©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation and Activation of the DNA Binding Activity of Purified Stat1 by the Janus Protein-tyrosine Kinases and the Epidermal Growth Factor Receptor (*)

(Received for publication, May 23, 1995)

Frederick W. Quelle (1) William Thierfelder (1) Bruce A. Witthuhn (1) Bo Tang (1) Stanley Cohen (2) James N. Ihle (1) (3)(§)

From the  (1)Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, the (2)Vanderbilt University School of Medicine, Department of Biochemistry, Nashville, Tennessee 37232-0146, and the (3)Department of Biochemistry, University of Tennessee Medical School, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The activation of Janus protein-tyrosine kinases (Jaks) and the subsequent phosphorylation and activation of latent signal transducers and activators of transcription (Stats) are common elements in signal transduction through the cytokine receptor superfamily. To assess the role and specificity of Jaks in Stat activation, we have utilized baculovirus expression systems to produce Stat1 and the Jaks. Co-expression of Stat1 with Tyk2, Jak1, or Jak2 resulted in the specific tyrosine phosphorylation of Stat1 at Tyr, the residue phosphorylated in mammalian cells stimulated with interferon . Alternatively, Stat1, purified to apparent homogeneity from insect cell extracts, was phosphorylated at Tyr in Jak immune complex kinase reactions. Phosphorylation of purified Stat1 was necessary and sufficient for the acquisition of DNA binding activity. The specificity in both systems was indicated by the inability of a Jak2 catalytically inactive mutant (Jak2-Glu) or the Tec protein-tyrosine kinase to phosphorylate Stat1. However, immune complex-purified epidermal growth factor receptor was capable of phosphorylating purified Stat1 at Tyr and activating its DNA binding activity in in vitro reactions.


INTRODUCTION

Cellular functions are regulated through the interaction of extracellular ligands with their cognate receptors. One group of ligands, variably termed growth factors, cytokines, or colony-stimulating factors, utilize structurally and functionally similar receptors of the cytokine receptor superfamily(1) . These receptors couple ligand binding to the activation of protein-tyrosine phosphorylation through their association with members of the Janus protein-tyrosine kinase (Jaks) family (reviewed in (2) and (3) ). The Jak family currently consists of Jak1, Jak2, Jak3, and Tyk2, which vary in size from 120-135 kDa. One or more of the Jaks associate with one or more of the cytokine receptor chains at a membrane proximal site of the cytoplasmic domain. The association and activation of Jaks is critical for signaling through these receptors as indicated by the inactivation of receptor function by deletions or point mutations in the receptors that disrupt their ability to associate with the Jaks. A critical role for Jaks in interferon (IFN) (^1)signaling has also been established through the identification of cell lines selected for the inability to respond to IFN that lack individual Jaks. In these cells, introduction of the appropriate Jak restores IFN signaling. Lastly, recent studies (4, 5) have shown that kinase inactive Jaks can function as dominant negatives in cytokine signaling.

The activation of Jaks is required for the subsequent tyrosine phosphorylation of a variety of proteins that are involved in signal transduction. In particular, analysis of receptor mutants has indicated that Jak activation is required for the tyrosine phosphorylation of SHC, the p85 subunit of phosphatidylinositol 3-kinase, Vav, as well as members of the Stat family of proteins. The Stats (signal transducers and activators of transcription) were initially identified in IFN signaling and have been shown to be essential for many of the responses to IFN (reviewed in (6) ). Individual cytokines induce the tyrosine phosphorylation of one or more of the seven known Stats (Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6). In the response to IFNalpha/beta, Jak1 and Tyk2 are activated, and Stat1 and Stat2 are subsequently tyrosine-phosphorylated. Following phosphorylation, Stat1 and Stat2 form a complex with a DNA-binding protein, p48, and the complex translocates to the nucleus, binds DNA, and is required for activation of transcription of IFN responsive genes. The absence of any of the components negates the response. In the response to IFN, Jak1 and Jak2 are activated, and Stat1 is tyrosine-phosphorylated and forms a DNA binding homodimer that translocates to the nucleus and is required for activation of the transcription of IFN responsive genes.

