©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Direct Association of STAT3 with the IFNAR-1 Chain of the Human Type I Interferon Receptor (*)

(Received for publication, October 19, 1995; and in revised form, February 9, 1996)

Chuan-He Yang (1) Wei Shi (1) Leela Basu (1) Aruna Murti (1) Stefan N. Constantinescu (1) Lawrence Blatt (2) Ed Croze (3) Jerald E. Mullersman (1) Lawrence M. Pfeffer (1)(§)

From the  (1)Department of Pathology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, (2)Interferon Program, AMGEN, Inc., Thousand Oaks, California 91320, and (3)Department of Protein Biochemistry and Biophysics, Berlex Biosciences, Inc., Richmond, California 94804

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Based on the reports of the activation of the transcription factor known as STAT3 (for signal transducers and activators of transcription) or APRF (for acute phase response factor) by various cytokines, we investigated the possible role of STAT3 in type I interferon (IFN) receptor signaling. We show that STAT3 undergoes IFNalphadependent tyrosine phosphorylation and IFNalpha treatment induces protein-DNA complexes that contain STAT3. In addition, STAT3 associates with the IFNAR-1 chain of the type I receptor in a tyrosine phosphorylation-dependent manner upon IFNalpha addition. The binding of STAT3 to the IFNAR-1 chain occurs through a direct interaction between the SH2 domain-containing portion of STAT3 and the tyrosine-phosphorylated IFNAR-1 chain. Furthermore, tyrosine-phosphorylated STAT3 bound to the IFNAR-1 chain also undergoes a secondary modification involving serine phosphorylation. This phosphorylation event is apparently mediated by protein kinase C, since it was blocked by low concentrations of the protein kinase inhibitor H-7. The biological relevance of IFN activation of STAT3 is further illustrated by the finding that STAT3 is not activated by IFN in a cell line resistant to the antiviral and antiproliferative actions of IFNalpha but in which other components of the JAK-STAT pathway are activated by IFNalpha.


INTRODUCTION

Cytokines are multifunctional mediators of the growth and differentiation of hematopoietic, lymphopoietic, and neural systems. They exert their effects through specific surface receptors expressed on target cells, triggering biological effects through the activation of specific gene transcription. One approach to the understanding of the molecular basis of transcriptional activation by cytokines is the identification of cis-responsive elements within genes and the transcription factors responsive to cytokine signals. For example, the type I interferons (IFNs), (^1)IFNalpha and IFNbeta, induce the transcription of the early IFN-stimulated gene (ISG) gene family(1) . IFNs are cytokines that have profound effects on cells, including antiviral protection, inhibition of the proliferation of normal and transformed cells, and modulation of the immune system. IFN signaling to the cell nucleus involves the tyrosine phosphorylation of STAT (signal transducers and activators of transcription) proteins. IFN-activated STAT1 and STAT2 translocate to the nucleus, where they recognize the conserved IFN stimulus response element (ISRE) within the promoter of ISGs, which is both necessary and sufficient for ISG transcription(2, 3) . Central to the type I IFN-activated pathway are two non-receptor protein tyrosine kinases, JAK1 and TYK2, which apparently mediate the tyrosine phosphorylation of IFN receptor chains and STATs(4, 5) .

The intracellular domains of type I IFN receptor chains contain conserved motifs that likely function in transmembrane signaling(6, 7) . The ligand-induced tyrosine phosphorylation of STAT transcription factors is one of the events most proximal to cytokine-dependent JAK activation(2, 3) . Recent studies indicate that many cytokines, including type I IFNs, induce tyrosine phosphorylation of the STAT3 transcription factor(8, 9) , also known as the acute phase response factor (APRF) involved in acute phase gene expression. We previously proposed that the intracellular domain of the IFNAR-1 chain plays a critical role in type I IFN signaling by specifically docking important SH2 domain-containing cytoplasmic proteins(6) . Therefore, we investigated the possible role of STAT3 in IFNalpha signaling through the IFNAR-1 chain.

