this report we demonstrate that there are functionally redundant phosphotyrosine-dependent and -independent binding sites for Stat2 in the alpha  and beta  subunits of the type I IFN-R. Expression of a type I IFN-R containing only the constitutive Stat2 site or the proximal tyrosines of beta L, but not the docking site on the alpha  chain (Tyr466 and Tyr481), supported low levels of Stat2 activation. However, the presence of only one intact Stat2 site did not lead to induction of interferon-stimulated gene factor 3 (ISGF3) or an antiviral state. Normal levels of Stat2 tyrosine phosphorylation, induction of ISGF3, and an antiviral effect always required the proximal tyrosines of beta L and at least one of the other Stat2 sites (Tyralpha 466, 481 or beta L404-462). These data suggest that a threshold of Stat2 tyrosine phosphorylation is required for complete activation of ISGF3. Interestingly, a receptor in which all tyrosines were mutated to phenylalanine shows normal Stat3 phosphorylation and low levels of activation of Stat1.

    INTRODUCTION
Top
Abstract
Introduction
References

The human type I interferon (IFN)1 family is composed of multiple subtypes of IFNalpha (IFNalpha 1, IFNalpha 2, etc.), IFNbeta , and IFNomega (1). The human type I IFN receptor (IFN-R) or IFNalpha beta R is composed of at least two subunits, termed alpha  and beta  (also designated as IFNAR1 and IFNAR2, respectively). The beta  subunit has two transmembrane forms beta Short (beta S) and beta Long (beta L), both of which can bind IFNs with low affinity (2). High affinity binding occurs when both alpha  and either form of beta  are coexpressed; however, in the absence of beta , the alpha  chain cannot bind IFN (reviewed in Ref. 3). Each chain also associates with a specific Jak kinase that is required for signaling; the alpha  chain docks Tyk2 (4, 5), whereas the beta L subunit interacts with Jak1 (6). Oligomerization of the receptor subunits occurs upon ligand binding and induces intra- and intermolecular phosphorylation of the Jak kinases (reviewed in Refs. 7-10). The activated kinases are then thought to phosphorylate the receptor chains and Stats 1, 2, and 3. The Stat molecules then homo- or heterodimerize and migrate to the nucleus where they stimulate transcription of the IFN-inducible genes (10-13).

In most cytokine systems tyrosine phosphorylation of the receptor subunits was originally thought to be required for recruitment and phosphorylation of the Stat factors by the receptor-Jak kinase complex. This model holds true for some cytokine systems such as the IFNgamma and interleukin 6 systems, where specific tyrosines in the alpha  and gp130 subunits were shown to be essential for recruitment and phosphorylation of Stat1 and Stat3, respectively (14, 15). However, recent reports indicate that not all cytokine systems require receptor phosphorylation for Stat activation, i.e. growth hormone and granulocyte-colony-stimulating growth factor receptors devoid of tyrosines can still activate Stat proteins (16-18).

In the type I IFN system, the role of tyrosine phosphorylation of the type I IFN-R in Stat binding and activation is not clear (19-21). For example, certain cell lines expressing a variant form of the type I receptor, which fails to phosphorylate the alpha  subunit, are still capable of producing an antiviral and antiproliferative effect in response to IFNalpha 2 (22). Moreover, Gibbs et al. (23) showed that expression of a human alpha  chain devoid of tyrosines in mouse L-929 cells did not impair formation of the Stat1- and Stat2-containing ISGF3 complex. However, it is worth mentioning that the role of the beta  chain of the receptor in Stat activation has not been explored.

This report seeks to answer several related questions regarding the mechanisms of Stat activation by type I IFNs. First, what receptor domains are required for Stats 1 and 2 activation? Second, what is the importance of tyrosine phosphorylation of the receptor subunits in signaling? Third, does activation of Stat3 require tyrosine phosphorylation of the alpha  chain? In addition to the previously reported docking site for Stat2 on the alpha  chain (Tyr466 and Tyr481), we have defined two new Stat2-binding sites in the beta L chain as follows: a phosphotyrosine-independent constitutive binding region located at amino acids 404-462, and a phosphotyrosine-dependent docking site formed by one or more of the five tyrosines N-terminal to amino acid 346 (Tyr269, Tyr306, Tyr316, Tyr318, and Tyr337).

Our data indicate the following. 1) Low levels of tyrosine phosphorylation of Stat2 can be supported by the phosphotyrosine-independent binding site on residues 404-462 of the beta L chain and the phosphotyrosine-dependent docking site on the proximal tyrosines of this chain but not by Tyr466 and Tyr481 of the alpha  chain. However, formation of ISGF3 and the induction of an antiviral state require the proximal tyrosines of beta L and at least one of the remaining Stat2 sites (Tyr466 and Tyr481 of the alpha  chain or amino acids 404-462 of beta L). 2) There appears to be a threshold of Stat2 tyrosine phosphorylation required for complete ISGF3 activation and induction of an antiviral state. This threshold is reached when at least two Stat2-docking sites are present in the receptor complex. 3) Normal levels of tyrosine phosphorylation of Stat1 require activation of Stat2. However, low levels of Stat1 tyrosine phosphorylation can be achieved in a receptor devoid of tyrosines and Stat2-binding sites. 4) The presence of functional tyrosine-independent docking sites on the IFNalpha beta R suggests that other cytokine systems, i.e. growth hormone, may rely on this mechanism to activate some Stat factors. 5) Tyrosine phosphorylation of Stat3 does not appear to require tyrosine phosphorylation of the alpha  or beta L subunits. Finally, we explored whether the expression of only beta L would support tyrosine phosphorylation of Stat2. Our data indicate that beta L alone can support low levels of tyrosine phosphorylation of Jak1 and Stat2 but not Tyk2 and Stat1.