Although a strong correlation exists between Jak activation and the tyrosine phosphorylation of the Stats, there has not been direct evidence to support the hypothesis that the Stats are immediate substrates for the Jaks. Indeed, Stat activation has been seen with other receptor systems, including the epidermal growth factor receptor (EGFR)(7, 8, 9) . Nevertheless, the utilization of specific Jaks by individual cytokines and activation of specific Stats have suggested the possibility that individual Jaks may specifically phosphorylate Stats. The studies presented here were designed to address these questions. Using baculovirus expressions systems, we demonstrate that Stat1, either co-expressed with the Jaks or as a purified protein added to immune complex reactions, is a substrate for Jak1, Jak2, or Tyk2. Phosphorylation occurs at the single site previously identified as the in vivo site of tyrosine phosphorylation and as a consequence, Stat1 acquires DNA binding activity. Lastly we demonstrate that Stat1 is similarly a substrate for EGFR but not for the cytoplasmic protein-tyrosine kinase Tec.


MATERIALS AND METHODS

Protein Expression in Insect Cells

Insect SF9 cells were maintained in Grace's insect media. Baculovirus expression vectors were prepared as described previously (10) using cDNAs for human Stat1(11) , murine Jak1(12) , and murine Jak2(13) . A human Tyk2 baculovirus expression construct was kindly provided by H. Yan and J. Krolewski from Columbia University, New York. Jak2-Glu was derived from the Jak2 cDNA by introducing a point mutation at codon 882 resulting in its alteration from a lysine to glutamic acid. For expression of proteins in insect cells, SF9 cells were infected with one or more viruses (multiplicity of infection, 5-10). 50-60 h after infection, cells were disrupted in lysis buffer (0.5% Nonidet P-40, 20 mM Tris, pH 8.0, 100 mM NaCl, 50 mM NaF, 100 µM Na(3)VO(4), 1 mM dithiothreitol, 50 µg/ml phenylmethylsulfonyl fluoride, 10% glycerol). Lysates were cleared of insoluble material by centrifugation at 10,000 g for 10 min and were then used directly for immunoprecipitation or gel mobility shift assays.

Immunoprecipitation and Western Blotting

Antisera specific for Stat1(14) , Jak1(13) , and Jak2 (15) have been described. Anti-Tyk2 serum was obtained from Santa Cruz Biotechnology. Anti-phosphotyrosine antibodies (PY20) were obtained from ICN. Rabbit anti-EGFR serum was prepared using the receptor isolated from mouse liver as an antigen (16) . Specific proteins were immunoprecipitated from cell lysates using the indicated antiserum and protein A-Sepharose (Sigma). Precipitated proteins were washed in lysis buffer and were eluted with sample buffer for SDS-PAGE. Eluted proteins were separated on 7% gels and transferred to nitrocellulose. Filters were probed using designated sera or antibodies and visualized with the ECL detection system (Amersham Corp.) as directed by the manufacturer.

Stat1 Purification

For Stat1 purification, SF9 cells infected with Stat1 virus were lysed by sonication in 10 mM HEPES, pH 7.6, 1 mM EDTA, 5 µg/ml phenylmethylsulfonyl fluoride, 10% glycerol. Lysates were passed over a DEAE-Sepharose column (Pharmacia Biotech Inc.) equilibrated with 10 mM HEPES, pH 7.6, 10% glycerol, and the column was eluted with a linear gradient of NaCl (0-500 mM). Elution fractions containing Stat1 were identified by SDS-PAGE and Western blotting and were pooled, concentrated, and subsequently separated on a Sephacryl S-300 column (Pharmacia) equilibrated with 10 mM HEPES, pH 7.4, 100 µM Na(3)VO(4), 50 mM NaCl, 10% glycerol. Peak Stat1-containing Sephacryl S-300 fractions were identified by SDS-PAGE and Western blotting, pooled, and concentrated for use in in vitro kinase reactions.