The type I IFN receptor apparently consists of multiple glycoprotein subunits(6, 10, 11) . The cDNAs coding for two subunits, the IFNAR-1 and IFNAR-2 chains, have recently been cloned(12, 13, 14, 15) . We show that STAT3 associates with the IFNAR-1 chain in a tyrosine phosphorylation-dependent manner after exposure of cells to IFN. The binding of STAT3 to the IFNAR-1 chain can occur through a direct interaction between the SH2 domain-containing portion of STAT3 and tyrosine-phosphorylated IFNAR-1 chain. The biological significance of IFN activation of STAT3 is further borne out by the finding that STAT3 is the only signaling molecule in the JAK-STAT pathway not activated by IFNalpha in an IFNalpha-resistant cell line.


MATERIALS AND METHODS

Cells

Human Daudi and U266 cells were maintained at 2.5-10 times 10^5 cells/ml in RPMI 1640 containing 10% defined bovine calf serum (Hyclone). Human HeLa-S3 cells were grown in stirred suspension culture in spinner medium supplemented with 10% bovine calf serum.

IFN and mAbs

The activity of recombinant human IFNalpha (IFNCon1), provided by AMGEN, is expressed in international reference units (IU)/ml as assayed by protection against the cytopathic effect of vesicular stomatitis virus on human fibroblasts, using the NIH human IFNalpha standard for reference. mAbs directed against the extracellular domain of the IFNAR-1 chain have been described previously(6) .

Nuclear Extracts and Gel Shift Assays

Nuclei were extracted with buffer containing 20 mM Tris-HCl, pH 7.85, 250 mM sucrose, 0.4 M KCl, 1.1 mM MgCl(2), 5 mM beta-mercaptoethanol, and 0.4 mM phenylmethylsulfonyl fluoride, and extracts were frozen on dry ice and stored at -80 °C(16, 17, 18) . For gel shift analysis, the nuclear extracts were incubated with a P-end-labeled promoter probe for either the high affinity sis-inducible element (SIE) from the c-fos gene (5`-AGCTTCATTTCCCGTAATCCCTAAAGCT-3`) or the ISRE from ISG15 (5`-GATCCATGCCTCGGGAAGGGAAACCGAAACTGAAGCC-3`) (19, 20) at 25 °C for 30 min, and the free probe was separated from protein-DNA complexes on 5% polyacrylamide gels. For supershift assays, nuclear extracts were preincubated with a 1:50 dilution of normal rabbit serum, anti-STAT1 (alphaSTAT1, Santa Cruz Laboratories), anti-STAT2 (alphaSTAT2, Santa Cruz Laboratories), or anti-STAT3 (alphaSTAT3) at 25 °C for 0.5 h. Gels were quantitated by PhosphorImager autoradiography.

GST Fusion Construct

A segment encoding residues 498-770 from a human STAT3 cDNA was subcloned into the EcoRI and SacI sites of pGEX-KG(21) . The construct was confirmed by restriction enzyme digestion. The fusion protein (STAT3-GST) was obtained from Escherichia coli transformed with the plasmid construct and affinity-purified on glutathione-Sepharose (Pharmacia Biotech Inc.) as described previously(22) .

Immunoprecipitations and Immunoblot Analysis

For immunoprecipitation studies, 1 times 10^8 cells were treated with IFNalpha (5,000 IU/ml) at 37 °C for the indicated periods of time and then washed with ice-cold phosphate-buffered saline and lysed for 20 min in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 15% glycerol) containing 1 mM NaF, 1 mM Na(3)VO(4), 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml soybean trypsin inhibitor, 5 µg/ml leupeptin, and 1.75 µg/ml benzamidine(3) . Samples were centrifuged (12,000 times g, 15 min) at 4 °C, and supernatants were immunoprecipitated with alphaSTAT3, anti-phosphotyrosine (alphaTyr(P), Oncogene Sciences Ab-2), or anti-IFNAR-1 (alphaIFNAR-1) overnight at 4 °C. Immune complexes were collected using Protein A-Sepharose beads (Pharmacia) and eluted in sample buffer. Samples were run on SDS, 7.5% PAGE, transferred to PVDF membranes (Millipore), and probed with alphaSTAT3 mAb (dilution, 1:1000), followed by anti-mouse IgG coupled with horseradish peroxidase (Amersham Corp.). Blots were developed using enhanced chemiluminescence (ECL, Amersham Corp.).