    MATERIALS AND METHODS

Cell Lines, IFNs, Antibodies, and Antiviral Assays-- Human recombinant IFNalpha 2 (huIFNalpha 2), IFNCon1, and IFNbeta (murine and human) were kindly provided by Drs. Paul Trotta (Schering-Plough), Lawrence Blatt (Amgen Biologicals), and S. Goelz (Biogen), respectively. Murine IFNalpha beta was purchased from Access Biomedicals (San Diego, CA). The anti-phosphotyrosine antibody (4G10) was obtained from Upstate Biotechnology Inc. (Lake Placid, NY), and the anti-Stat2 serum was from Santa Cruz Biotechnology. Monoclonal antibodies against Jak1 and Stat1 were purchased from Transduction Laboratories (Lexington, KY). The anti-Stat1 and -Stat2 sera were kindly provided by Dr. A. Larner (Cleveland Clinic Foundation, Cleveland, OH), and the anti-Jak1 and -Stat3 sera were a generous gift of J. N. Ihle (St. Jude's Children's Hospital, Memphis, TN). The polyclonal and monoclonal antibodies against Tyk2 and the type I IFN-R subunits were described previously (4, 24, 25). The mouse fibrosarcoma L-929 and human myeloma U-266 cells were obtained from ATCC. Mouse embryonal fibroblast null for the alpha  chain (MEFalpha -/-) (26) were a kind gift of Dr. M. Aguet (Swiss Institute for Experimental Cancer Research). Antiviral assays were performed as described previously (27, 28).

GST Fusion Proteins-- Experiments were performed with GST fusion proteins encoding the whole cytoplasmic domain of beta S (GSTbeta S amino acids 265-331) and beta L (GSTbeta L265-515), as well as C-terminal truncations of the cytoplasmic domain of beta L at aa 462, 375, 346, and 299 (GSTbeta L265-462, GSTbeta L265-375, GSTbeta L265-346, and GSTbeta L265-299, respectively, see Ref. 6). The GSTbeta L300-515 was produced by deletion of an NcoI restriction fragment (from the NcoI site in the pGEX-KG cloning site to the NcoI site at position 959 of the beta L chain) from the GSTbeta L265-515 construct (6). The GST fusion proteins were produced in BL-21 cells (Novagen) and purified by affinity chromatography on glutathione-Sepharose (Amersham Pharmacia Biotech). Five to ten µg of the indicated GST fusion proteins were used for precipitations.

Expression of Different Mutants of the alpha  and beta L Subunit of Type I IFN-R in Mouse L-929 and MEFalpha -/- Cells-- These constructs were generated using a polymerase chain reaction-based protocol (Quickchange, Stratagene). All mutations were confirmed by sequencing. The alpha  and beta L chain constructs were subcloned into the pRep4 and pZipNeoSV(X) vectors, respectively, and used for expression in L-929 and MEFalpha -/- cells. L-929 transfectants were selected in medium containing G-418 (500 µg/ml) and hygromycin B (500 µg/ml), and MEFalpha -/- transfectants were grown only in hygromycin B (500 µg/ml). Table I is a summary of the transfectants produced. Fig. 2 shows a schematic representation of the wild type (A) and mutated forms (B-D, top panels) of the receptor used in this study. We produced the following cell lines carrying mutations in only one Stat2 site: 1) Lalpha YF526beta L clones .4 and .22, corresponding to L-929 cells expressing wild type beta L and the alpha  chain without the Stat2 sites (stop codon at position 526 and phenylalanine substituted for tyrosines 466 and 481); 2) Lalpha beta L346.4, L-929 cells expressing alpha  wild type and beta L truncated at position 346 to delete the distal constitutive Stat2 site of beta L (6); 3) Lalpha beta LYF clones .1 and .7, L-929 expressing wild type alpha  chain and beta L devoid of tyrosines and thus eliminating the proximal tyrosines that serve as a Stat2-docking site (see below). The following cell lines contain mutations in two Stat2 sites: 1) Lalpha YF526beta L346 clones .3 and .5, L-929 cells expressing beta L truncated at amino acid 346 to delete the constitutive Stat2-binding site, and substitution of the tyrosines on the alpha  chain (Tyr466 and Tyr481) for phenylalanines; 2) Lalpha beta L346YF.1, corresponding to L-929 cells expressing wild type alpha  chain and beta L truncated at amino acid 346 and with substitution of the remaining tyrosines of beta L to phenylalanines in order to delete both the constitutive site (aa 404-462) and the five proximal tyrosines of the cytoplasmic domain (Tyr269, Tyr306, Tyr316, Tyr318, and Tyr337) that serve as a docking site for Stat2 (see below); and 3) Malpha YFbeta LYF, corresponding to MEF null for the murine alpha  chain (MEFalpha -/-) that express human alpha  and beta L chains without tyrosines (alpha YF526 and beta LYF, respectively), but maintaining the constitutive Stat2 site of beta L. The latter constructs were expressed in MEFalpha -/- because they appear to be toxic for L-929 cells for reasons that are not clear. The Lalpha YFbeta L346YF clones 2 and 4 and Malpha YFbeta L346YF clones 7 and 9, corresponding to L-929 and MEFalpha -/-, express mutations of all three Stat2 sites. Finally, the Mbeta L.1 cell line corresponds to MEFalpha -/- transfected with wild type human beta L.