In Vitro Kinase Reactions

Immune complex Jak kinases were prepared by washing the products of anti-Jak immunoprecipitations in kinase buffer (10 mM HEPES, pH 7.6, 50 mM NaCl, 100 µM Na(3)VO(4), 5 mM MnCl(2), 5 mM MgCl(2)). For peptide kinase assays, immune complex Jak preparations were suspended in 15 µl of kinase buffer with 1 mg/ml peptide and 5 µCi of [-P]ATP. Reactions were incubated at 25 °C for 30 min and then stopped by the addition of SDS-PAGE sample buffer. Peptides were separated from unincorporated label on 20% gels and visualized by autoradiography. The Tyr peptide corresponds to amino acid 695-711 of Stat1 (GPKGTGYIKTELISVS). The Tyr peptide corresponds to amino acids 14-29 of Stat1 (FLEQVHQLYDDSFPME).

For Stat1 kinase reactions, immune complex Jak kinases were mixed with purified Stat1 (8 µg of protein) in kinase buffer plus 5 mM ATP. Following 30 min of incubation at 25 °C, reactions were stopped by the addition of EDTA to a 15 mM final concentration. The products of these reactions were separated by centrifugation into soluble (Stat1) and insoluble (Jak kinase) fractions for use in SDS-PAGE and gel mobility shift assays.

EGFR immune complexes were prepared by adding 4 µl of anti-EGFR serum to 1 ml of a centrifuged (12,000 g for 10 min) 10% homogenate of adult mouse liver prepared with a lysis buffer containing 20 mM HEPES, pH 7.4, 1% Trition X-1000, 50 mM sodium beta-glycerophosphate, 10 mM sodium fluoride, 100 µM sodium vanadate, 0.1 M sodium chloride. After standing for 2 h at 0 °C, 100 µl of a 50% slurry of protein A-Sepharose was added, and incubation, with mixing, was continued for 30 min. The suspension was centrifuged, washed three times with 1 ml of homogenizing buffer and stored frozen at -70 °C. Before use, the complex was washed once with 1 ml of 20 mM HEPES pH 7.4 buffer containing 1 mM dithiothreitol.

Gel Mobility Shift Assays

For the electrophoretic mobility shift assay, cell extracts (10 µg of total protein) were incubated with 2 µg of poly(dI-dC) for 30 min, followed by a 30-min incubation with 1 ng of Klenow-labeled probe DNA. The probe used (5`-CTAGCAGTGTTTCCCCGAAACACGCTAG-3`) contains a core sequence corresponding to the GAS element found in the interferon regulatory factor 1 promoter. Samples were run on a 4.5% polyacrylamide gel in 200 mM Tris borate, 2.2 mM EDTA. Gels were dried and visualized by autoradiography.

Phosphopeptide Mapping

Analysis of tryptic phosphopeptide was performed as described by Boyle et al.(17) . Briefly, SF9 cells were infected with appropriate viruses and cultured for 60 h. Infected cells were transferred to phosphate-free Grace's media and incubated with 1 mCi/ml [P]orthophosphate for 3 h. Labeled cells were lysed, and Stat1 was isolated by immunoprecipitation with Stat1-specific antisera and separation by SDS-PAGE. [P]Stat1 was identified by autoradiography, isolated from gel slices, and digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma). Tryptic peptides were mixed with a synthetic phosphopeptide corresponding to the Tyr peptide of Stat1 (GTGYIK). The peptide mixture was separated in two dimensions on thin layer cellulose plates. P-labeled peptides were visualized on a PhosphorImager (Molecular Dynamics). The position of the Tyr peptide was determined by ninhydrin staining.