For precipitation with GST fusion proteins, lysates from control or IFN-treated Daudi cells were precipitated with STAT3-GST or GST bound to glutathione-agarose beads. The precipitated proteins were resolved by SDS-PAGE (7.5%), blotted onto PVDF membranes, and probed with alphaIFNAR-1. For blotting with GST fusion proteins, alphaIFNAR-1 immunoprecipitates resolved by SDS-PAGE were probed with STAT3-GST or GST (purified after elution with glutathione from agarose beads). The IFNAR-1 chain was visualized by ECL using a hamster alphaGST mAb and a goat anti-hamster IgG horseradish peroxidase conjugate (Southern Biotechnology Associates).


RESULTS

IFNalpha Induction of STAT-related Proteins That Bind to Specific Promoter Elements

Nuclear extracts prepared from IFN-sensitive Daudi cells treated with IFNalpha were incubated with a labeled probe for either the high affinity SIE or the ISRE, and the resultant DNA-protein complexes were analyzed by an electrophoretic mobility shift assay (EMSA). Fig. 1shows that treatment with IFNalpha induced the STAT-related DNA binding factors, sis-inducible factor (SIF)-A, SIF-B, and SIF-C, as well as IFN-stimulated gene factors (ISGFs). No DNA binding to the SIE was detected in the presence of excess unlabeled SIE oligonucleotide, and binding was not competed by an excess of ISRE oligonucleotide. Similarly, no DNA binding to the ISRE probe was detected in the presence of excess ISRE oligonucleotide, and binding was not competed by the SIE oligonucleotide. Taken together these results indicate that the binding to each probe was specific. To detect specific STAT proteins in the IFN-inducible DNA-protein complexes, we performed gel supershift assays with various STAT-specific antisera (alphaSTAT). Previous studies showed that the ISRE-specific ISGF3 factor contains only STAT1 and STAT2 but not STAT3 (1) . Consistent with these results, we found that the ISGF3 complex was not supershifted with alphaSTAT3 but was supershifted by either alphaSTAT1 or alphaSTAT2 (data not shown). However, a distinct slowly migrating DNA-protein complex formed with the ISRE, denoted ISGFX in Fig. 1, was supershifted by alphaSTAT3. In addition, alphaSTAT3 supershifted SIF-A and SIF-B formed with the SIE in IFN-treated Daudi cells, while alphaSTAT1 supershifted SIF-C(23, 24) . Neither control normal rabbit serum nor alphaSTAT2 shifted any of the IFN-induced SIE DNA-protein complexes. Thus, IFNalpha treatment of Daudi cells induced DNA binding activities attributable to STAT1, STAT2, STAT3, and possibly additional unknown factor(s).


Figure 1: The presence of STAT-related proteins in ISRE and SIE protein-DNA complexes induced by IFN. A, nuclear extracts were prepared from control and IFN-treated (5,000 IU/ml) IFN-sensitive Daudi cells and then subjected to EMSA with a P-labeled ISRE or SIE probe in the absence or presence of a 50-fold excess of unlabeled oligonucleotide probes. In addition, one set of nuclear extracts from IFN-treated cells was preincubated with alphaSTAT3 prior to EMSA analysis. The positions of ISGF complexes and the SIF complexes are indicated. B, nuclear extracts from IFN-treated (5,000 IU/ml) Daudi cells were incubated with normal rabbit serum (NRS), alphaSTAT1, alphaSTAT2, or alphaSTAT3 prior to EMSA analysis with a P-labeled SIE probe.