Immunoblotting-- Cells were treated with different concentrations of the indicated IFNs for 10 min and rapidly solubilized in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 10 mM sodium pyrophosphate, 20 mM NaF, 1 mM EDTA, 1 mM MgCl2, 1 mM dithiothreitol, 0.5% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 100 mM phenylmethylsulfonyl fluoride, 200 µM sodium orthovanadate). Lower concentrations of NaCl (50 mM) were used for coprecipitation of Stat2 with the receptor. Immunoprecipitation and immunoblotting were performed as described previously (4).

Radioiodination of Type I IFNs and Affinity Cross-linking-- Radioiodination of IFNalpha 2 and affinity cross-linking were performed as described previously (24).

Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts were prepared as described by Ghislain et al. (29) and analyzed by EMSA using end-labeled ISRE and m67SIE oligonucleotides to detect ISGF3 and c-sis-inducible factor complexes, respectively.

    RESULTS

Stat2 Interacts with the 404-462 Region of beta L-- To determine whether Stat2 was able to interact with the beta L chain in vivo, we performed coimmunoprecipitation experiments followed by Western blotting with an anti-Stat2 serum. Fig. 1A shows that a polyclonal serum against beta L coprecipitates low levels of Stat2 in control U-266 cell lysates (lane 3, beta L375-515 serum). However, the interaction between beta L and Stat2 appears to be enhanced after IFNbeta treatment, since the levels of Stat2 coprecipitated by the anti-beta L serum relative to those precipitated by the anti-Stat2 antibody used as positive control are higher in IFN-treated (lanes 7 and 9) than in control cells (lanes 3 and 4). As expected, Stat2 is coprecipitated by an anti-Stat1 serum after IFNbeta treatment (lane 8). No interaction between Stat2 and the alpha  chain was detected before or after type I IFN treatment (lanes 2 and 6), probably because this interaction is transitory and weak as previously reported (Ref. 19, see also "Discussion").


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Fig. 1.   Direct association of Stat2 with beta L. A, lysates obtained from IFNbeta -treated (lanes 5-9) or untreated (lanes 1-4) U-266 cells were immunoprecipitated with antibodies against the alpha  (alpha 511-557) or beta L (beta L375-515) chains. Anti-Stat1 (lane 8) and -Stat2 (lanes 4 and 9) antibodies and normal rabbit serum (NR, lanes 1 and 5) were used as positive and negative controls, respectively. The control and IFN-treated samples were analyzed in different gels. This accounts for the apparent lower amounts of Stat2 immunoprecipitated after IFNbeta treatment. B, lysates from IFNalpha 2-treated (lower panel) or untreated (upper panel) U-266 cells were precipitated with GST fusion proteins encoding wild type beta L (beta L265-515) or the indicated deletions of the cytoplasmic domain. Precipitations with GST alone and GSTbeta S (lanes 1 and 2) as well as an anti-Stat2 serum were used as negative and positive controls, respectively. The lower signal for Stat2 precipitated with the GSTbeta L265-462 from control cells (lane 3, top panel) is due to lower amounts of the fusion protein present in this lane. C, GST fusion proteins encoding entire cytoplasmic domains of beta S, beta L, alpha , and IL10Rbeta (GST-B4) chains were used to precipitate [35S]methionine-labeled Stat1 (lanes 1-7) and Stat2 (lanes 8-14) produced by in vitro transcription/translation using a wheat germ lectin kit. Anti-Stat1 and -Stat2 antibodies were used as positive control. An aliquot equivalent to the input used for each precipitation was run directly in the gels (Lysate). The migration of Stat1 and Stat2 is indicated.

To map the Stat2-docking site on beta L, we performed binding experiments with GST fusion proteins encoding the wild type form or deletions of the cytoplasmic domain of beta L. As a source of Stat2 protein we used lysates from IFNalpha 2-treated and control U-266 cells. Fig. 1B shows that Stat2 associates with wild type GSTbeta L (lane 7) and beta L truncated at amino acid 462 (lanes 3 and 10). However, more proximal deletions at amino acids 404, 375, 346, and 299 (lanes 4-6 and 11) completely abrogated binding. Deletion of the first 45 amino acids of the cytoplasmic domain that include the Box 1 motif (2 and 6), did not affect the interaction with Stat2 (GSTbeta L300-515, lane 8). It is worth mentioning that beta L truncated at amino acid 462 bound Stat2 with slightly less efficiency than the full-length cytoplasmic domain. This may reflect a minor conformational change in the Stat2-binding site produced by the deletion of the last 53 aa of beta L. These results map the constitutive Stat2 binding region to amino acids 404-462 of beta L. Treatment with IFNalpha 2 did not increase the association of Stat2 with GSTbeta L as detected by coprecipitation (Fig. 1A). This is probably due to fact that the GST fusion proteins are not phosphorylated, and the increase in binding of Stat2 to beta L after IFN treatment is accounted for by an interaction between one or more phosphorylated tyrosines of beta L and the SH2 domain of Stat2 (see below). No binding of Stat2 to beta S or a GST control was detected.