RESULTS

Cytokine-dependent activation of the Jaks is associated with the phosphorylation and activation of one or more of the known Stats. To initially explore the role of the Jaks in Stat phosphorylation, the ability of various Jaks to phosphorylate Stat1 in insect cells was examined. Baculovirus expression constructs were developed for Jak1, Jak2, Tyk2, and a kinase inactive mutant Jak2-Glu as well as for Stat1. An expression construct containing the Tec kinase (18) was also utilized to determine specificity relative to the Jaks. Each of the wild type kinases was expressed at comparable levels and was tyrosine-phosphorylated constitutively and catalytically active in immune kinase reactions (data not shown). As illustrated in Fig. 1, when expressed alone, Stat1 was not detectably tyrosine-phosphorylated. However co-expression of Stat1 with Jak1, Jak2, or Tyk2 resulted in the tyrosine phosphorylation of Stat1. Moreover, over several experiments, the levels of phosphorylation with the various Jaks were comparable with the levels of Jak expression, indicating that Stat1 was not preferentially phosphorylated by a particular Jak. The specificity was demonstrated by a lack of Stat1 phosphorylation by the kinase inactive Jak2 mutant (Jak2-Glu) or by Tec.


Figure 1: Phosphorylation of Stat1 by Jak kinases. SF9 cells were infected with baculovirus expression vectors for Stat1, Tyk2, Jak1, Jak2, an inactive point mutant of Jak2 (Jak2-E), or Tec. Lysates from infected cells were immunoprecipitated (IP) with Stat1 specific antisera (A), pooled antisera specific for Tyk2, Jak1, or Jak2 (B), or antisera specific for Tec (C). Immunoprecipitated proteins were separated on 7% SDS-polyacrylamide gels, transferred to nitrocellulose filters, and probed with the anti-phosphotyrosine antibody PY20. Alternatively, filters were probed with the same sera used for immunoprecipitation.



In the cellular response to IFN, tyrosine phosphorylation of Stat1 occurs on a single residue (Tyr)(19) . To determine whether the Jaks phosphorylate this site and/or additional sites in Stat1, two-dimensional tryptic peptide analysis was done (Fig. 2). Stat1 expressed alone contained a single phosphopeptide that based on the above data is not a phosphotyrosine-containing peptide. When expressed with Jak1, Jak2, or Tyk2, a single additional phosphopeptide was detected. This phosphopeptide co-migrated with a synthetic tryptic peptide containing phosphotyrosine at position Tyr (data not shown).


Figure 2: Phosphopeptide mapping of Stat1. SF9 cells were infected with Stat1 virus (A) or co-infected with virus for Stat1 and Tyk2 (B), Stat1 and Jak1 (C), or Stat1 and Jak2 (D). 60 h after infection, cells were labeled with [P]orthophosphate. Subsequently, Stat1 was isolated by immunoprecipitation and SDS-PAGE separation. Tryptic peptides of the isolated Stat1 were prepared and mixed with a synthetic phosphopeptide corresponding to the predicted Tyr peptide of Stat1. Separation was performed in two dimensions, and labeled phosphopeptides were visualized using a PhosphorImager. The positions of the synthetic peptides were determined by ninhydrin staining and are indicated by the broken circles. The origin of separation is indicated by the arrowhead.



The tyrosine phosphorylation of Stat1 in mammalian cells is associated with the acquisition of the ability to bind DNA. In the response to IFNalpha/beta, Stat1 participates in a complex containing an additional protein, p48, that is essential for DNA binding, whereas in the response to IFN p48 is not required for DNA binding. It was therefore important to determine whether Stat1, phosphorylated in a non-mammalian cell where associating proteins are less likely to be present, would bind its cognate sequence. As illustrated in Fig. 3, expression of Stat1 alone resulted in the appearance of no detectable GAS binding activity. However, co-expression of Stat1 with any of the wild type Jaks resulted in the appearance of DNA binding activity. The contribution of Stat1 to this activity is evident from the ability of antisera to Stat1 to supershift the complexes (Fig. 3B).