IFNalpha-dependent Tyrosine Phosphorylation of STAT3 and the Coprecipitation of STAT3 with the IFNAR-1 Chain

Tyrosine phosphorylation of STATs is essential for their DNA binding activity and their translocation from the cytoplasm into the nucleus(1) . To determine whether STAT3 is tyrosine-phosphorylated in response to IFN, cell lysates were precipitated with alphaSTAT3 and analyzed by blotting with alphaTyr(P) (Fig. 2). Although similar amounts of STAT3 from both IFN-treated and untreated cells were precipitated by alphaSTAT3, only alphaSTAT3 precipitates from IFN-treated cells contained tyrosine-phosphorylated STAT3. Furthermore, a doublet of STAT3 was observed, as illustrated in Fig. 2B. The more slowly migrating STAT3 band reportedly represents a secondary modification of STAT3 involving serine phosphorylation(25, 26) .


Figure 2: Coprecipitation of tyrosine-phosphorylated STAT3 with the IFNAR-1 chain in IFN-sensitive Daudi cells. A, lysates prepared from control or IFN-treated (5,000 IU/ml) cells were immunoprecipitated with either alphaIFNAR-1, alphaTyr(P) (alphapTyr) or alphaSTAT3. The proteins were resolved by SDS-PAGE, blotted onto PVDF membranes, and probed with alphaSTAT3. IP, immunoprecipitate; WB, Western blot. B, to show serine phosphorylation of STAT3, cells were treated with H-7 at varying concentrations (0-100 µM) or staurosporine (st, 300 nM) for 30 min prior to IFNalpha addition. Lysates prepared from IFN-treated (5,000 IU/ml; 15 min) cells were precipitated with alphaIFNAR-1 and blotted with alphaSTAT3. The faster and slower migrating forms of STAT3 are indicated on the figure as STAT3 and STAT3, respectively.



We have previously shown that the IFNAR-1 chain undergoes IFN-dependent tyrosine phosphorylation(6) . In addition, several tyrosine-phosphorylated proteins coprecipitate with the IFNAR-1 chain. To determine if STAT3 coprecipitated with IFNAR-1, lysates from control and IFN-treated Daudi cells were precipitated with alphaIFNAR-1 and analyzed by blotting with alphaSTAT3 (Fig. 2). Although similar amounts of IFNAR-1 chain were precipitated by alphaIFNAR-1 (data not shown), only alphaIFNAR-1 precipitates from IFN-treated cells contained STAT3 protein (Fig. 2). Furthermore, blotting with alphaTyr(P) indicated that the kinetics of tyrosine phosphorylation of IFNAR-1 and STAT3 were remarkably similar. IFN-dependent coprecipitation of STAT3 with the IFNAR-1 chain was also observed in HeLa epithelioid carcinoma cells and U-266 lymphoblastoid cells (data not shown). As observed in alphaSTAT3 precipitates, the STAT3 band precipitated by alphaIFNAR-1 resolved as a doublet. This result directly places the kinase responsible for the secondary modification of STAT3 (serine phosphorylation) in close proximity to the IFNAR-1 chain of the receptor.