We next studied whether Stat2 interacts directly with beta L using a cell-free system that lacks adaptor proteins and tyrosine kinases. Stat2 was produced using a wheat germ in vitro transcription/translation system, labeled with [35S]methionine, and precipitated with different GST fusion proteins (lanes 8-14). Stat2 produced in the wheat germ system was precipitated by GSTbeta L and the anti-Stat2 serum (Fig. 1C, lanes 10 and 13). No binding of Stat2 to GST control or GST fusion proteins encoding the alpha , beta S, or the IL10Rbeta (CRFB4, Ref. 30) subunits was observed (Fig. 1C, lanes 8, 9, 11, and 12, respectively). In vitro translated Stat1 (Fig. 1C, lanes 1-7) did not interact with GSTbeta L, GSTalpha , GSTbeta S, or GST-CRFB4 (lanes 2-5). These results indicate that the interaction between Stat2 and beta L is direct, since Stat2 and the GST fusion proteins were produced in cell-free systems (wheat germ lysates and bacteria, respectively) which should not contain adaptor proteins.

Heterologous Expression in L-929 and MEFalpha -/- of Mutant Human alpha  and beta L Chains-- To determine the role of the different Stat2-binding sites in IFNalpha beta signaling, we developed stable transfectants expressing receptors containing single or combined mutations of the Stat2 sites (Table I). Fig. 2 shows a schematic representation of the wild type receptor (A) and the different mutations expressed in mouse L-929 or MEFalpha -/- cells (Fig. 2, B-D, upper panels). Fig. 2B and Table I show stable transfectants expressing mutations of only one of the Stat2-binding sites: (i) Lalpha YFbeta L lacking the tyrosine-docking sites on the alpha  chain (Tyr466 and Tyr481); (ii) Lalpha beta L346 cells that lack the constitutive Stat2-binding site on beta L (aa 404-462) (6); and (iii) Lalpha beta LYF cells miss the proximal tyrosines of the beta L chain, which in the course of this work were shown to serve as docking sites for Stat2 (see below). We also produced transfectants in which different combinations of two Stat2-docking sites on the receptor chains have been mutated (Fig. 2C): (i) Lalpha beta L346YF lacking the constitutive docking site and the proximal tyrosines of beta L; (ii) Lalpha YFbeta L346, lacking the constitutive site of beta L (aa 404-462) and the tyrosines on the alpha  chain (Tyr466 and Tyr481); and (iii) Malpha YFbeta LYF, corresponding to MEFalpha -/- expressing human receptors in which all the tyrosines were mutated to phenylalanine but maintaining the constitutive Stat2-docking site on beta L. For reasons that are not clear, we could not isolate L-929 transfectants expressing the latter receptor combination. In addition, we produced L-929 (Fig. 2C, Lalpha YFbeta L346YF) and MEFalpha -/- (data not shown) cell lines lacking all the Stat2-docking sites. Cell-surface expression of the human receptor subunits was studied by cross-linking 125I-IFNalpha 2 to the receptor, followed by immunoprecipitation with antibodies against the human alpha  and beta L chains, as well as mouse alpha  subunit. Fig. 2, B and C, shows that the anti-human alpha  subunit antibody IFNaR3 immunoprecipitated the appropriate form of the alpha  chain, and in most cases a high molecular weight complex that corresponds to the association of the alpha  and beta L (31) subunits in L-929 and MEFalpha -/- transfectants (Fig. 2, B and C, lanes 2, 5, 8, 11, 13, 17, and 19). The anti-beta L serum detects the beta L chain (wild type or truncated, lanes 3, 9, 12, 15, 18, and 20), the complex formed by alpha  and beta L, and coprecipitates the alpha  chain as described previously (31). The anti-mouse alpha  subunit antibody failed to detect any receptor component in all cell lines tested (lanes 4, 6, 7, 10, and 14) demonstrating that no interaction between this mouse chain and human IFNalpha 2 takes place in these transfectants. The anti-human alpha 511-557 serum that recognizes an epitope in the human alpha  chain between amino acids 526 and 557 (6), and therefore not present in the alpha YF526 construct, fails to detect this mutated chain (lanes 1 and 16).

                              
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Table I
Summary of stable transfectants


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Fig. 2.   Expression of alpha  and beta L constructs in L-929 and MEFalpha -/- cells. Expression of the different constructs was assessed by cross-linking 125I-IFNalpha 2 to the receptor followed by immunoprecipitation with specific antibodies against the different receptor subunits. A, diagram of the wild type receptor subunits including sites for interaction with Jak kinases and Stat2. B, L-929 transfectants expressing mutations of only one Stat2 site (Lalpha YFbeta L, Lalpha beta L346, and Lalpha beta LYF). C, L-929 and MEFalpha -/- transfectants expressing receptor complexes with mutations of two (Lalpha beta L346YF, Lalpha YFbeta L346, and Malpha YFbeta LYF) and three (Lalpha YFbeta L346YF) Stat2-binding sites. The top panels (B and C) show a schematic representation of the constructs expressed in murine L-929 and MEFalpha [-/[- (transfectants whose designation starts with L or M, respectively) cells. Bottom panels show expression in mouse L-929 and MEFalpha -/- cells of human receptor subunits. One clone of the various transfectant lines is shown as an example. The following antibodies were used for immunoprecipitation: alpha 511-557 (alpha 511) and IFNaR3 (6), recognize different epitopes on the alpha  chain (24, 31); beta L515, recognizes epitopes between amino acids 300 and 515 of the beta L chain (31); and an antiserum against the murine alpha  chain (mualpha ). Migration of the alpha , beta L (*), and the association of the alpha  and beta L chains (alpha +beta L) is indicated. A complete characterization of the Lalpha beta L346 cell line was previously reported (6); therefore, only immunoprecipitations with IFNaR3 and murine alpha  subunit antibodies are shown to demonstrate that 125I-IFNalpha 2 does not interact with the mouse alpha  chain. The upper part shows the constructs used for expression. D, expression of human beta L in MEFalpha -/- cells. The electrophoretic mobility of previously unidentified complexes is indicated (left panel, arrowhead and 170). The dashed lines correspond to molecular mass markers, 184, 116, and 84 kDa (from top to bottom).