Figure 3: GAS binding activity of phosphorylated Stat1. A, the cell lysates prepared for Fig. 1were used directly for gel shift analysis using an oligonucleotide containing the core GAS element of the interferon regulatory factor 1 promotor. B, alternatively, lysates were mixed with normal rabbit serum (control antibody) or Stat1 specific serum prior to gel shift analysis.



To further establish the ability of Jaks to directly phosphorylate Stat1, Jaks were purified by immunoprecipitation and examined for their ability to phosphorylate either a peptide containing Stat1 Tyr or purified Stat1. As shown in Fig. 4A, Jak2, but not the kinase inactive mutant Jak2-Glu, phosphorylated the Stat1 peptide. The specificity is indicated by the lack of phosphorylation of a peptide containing Stat1 Tyr, a tyrosine that is conserved in all the Stats. Comparable results were obtained with Jak1 and Tyk2 (data not shown). Jak2 was also able to phosphorylate peptides containing the predicted phosphorylation sites of Stat2 and Stat4 (data not shown).


Figure 4: Jak2 phosphorylation of a Stat1-Tyr peptide. Jak2 immunoprecipitates were prepared for SF9 cells infected with a control virus, Jak2 virus, or Jak2-Glu virus. A portion of each immunoprecipitation was used for in vitro kinase reactions with the Tyr-peptide substrate (A) or the Y-peptide substrate (B). A portion of the immunoprecipitation was also analyzed by SDS-PAGE and Western blotting with Jak2 antisera (C).



The ability of Jaks to phosphorylate purified Stat1 is shown in Fig. 5. For these experiments, Stat1 was purified to apparent homogeneity (Fig. 5A) by sequential ion exchange and gel filtration chromatography as detailed under ``Materials and Methods.'' The purified Stat1 was added to the immune complex kinase reactions, and the products were resolved by SDS-PAGE and examined by autoradiography (Fig. 5B). As indicated, the reactions were separated into a fraction containing the immune complexes (insoluble) and the supernatant fraction (soluble). Jak1 and Jak2 autophosphorylations were seen in the immune complexes as expected. In addition, a fraction of the phosphorylated Stat1 was co-isolated with the immune complexes. However, phosphorylated Stat1 was also seen in the soluble fraction. The requirements for the Jaks is indicated by the lack of Stat1 phosphorylation by the kinase inactive Jak2-Glu. Tryptic peptide analysis of in vitro phosphorylated Stat1 demonstrated the phosphorylation of a single tryptic fragment that co-migrated with the Tyr tryptic peptide (Fig. 5, C and D). Lastly, in vitro phosphorylation of purified Stat1 activated its latent DNA binding activity (Fig. 6A), and this activation was dependent upon the addition of ATP (Fig. 6D). Interestingly, two distinct GAS binding complexes were seen with in vitro phosphorylated Stat1. The major, faster migrating complex co-migrated with the complex seen in the co-expression studies above. The slower migrating complex was more variably observed. The basis for the second complex is unknown.


Figure 5: In vitro phosphorylation of Stat1. Stat1 expressed in SF9 cells was purified by sequential ion exchange and gel filtration chromatography. Purified Stat1 was separated by SDS-PAGE, and the gel was stained with Coomassie Blue (A). Immune complex kinase preparations were made for Jak1, Jak2, Jak2-Glu, Tec, EGFR, or control (no kinase) as described under ``Materials and Methods.'' Each kinase preparation was reacted with purified Stat1 in the presence of [-P]ATP. The reaction products were separated by centrifugation into soluble (Stat1) and insoluble (kinase) fractions. Each fraction was further separated by SDS-PAGE and visualized by autoradiography (B). Stat1 phosphorylated by Jak1 (C), Jak2 (D), or EGF-R (E) was then excised from gel slices of the soluble reaction product and used for tryptic phosphopeptide mapping as described in the legend to Fig. 2.