The Effects of PKC Inhibitors on IFN-induced Serine Phosphorylation of STAT3

Recently, it has been shown that IFN also induces the serine phosphorylation of both STAT1 and STAT3(25, 26) , an event that is necessary for maximal activation of transcription(26) . Since we previously showed that IFNalpha activates the and subspecies of PKC in IFN-sensitive Daudi cells and that PKC plays a role in IFNalpha action in these cells(27) , (^2)we investigated the role of PKC in the serine phosphorylation of STAT3. The serine phosphorylation of STAT3 is readily detectable because it results in the presence of a doublet of tyrosine-phosphorylated STAT3 in SDS-PAGE (Fig. 2). Since at low concentrations H-7 is a selective inhibitor of PKC(29) , we examined the effect of pretreatment of Daudi cells with various concentrations of H-7 on the IFN-induced tyrosine-phosphorylated STAT3 doublet. As shown in Fig. 2B, H-7 caused a dramatic disappearance of the slower mobility, upper STAT3 band at concentrations of 10 µM or less, with a 50% inhibition observed at 3 µM, which is consistent with an inhibitory effect of H-7 on PKC. In addition, pretreatment with the PKC inhibitor staurosporine also blocked the secondary modification of STAT3, and thus only the faster mobility tyrosine-phosphorylated form of STAT3 is detected. At high concentrations staurosporine (200 nM) and H-7 (30 µM) also block the tyrosine phosphorylation of JAK protein tyrosine kinases (^3)and thus inhibit the tyrosine phosphorylation of STAT3 (Fig. 2B). Thus, these results suggest that an IFN-activated PKC may mediate the serine phosphorylation of STAT3. We are currently evaluating whether PKC subspecies or other serine kinases are physically associated with the type I IFN receptor.

Precipitation and Direct Blotting of the Tyrosine-phosphorylated IFNAR-1 Chain by a STAT3-GST Fusion Protein

STAT3 contains an SH2 domain(3) , a modular noncatalytic domain of about 100 amino acid residues, that mediates high affinity interactions of cytoplasmic effectors with specific phosphotyrosine motifs in activated cell surface receptors(30, 31) . The phosphorylation of conserved motifs present in cytokine receptor subunits creates multifunctional docking sites for SH2 domain-containing cytoplasmic effectors(6, 32, 33) . Since the IFNAR-1 chain undergoes rapid tyrosine phosphorylation and associates with STAT3, one likely possibility is that this interaction involves the SH2 domain of STAT3. Although the phosphopeptide specificity of many SH2 domain-containing proteins has been determined, attempts to define the phosphopeptide specificity of the SH2 domains of STATs have been unsuccessful(34) . Recent structural and functional studies on the Src family tyrosine kinases Lck and Fyn have demonstrated important interactions between SH2 domains and adjacent protein structures (i.e. SH3 domains)(35, 36) . Therefore, we prepared a GST fusion protein (STAT3-GST) that encompasses the SH2 and SH3 domains of STAT3, as well as carboxyl-terminal residues. Lysates were prepared from IFN-treated Daudi cells and incubated with STAT3-GST bound to glutathione-agarose beads. The material bound was analyzed by blotting with alphaIFNAR-1. As shown in Fig. 3, IFN treatment resulted in binding of the IFNAR-1 chain to the SH2 domain-containing STAT3-GST. Pretreatment of cells with genistein, a tyrosine kinase inhibitor, blocked interaction of the IFNAR-1 chain with STAT3-GST, demonstrating that tyrosine phosphorylation of IFNAR-1 is required for interaction with the fusion protein. The specificity of the interaction is further illustrated by the finding that IFNAR-1 chain was not bound to GST protein alone. To show that STAT3 can directly interact with the tyrosine-phosphorylated IFNAR-1 chain, alphaIFNAR-1 precipitates were blotted with STAT3-GST. STAT3-GST bound only to tyrosine-phosphorylated IFNAR-1 chain, since binding was only detected after IFN treatment and was abolished by pretreatment with genistein. In contrast, blotting with GST fusion proteins containing the SH2 domains of Abl, Crk, or GTPase-activating protein did not show any interaction with tyrosine-phosphorylated IFNAR-1 chains (data not shown).


Figure 3: Precipitation and direct blotting of the tyrosine-phosphorylated IFNAR-1 chain in IFN-sensitive Daudi cells by the STAT3-GST fusion protein. A, lysates from control or IFN-treated (5,000 IU/ml; 15 min) cells were precipitated with STAT3-GST, GST, or by alphaTyr(P) (alphapTyr). The precipitated proteins were resolved by SDS-PAGE, blotted onto PVDF membranes, and probed with alphaIFNAR-1. B, lysates prepared from control or IFN-treated (5,000 IU/ml; 15 min) cells were precipitated with alphaIFNAR-1. The proteins were resolved by SDS-PAGE, blotted onto PVDF membranes, and probed with STAT3-GST or GST. To test for the role of tyrosine phosphorylation of the IFNAR-1 chain in its interaction with STAT3, cells were treated in the presence or absence of genistein (gen, 100 µM) for 30 min prior to IFNalpha addition.