Finally, to explore the role of beta L in Stat and Jak activation, we developed a MEFalpha -/- cell line expressing only the human beta L chain (Fig. 2D, Mbeta L). Fig. 2D (lane 3) shows that the beta L antisera detects this subunit in cells expressing beta L wild type, whereas the anti-mouse or -human alpha  chain antibodies (lanes 1 and 2, respectively) do not detect any affinity cross-linking complexes in these cells. Interestingly, a 170-kDa and a higher molecular weight complex are detected in Mbeta L cells (Fig. 2D, 170 and arrow). The identity of these complexes is not clear, but they may correspond to beta  chain dimers, cross-linking of 125I-IFNalpha 2 to a surface protein in the proximity of the receptor complex, or novel receptor components. It is worth mentioning that these complexes may have not been detected previously because they show similar electrophoretic mobility as those formed by the association of alpha  and beta L and alpha  and beta S.

Activation of the Stat Pathway in Transfectants Expressing IFNalpha R Chains with Mutations of the Stat2-docking Sites-- We initially studied stable transfectants expressing a single mutation of the previously identified Stat2-binding sites, i.e. Tyralpha 466, 481 (19) and the constitutive site (20) on beta L404-462 (Fig. 3, Lalpha YFbeta L and Lalpha beta L346, lanes 1-6). We also studied mutations of a Stat2-docking site on the proximal tyrosines of beta L (Tyr269, Tyr306, Tyr316, Tyr318, and Tyr337) whose presence became evident in the course of these experiments (Lalpha beta LYF, lanes 7-9). In this context, transfectants lacking only one Stat2-binding site in the receptor complex would be informative regarding the role of that particular site on Stat2 tyrosine phosphorylation. HuIFNalpha 2 (lanes 3 and 6) can induce tyrosine phosphorylation of Stat2 (A) and Stat1 (B) in transfectants carrying mutations of only the constitutive site of beta L (beta L404-462) or the alpha  chain (Tyr466 and Tyr481). More importantly, the levels of tyrosine phosphorylation were comparable with those induced by murine IFNalpha beta (malpha beta ) used as positive control (lanes 2 and 5). However, mutation of the tyrosines of beta L (Lalpha beta LYF, lanes 7-9) significantly decreased huIFNalpha 2-induced tyrosine phosphorylation of Stat2 (compare human and murine IFNs in Lalpha YFbeta L and Lalpha beta L346 with Lalpha beta LYF, lanes 1-9). These data suggest that the tyrosines N-terminal to aa 346 of beta L play a more important role in Stat2 tyrosine phosphorylation than the remaining sites.


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Fig. 3.   Tyrosine phosphorylation of Stat1, Stat2, and Stat3 in L-929 cells expressing mutant alpha  and beta L chains. A and B, cells expressing mutations of only one (Lalpha YFbeta L, Lalpha beta L346, and Lalpha beta LYF cells, lanes 1-9), two (Lalpha YFbeta L346, Malpha YFbeta LYF, and Lalpha beta L346YF, lanes 10-17), or three (Lalpha YFbeta L346YF, lanes 18-20) Stat2-binding sites were stimulated with muIFNalpha beta (lanes 2, 5, 8, 11, 16, and 19), huIFNalpha 2 (lanes 3, 6, 9, 12, 14, 17, and 20), or left untreated (lanes 1, 4, 7, 10, 13, 15, and 18) at 37 °C for 10 min. Cell lysates were immunoprecipitated with an anti-Stat2 (A), anti-Stat1 (B) sera, resolved by SDS-polyacrylamide gel electrophoresis, and immunoblotted with an anti-phosphotyrosine antibody (4G10, Blot, pTyr, upper panel). Membranes were stripped and reprobed with an anti-Stat2 serum or anti-Stat1 monoclonal antibody (A and B, lower panels). C, a similar experiment as in A and B, but lysates were immunoprecipitated with an anti-Stat3 serum and immunoblotted with anti-phosphotyrosine. The lower panel shows an immunoblotting with an anti-Stat3 antibody after stripping of the filters.

We next studied transfectants carrying simultaneous mutations of two Stat2 sites. Therefore, these transfectants would be important to determine if the remaining Stat2 site is enough to support Stat2 tyrosine phosphorylation. Fig. 3 shows that Lalpha YFbeta L346 (lanes 10-12) containing only the docking site on the proximal tyrosines of beta L and Malpha YFbeta LYF cells (lanes 13 and 14) expressing only the constitutive Stat2-binding site on the beta  chain can support low levels of tyrosine phosphorylation of Stat2 in response to huIFNalpha 2. By contrast, Lalpha beta L346YF cells (lanes 15-17) expressing only the alpha  subunit-docking site (Tyr466 and Tyr481) are unable to support huIFNalpha 2-induced Stat2 tyrosine phosphorylation. As expected, deletion of all three Stat2 sites completely abolished tyrosine phosphorylation of Stat2 (Fig. 3A, lanes 18-20) but allowed low levels of tyrosine phosphorylation of Stat1 (Fig. 3B, lanes 18-20) in response to huIFNalpha .