Figure 6: In vitro activation of Stat1. Immune complex kinase reactions were preformed in the presence or the absence of purified Stat1. Following the kinase reaction, the soluble (Stat1) and insoluble (kinase) components were separated by centrifugation. An aliquot of the soluble portion of each in vitro kinase reaction was assayed for GAS binding activity (A). Aliquots of the insoluble fractions were separated by SDS-PAGE, transferred to nitrocellulose and probed with antibodies for phosphotyrosine (B) or with a pool of antisera specific for Tyk2, Jak1, Jak2, and Jak3 (C). Alternatively, Jak2 immune complex kinase reactions were performed with purified Stat1 in the presence or absence of ATP. Aliquots of the soluble fraction of each reaction were incubated with non-reactive rabbit serum (NRS) or anti-Stat1 serum prior to the gel mobility shift assay (D).



Previous studies (7, 8, 9) have shown that EGF stimulation, both in tissue culture cell lines and in vivo, can induce the tyrosine phosphorylation of Stat1 as well as Stat3. However, it was not determined whether this occurred directly or through the activation of a Jak by the EGFR. As illustrated in Fig. 5B, immune complex purified EGFR readily phosphorylated purified Stat1 and this phosphorylation occurred on a single tryptic peptide corresponding to the peptide containing Tyr (Fig. 5E). The specificity is indicated by the inability of Tec to phosphorylate Stat1, although autophosphorylation was readily detectable in these reactions (Fig. 6B). It should also be noted that the immune complex purified Tec and EGFR contained no detectable Jak1 or Jak2 by Western blotting of the preparations (Fig. 6, B and C). As with the Jaks, phosphorylation of Stat1 by EGFR resulted in the activation of its latent DNA binding activity (Fig. 6A).


DISCUSSION

The Jaks are required for cytokine-stimulated signal transduction pathways leading to the tyrosine phosphorylation and activation of the Stats. However, whether the Stats are immediate substrates of the Jaks or substrates of a Jak-activated kinase has not been established. The studies described here address this question and the related question of the specificity of the kinases for Stats. Our results demonstrate that the Jaks are capable of mediating the specific tyrosine phosphorylation of Stat1 in vivo in insect cells and in in vitro reactions with purified proteins. Similar to Stat1, co-expression of Jak2 with Stat2, Stat3, Stat4, Stat5, or Stat6 resulted in their tyrosine phosphorylation (data not shown), although these results have not as yet been done with the respective purified proteins. Therefore Stats are potentially immediate substrates of the activated Jaks in vivo.

Strikingly, tyrosine phosphorylation, both in insect cells and in vitro, occurs at a single site, Tyr, although numerous tyrosines are present in Stat1. This specificity is particularly noteworthy because it occurs in the absence of any relevant receptor complex. The specificity for Tyr may be determined solely by the immediate flanking sequences and/or other structural determinants of Stat1 that contribute to the tertiary structure and position Tyr to be accessible for phosphorylation. The ability of Jaks to phosphorylate a peptide containing Tyr demonstrates the ability of the catalytic pocket to bind the site of phosphorylation in the absence of other structural determinants. In this regard it should be noted that considerable similarity exists at comparable sites in the other Stats, as indicated in Table 1. Among these peptides, those for Stat4 and Stat2 peptides are also substrates of Jak2 (data not shown); the remainder have not been examined.



In addition to the Jak-dependent tyrosine phosphorylation of Tyr, a second site of phosphorylation was seen in Stat1 produced in insect cells. This phosphorylation was seen without co-expression of the Jaks, suggesting that an endogenous insect cell kinase is involved. The lack of reactivity of Stat1 produced alone in insect cells with antiphosphotyrosine reagents would suggest that this second site of phosphorylation is either a serine or threonine residue. The potential significance of this phosphorylation site is unclear. Because the insect cell-produced Stat1 is constitutively phosphorylated at this site, we cannot rule out a role in DNA binding. In this regard, recent studies have indicated that phosphorylation of Stat1 S may be required for maximal transcriptional activation (20) .