IFNalpha Fails to Induce STAT3 Tyrosine Phosphorylation and STAT3 DNA Binding Activity in IFN-resistant Daudi Cells

We then examined the activation of STAT3 by IFNalpha in a Daudi subline, which is highly resistant to the antiviral and antiproliferative action of IFNalpha. In contrast to the results obtained with IFN-sensitive cells, IFNalpha did not induce the tyrosine phosphorylation of STAT3 (Fig. 4). This is in spite of the fact that IFN-resistant Daudi cells contain similar levels of STAT3 when compared with IFN-sensitive Daudi cells ( Fig. 2and Fig. 4). The failure of IFN to activate STAT3 in IFN-resistant cells was also demonstrated in gel shift assays. Nuclear extracts prepared from IFN-resistant Daudi cells treated with IFNalpha were incubated with a labeled probe for the high affinity SIE, and the resultant DNA-protein complexes were analyzed by the EMSA. Fig. 4shows that treatment of IFN-resistant Daudi with IFNalpha only induced the STAT1-related DNA binding factor SIF-C. SIF-C was supershifted with alphaSTAT1 sera demonstrating its presence in the protein-DNA complex. This is in sharp contrast to the findings with IFN-sensitive Daudi cells where STAT3-containing protein-DNA complexes SIF-A and SIF-B were also induced by IFN treatment.


Figure 4: The failure of IFNalpha to activate STAT3 in IFN-resistant Daudi cells. A, lysates prepared from control or IFN-treated (5,000 IU/ml) cells were precipitated with alphaSTAT3. The proteins were resolved by SDS-PAGE, blotted onto PVDF membranes, and probed with alphaTyr(P) (pTyr) or alphaSTAT3. B, nuclear extracts from Daudi cells treated with IFNalpha (5,000 IU/ml) were preincubated with alphaSTAT1, alphaSTAT2, or alphaSTAT3 and then subjected to EMSA with a P-labeled SIE probe. The positions of SIF complexes formed in extracts from IFN-sensitive cells are presented for reference. gen, genistein.




DISCUSSION

The binding of cytokines to cell surface receptors on target cells induces the transcription of specific sets of genes. This new gene expression involves the cytokine-induced tyrosine phosphorylation of specific subsets of STAT transcription factors and the formation of phosphorylation-dependent STAT complexes. The mechanism by which a cytokine and its cognate receptor selectively activate only certain STATs is poorly understood, but several lines of evidence indicate that it involves intermediate phosphotyrosine- and SH2 domain-dependent complexes between the receptor chain and specific STAT factors. For example, IFN induces the tyrosine phosphorylation of the IFNGR-1 chain of the multisubunit IFN receptor as well as of STAT1(37) . A functionally critical tyrosine residue in the membrane distal region of the IFNGR-1 chain is involved in STAT1 activation. Furthermore, phosphopeptides corresponding to this region interact with STAT1 and block its activation. These results were the first to show that a specific tyrosine-based activation motif (TBM) in the cytosolic tail of an IFN receptor subunit dictated specific STAT activation. The results reported herein establish that IFNalpha activates STAT3 directly through an interaction between the tyrosine-phosphorylated IFNAR-1 chain and the SH2 domain-containing half of STAT3. It is likely that STAT3 becomes tyrosine-phosphorylated by the receptor-associated JAK1 or TYK2 kinases. Most importantly, we demonstrate a direct association of a STAT with a cytokine receptor chain and thus provide a mechanism whereby cytokine receptors dictate signaling specificity.