In summary, induction of normal levels of Stat2 tyrosine phosphorylation by IFNalpha 2 requires the proximal tyrosines of beta L and at least one of the other Stat2 sites, the constitutive site of beta L or Tyr466 and Tyr481 on the alpha  chain. The proximal tyrosines and the constitutive site of beta L are probably the most important sites since they alone can support low levels of tyrosine phosphorylation, whereas Tyr466 and Tyr481 on the alpha  chain may play an accessory role in tyrosine phosphorylation of Stat2. A decrease in tyrosine phosphorylation of Stat1 (Fig. 3B) was observed in transfectants that have impaired tyrosine phosphorylation of Stat2 as previously reported (32). It is worth mentioning that huIFNalpha 2 was at least as efficient as muIFNalpha beta in inducing tyrosine phosphorylation of Jak1 and Tyk2 in all transfectant cell lines (data not shown) indicating that the levels of human receptor subunits expressed were sufficient to achieve full activation of the system.

We also studied the role of the different tyrosines in the receptor complex on the activation of Stat3. Interestingly, tyrosine phosphorylation of Stat3 was not affected by the absence of tyrosines on the alpha  (Fig. 3C, lanes 1-3), beta L (lanes 4-6), or both (lanes 7-9) chains strongly arguing against the requirement of tyrosine phosphorylation of the receptor for Stat3 activation and the role of this factor as an adaptor for PI3K (see "Discussion").

To determine whether the levels of activation of Stat2 and Stat1 detected in cells with mutations of the different Stat2 sites correlated with the formation of ISGF3, we performed EMSA using an ISRE probe. Analysis of transfectants expressing mutation of only one Stat2 site revealed that mutation of Tyralpha 466, 481 (Fig. 4, lanes 1-3) or the constitutive site in beta L (see Ref. 6) did not affect ISGF3 induction by huIFNalpha 2. On the contrary, mutation of the proximal tyrosines of beta L (Fig. 4, Lalpha beta LYF, lane 5) significantly impaired the formation of ISGF3. Similarly, a significant decrease (Fig. 4, Lalpha YFbeta L346, lane 8) or complete abrogation of the induction of the ISGF3 complex in response to huIFNs is observed in transfectants carrying mutations of two (Lalpha beta L346YF and Malpha YFbeta LYF, lanes 12 and 18, respectively) or three (Lalpha YFbeta L346YF, lane 15) Stat2-binding sites. Formation of the ISGF3 complex was blocked by an excess of cold ISRE oligonucleotide (lane 10). Interestingly, although low levels of tyrosine phosphorylation of Stat2 were observed in some cases (Fig. 2, A and B, Lalpha beta LYF, Lalpha YFbeta L346, and Malpha YFbeta LYF cells), this was not sufficient to produce significant amounts of ISGF3, suggesting that a threshold of Stat2 tyrosine phosphorylation has to be reached to obtain ISGF3 formation. The formation of Stat3 containing c-sis-inducible factor complexes in Lalpha YFbeta L and Lalpha YFbeta L346YF cells treated with human type I IFNs was not affected (data not shown).


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Fig. 4.   EMSA with an ISRE probe. Cell extracts from different transfectants were treated as indicated and used for EMSA with an ISRE probe. The migration of the ISGF3 complex is indicated. In some experiments a preparation of huIFNalpha Con1 (huCon, lane 2) that has similar biological activity as IFNalpha 2 was used. L-929 cells were used as positive control. Formation of ISGF3 was also competed by a 50-fold excess of unlabeled ISRE oligonucleotide.

Mutation of the Stat-binding Sites Impairs the Induction of an Antiviral State-- We next explored the ability of the different transfectants to support an IFN-induced antiviral state. We have previously reported that deletion of the region of beta L containing the Stat2-binding site (alpha beta L346 cells) did not affect the induction of an antiviral state by IFNalpha 2 (6). Similarly, 50% protection against the encephalomyocarditis virus was observed with 5 and 14 units/ml huIFNalpha 2 and muIFNalpha , respectively, in alpha YFbeta L cells (Table II) expressing mutation of the Stat2-docking sites (Tyr466 and Tyr481) on the alpha  chain. However, mutation of the proximal tyrosines of beta L (Table II, Lalpha beta LYF cells) or simultaneous mutation of two Stat2-binding sites (Lalpha YFbeta L346, Lalpha beta L346YF, Malpha YFbeta LYF, and Lalpha YFbeta L346YF cells) significantly impaired the antiviral response to huIFNalpha 2 but did not affect the antiviral effect of muIFNalpha (except in the MEFalpha -/- transfectants that are null for the alpha  chain and therefore unresponsive to muIFNalpha 4). The antiviral data correlate with the levels of tyrosine phosphorylation and ISGF3 induction observed in these cells.

                              
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Table II
Antiviral response

beta L Is Sufficient to Support Tyrosine Phosphorylation of Jak1 and Stat2 but Not Tyk2 or Stat1-- To investigate further the role of beta L in activation of Stat2, we studied the induction of tyrosine phosphorylation of this factor through the endogenous murine beta L chain expressed in MEF cells null for the alpha  chain (MEFalpha -/-) using muIFNalpha beta , and in MEFalpha -/- transfected with the human beta L chain using human type I IFNs. This system allows us to completely eliminate any role that the alpha  chain, human or murine, could play in the activation of Stat2. Moreover, it allows us to determine if beta L alone is sufficient to activate other proteins of the Jak-Stat pathway. Fig. 5A shows that stimulation of the murine beta L chain with recombinant muIFNbeta induces Jak1 tyrosine phosphorylation, although to a slightly lower extent than in L-929 cells (lane 2 and 4). As expected, treatment of MEFalpha -/- cells with recombinant muIFNalpha 4 or muIFNbeta (Fig. 5A, lanes 5-7) did not induce significant tyrosine phosphorylation of Tyk2 above base-line levels. Similarly, huIFNalpha 2 induced tyrosine phosphorylation of Jak1 in MEFalpha -/- cells expressing the human beta L chain (Mbeta L cells, lane 9) but failed to activate Tyk2 (data not shown).


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Fig. 5.   Activation of the Jak-Stat pathway through the beta L chain. A, lysates from MEFalpha -/- (lanes 1, 2, and 5-7), L-929 (lanes 3 and 4), or Mbeta L (lanes 8 and 9) cells stimulated with muIFNbeta (mbeta , lanes 2, 4, and 7), muIFNalpha 4 (lane 6), and huIFNalpha 2 (halpha , lane 9), or left untreated (CT, lanes 1, 3 and 8) were immunoprecipitated with an anti-Jak1 antiserum (lanes 1-4, 8, and 9) and immunoblotted with anti-phosphotyrosine antibodies (upper panel). Filters were stripped and blotted with an anti-Jak1 antibody (lower panel). Lanes 5-7 show a similar experiment performed with MEFalpha -/-, but immunoprecipitations were performed with an anti-Tyk2 antiserum. B, a similar experiment as in A in which lysates from MEFalpha -/- (lanes 1-3), L-929 (lanes 4-6), or Mbeta L (lanes 7-9) were immunoprecipitated with anti-Stat1 (lanes 1-6) and anti-Stat2 (lanes 7-9) antibodies. Upper and lower panels correspond to anti-phosphotyrosine (pTyr) and Stat1 or Stat2 immunoblots, respectively.

Murine type I IFNs also failed to induce tyrosine phosphorylation of Stat1 in MEFalpha -/- (Fig. 5B, lanes 2 and 3). The experiments aimed to determine whether different preparations of murine IFN, alpha  or beta , induced tyrosine phosphorylation of Stat2 in MEFalpha -/- cells were inconclusive (data not shown). Similar experiments performed with Mbeta L cells, however, showed that human beta L alone can support at least low levels of Stat2 tyrosine phosphorylation (Fig. 5B, lanes 8 and 9). These results clearly demonstrate that beta L alone can support low levels of activation of Jak1 and Stat2 but not tyrosine phosphorylation of Tyk2 and Stat1 which require the presence of the alpha  chain.

    DISCUSSION

It is evident that tyrosine phosphorylation of receptor subunits plays an important role in transmembrane signaling by some cytokines, i.e. interleukin 6 and IFNgamma (14, 15). More precisely, tyrosines in different receptor subunits serve as docking sites for Stat factors that are recruited to the receptor complex where they serve as targets for tyrosine kinases, probably of the Jak family. It is worth noting, however, that in some cytokine systems, i.e. growth hormone and granulocyte-colony-stimulating growth factor, activation of Stat factors appears to occur in the absence of tyrosines in the receptor (16-18). In the type I IFN system there are contradicting data regarding the role of the receptor subunits in the activation of Stat factors. For example, one report indicated that Tyr466 and Tyr481 of the alpha  chain served as docking sites only for Stat2 (19), whereas others indicated that the same tyrosines were able to bind not only Stat2 but also Stat1 and Stat3 (20). In addition, Gibbs et al. (23) indicated that cells expressing alpha  chains devoid of tyrosines have normal levels of activation of ISGF3.

We sought to test the hypothesis that there are redundant Stat2-docking sites present on the alpha  and beta  chains of the receptor. We first mapped a constitutive docking site in beta L (20). Surprisingly, elimination of both the constitutive site of beta L and Tyr466 and Tyr481 of the alpha  chain decreased, but did not abolish, Stat2 activation. The additional elimination of the five proximal tyrosines of beta L was required to completely abrogate activation of Stat2. Therefore, there are redundant, yet still distinct mechanisms of docking for Stat2, i.e. phosphotyrosine-dependent and -independent, in the alpha  and beta  chains of the receptor. Our results clearly demonstrate that there are three Stat2-docking sites in the receptor complex as follows: (a) a constitutive, phosphotyrosine-independent site on beta L (aa 404-462); (b) one or more of the proximal tyrosines (Tyr269, Tyr306, Tyr316, Tyr318, and Tyr337) also in beta L; and (c) Tyr466 and Tyr481 in the alpha  chain. Interestingly, isolated expression of either the constitutive site or proximal tyrosines of beta L was enough to support low levels of tyrosine phosphorylation of Stat2/Stat1 but not for induction of ISGF3 or an antiviral response. A receptor complex containing only the Stat2-docking site in the alpha  chain was unable to phosphorylate Stat2 suggesting, contrary to previous reports (19, 20), that this site may only play a secondary role in activation of Stat2. This is also supported by the detection of ISGF3 induction in cells that do not phosphorylate the alpha  chain (22) and by the finding that Stat2 is not coprecipitated with the alpha  chain after IFNalpha or IFNbeta treatment (Fig. 1A). The presence of the proximal tyrosines of beta L and at least one other Stat2 site within the receptor complex was required to obtain significant phosphorylation activation of Stat2/1, ISGF3 induction, and an antiviral response. These results suggest that the proximal tyrosines of beta L are absolutely required, but not sufficient, for full phosphorylation and activation of Stat2. Moreover, a threshold of Stat2 tyrosine phosphorylation should be reached to achieve full activation of this factor, leading to efficient formation of the ISGF3 complex and an antiviral state. In this respect, the activation of the type I IFN system appears to be different from other cytokine systems in which the presence of only one Stat-docking site is enough to support Stat-mediated DNA binding activity (14, 15). These results also strongly argue against a mechanism in which Stat2 is transferred from the constitutive docking site on beta L to a phosphorylated tyrosine on the alpha  chain, to be finally phosphorylated by a Jak kinase (20). It appears that, at least in part, the redundancy of Stat2-docking sites serves as a safety net to preserve the response of a system that serves as the first line of defense against viral infections. A decrease in activation of Stat1 accompanied the diminished activation of Stat2 as previously reported (32).