The IFNAR-1 chain of the type I IFN receptor undergoes ligand-dependent tyrosine phosphorylation and plays a crucial role in signal transduction(6, 38, 39) . The cytoplasmic tails of the mouse, human, and bovine IFNAR-1 chains contain a perfectly conserved membrane distal amino acid motif, KYSSQTSQDSGNYSNE(6, 7) . We previously proposed that this TBM plays a critical role in the signaling of type I IFN through its receptor by specifically docking important SH2 domain-containing cytoplasmic proteins(6) . Recently, it was reported that a TBM of YXXQ in the cytosolic tail of the shared signal-transducing gp130 chain of the IL6 receptor family is required for cytokine-dependent STAT3 activation(40) . Thus, it is possible that the conserved YSSQ motif of the cytosolic tail of the IFNAR-1 chain may also serve as a docking site for STAT3. We are presently investigating whether this motif in fact is the STAT3 docking site on the IFNAR-1 chain.

STAT3 also undergoes serine phosphorylation(25, 26) , a modification that was blocked in Daudi cells by serine kinase inhibitors (H-7 and staurosporine) in IFN-treated cells. Thus, we can place both serine and tyrosine protein kinases in the vicinity of the type I IFN receptor and in early transmembrane signaling events. Furthermore, the IC for inhibitory action of H-7 on IFN-induced STAT3 serine phosphorylation suggests that PKC may mediate the serine phosphorylation of STAT3. We have previously shown that, although Daudi cells express PKCalpha, -beta, -, -, -, and -, IFNalpha selectively activates PKC and PKC(27) .^2 Thus, it is tempting to suggest that either one or both IFN-activated PKC subspecies may mediate the phosphorylation of STAT3. Recently, it has been shown that the serine phosphorylation of STAT1 and STAT3 is necessary for maximal activation of transcription(26) .

Finally, although much attention has been focused on the role of STAT1 and STAT2 in IFNalpha action, our results suggest that STAT3 is also involved in the biological actions of IFN. We previously reported that IFNalpha rapidly induces ISG transcription in IFN-sensitive and IFN-resistant Daudi cells(17, 28) . However, while ISG transcription persists at high levels in the IFN-sensitive Daudi line, the activation of ISG transcription is only transient in IFN-resistant Daudi cells. IFN-resistant Daudi cells undergo the normal activation of other components of the JAK-STAT pathway, as determined by the ligand-dependent tyrosine phosphorylation of STAT1, STAT2, JAK1, and TYK2.^2 It is of note that the ligand-dependent tyrosine phosphorylation of the IFNAR-1 chain was reduced in IFN-resistant Daudi cells when compared with IFN-sensitive cells. In the present report we show that, although IFN activates STAT3 in IFN-sensitive Daudi cells, STAT3 was not activated in the IFN-resistant line when assayed by STAT3 tyrosine phosphorylation or by formation of STAT3-containing protein-DNA complexes in gel supershift assays. We provide evidence for a direct phosphotyrosine-dependent interaction between the IFNAR-1 chain of the human type I interferon receptor and the SH2 domain-containing portion of STAT3. This association is required for the formation of functional STAT3-containing transcription factors in response to interferon-alpha.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM36716 and by a grant from AMGEN, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: IFN, interferon; ISG, IFN-stimulated gene; ISRE, IFN stimulus response element; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; GST, glutathione S-transferase; SIE, sis-inducible element; EMSA, electrophoretic mobility shift assay; SIF, sis-inducible factor; ISGF, IFN-stimulated gene factor; PKC, protein kinase C; TBM, tyrosine-based activation motif.

(^2)
L. M. Pfeffer, M. K. Dahmer, C. Wang, R. Pine, N. C. Reich, A. Murti, D. J. MacEwan, and S. N. Constantinescu, submitted for publication.

(^3)
C-H. Yang and L. M. Pfeffer, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. J. E. Darnell, Jr. (Rockefeller University) for providing alphaSTAT3 and B. Mayer (Harvard University) for providing GST fusion proteins containing the SH2 domains of Abl, Crk, or GTPase-activating protein.


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