Interestingly, mutations of all the tyrosines in the cytoplasmic domain of the alpha  chain did not affect activation of Stat3. Moreover, tyrosine phosphorylation of Stat3 was even detected at normal levels in a receptor complex completely devoid of tyrosines. A previous report suggested that tyrosines 527 and 538 of the alpha  chain are required for Stat3 phosphorylation and subsequent activation of PI3K (21). Several lines of evidence argue against the role of phosphorylation of these tyrosines in IFN signaling. First, all the evidence suggests that Tyr466 and Tyr481, rather than Tyr527 and Tyr538 are the targets for tyrosine phosphorylation (4, 19). Therefore, even if Tyr527 is in a region containing a consensus sequence for the SH2 domain of Stat3, phosphorylation of this residue should occur for SH2 binding. Neither the mutational analysis (Fig. 3 and Refs. 4, 19, and 23) nor the in vivo data (22) provide evidence that Tyr527 and Tyr538 are phosphorylated. Second, mutation of Tyr527 and Tyr538 or deletion of the region that includes these tyrosines does not affect IFNalpha signaling (Fig. 3 and Ref. 23). Third, PI3K appears to be associated with and activated through IRS1/2 rather than Stat3 (33, 34).2 Fourth, L-929 cells expressing receptors without tyrosines and beta L346 (Lalpha YFbeta L346YF) have normal IFNalpha -induced PI3K-mediated serine phosphorylation.2 Therefore, the finding that activation of Stat3 persists after mutation of the two proximal tyrosines and deletion of the distal region containing the putative Stat3-docking site of the alpha  chain strongly argues against a role for tyrosine phosphorylation of this receptor subunit in Stat3 activation and the role of this factor as an adaptor.

Our results also indicate that the study of Stat-docking sites using phosphorylated peptides alone, without stable expression of the mutant receptors in the appropriate cell lines, may lead to ambiguous conclusions (19-21). The high concentrations of phosphorylated peptides used in some cases may result in non-physiological interactions between SH2 domains and phosphopeptides. Songyang et al. (35, 36) indicated that although SH2 domains have higher affinities for a specific sequence, they could interact with more than one sequence C-terminal to the phosphorylated tyrosine. Therefore, the concentrations of phosphorylated peptides used in most studies (in the order of 100 µM) may not represent physiological interactions, unless the interactions are confirmed by mutational analysis and expression in mammalian cells. In this scenario, stable expression of the alpha  chain with mutations of Tyr466 and Tyr481 (Stat2-docking site) and deletion of the distal tyrosines (Stat3-docking sites) reveals that the results with peptides in permeabilized cells belied the direct effect expected from disrupting the interaction of the alpha  chain with Stat2 or Stat3 (19, 21).

It has been proposed that Jak1 and Tyk2 are activated by transphosphorylation (37-39) after heterodimerization of the alpha  and beta  chains of the receptor. It is clear that activation of both kinases is required for IFNalpha 2 signaling, although a residual response for IFNbeta can be observed in the absence of Tyk2 (38-40). Our results clearly indicate that activation of Jak1 occurs in the complete absence of alpha  chain and Tyk2 activation. Moreover, this activation is sufficient to support low levels of Stat2 phosphorylation but not Stat1 by both IFNalpha and IFNbeta . Activation of Jak1 could be explained by homodimerization of two beta L chains or by heterodimerization with a novel receptor component. The finding of a novel 170-kDa complex in affinity cross-linking experiments performed with MEFalpha -/- transfected with beta L wild type (Fig. 2D) suggest that there are novel receptor chains, whereas the higher molecular weight complex may be indicative of beta  chain dimers. Alternatively, the beta L chain alone may be sufficient to activate Jak1. Whatever the mechanism involved, we did not find differences in Jak1 or Stat2 activation by IFNalpha and IFNbeta as previously reported for mutants lacking Tyk2. This is not surprising because the activation of this pathway by these IFNs relies on the selective requirement by IFNbeta of the 417-462 region of beta L (41).

    ACKNOWLEDGEMENTS

We thank Drs. Michell Aguet and James Ihle for providing the MEF null for the alpha  chain and the anti-Jak1 and Stat3 sera, respectively.

    FOOTNOTES

* This work was supported by National lnstitutes of Health Grants GM54709 (to O. R. C.) and CA73381 (to L. C. P).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

b Both authors contributed equally to this work.

h To whom correspondence should be addressed: Dept. of Pharmacology, University of Illinois, 835 S. Wolcott, Rm. E403 (M/C 868), Chicago, IL 60612. Tel.: 312-413-4113; Fax: 312-996-1225; E-mail: ocolamon{at}uic.edu.

The abbreviations used are: IFN, interferon; Stat, signal transducer and activator of transcription; aa, amino acids; ISGF3, interferon-stimulated gene factor 3; GST, glutathione S-transferase; ISRE, interferon-stimulated response element; EMSA, electrophoretic mobility shift assay; hu, human; mu, murine; PI3K, phosphatidylinositol 3-kinase.

2 S. Uddin, O. R. Colamonici, and L. C. Platanias, manuscript in preparation.

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