The above data are consistent with a relatively simple recognition of Stat1 by the Jaks. However, we have also begun to examine the ability of various mutants of Stat proteins to be phosphorylated when co-expressed with Jaks. Strikingly many mutations, deletions, or truncations eliminate the ability of Stats to be recognized and phosphorylated by the Jaks (data not shown). For example, relatively small deletions in the amino-terminal region of Stat4 eliminate its ability to be phosphorylated by Jak2. The basis for these effects are not known and are currently being examined, although they demonstrate that the interaction of Jaks and Stats may be more complex and may be dependent upon Stat tertiary structure.

The complexity of Stat activation is further indicated by recent studies that have demonstrated that Stat2 is required for IFNalpha-induced tyrosine phosphorylation of Stat1(21) . The basis for this dependence is not known; however, it can be hypothesized that Stat2 is required to recruit Stat1 to the receptor complex. Recruitment may rely on the SH2 domain of Stat1 interacting with phosphorylated Stat2. Alternatively, recruitment may rely on heterodimerization of the Stat1 and Stat2 through other domains that affect the ability of the complex to associate with the activated Jaks.

When co-expressed in insect cells, Jak1, Jak2, or Tyk2 were equally capable of phosphorylating and activating Stat1. Therefore the specificity with which different cytokines induce the tyrosine phosphorylation of specific Stat proteins in vivo cannot be ascribed to the Jaks. Rather, the specificity must reside in the ability of individual receptor complexes to recruit specific Stat proteins to the associated Jaks. Recent studies have provided compelling evidence that this recruitment involves the interaction of the SH2 domains of the Stats with sites of tyrosine phosphorylation on the receptor. Specifically, a single tyrosine in the IFN receptor alpha (R1) chain has been implicated in recruiting Stat1(22) . Similarly, tyrosine residues within gp130 or the IL-4 receptor alpha chain have been shown to be docking sites for Stat3 (23) or Stat6(24, 25) , respectively. Lastly, exchanging SH2 domains has been shown to alter the specificity with which Stat proteins associate with receptor complexes(26) . It is important to note that in this model and based on our results it is essential that the activated Jaks be restricted to the activated receptor complex to maintain specificity. Indeed, overexpression of Jaks in mammalian cells results in their constitutive, ligand-independent activation and the concommitant constitutive activation of the endogenous Stats(27, 28) .

The phosphorylation of Stat1, either in vivo in insect cells or in vitro, resulted in the acquisition of GAS DNA binding activity. This result supports the hypothesis that unlike the ISGF3 complex, Stat1 is capable of binding DNA in the absence of any additional factors. Because of this result we have also examined the properties of Stat2 phosphorylated in insect cells by the Jaks (data not shown). Consistent with the current models, no DNA binding activity was detected against either various GAS sequences or the ISRE sequence.

The Stat proteins are utilized in signaling pathways other than those activated by receptors of the cytokine receptor superfamily. In particular, EGF has been shown to induce the tyrosine phosphorylation of Stat1 as well as Stat3(7, 8, 9) . Stat1 is hypothesized to be a necessary component of an EGF induced DNA binding activity that recognizes the SIE (c-sis-inducible element) in the c-fos gene promoter. From these studies, however, it was not clear whether Stat1 phosphorylation was mediated by the EGF receptor or through activation of a Jak. We therefore examined the ability of immuno-purified EGFR to phosphorylate purified Stat1. As demonstrated, EGFR catalyzed the tyrosine phosphorylation of Stat1 at Tyr. Conversely, Stat1 was not detectably tyrosine-phosphorylated when co-expressed with the Tec protein-tyrosine kinase. Based on these results it will be of some interest to define the spectrum of kinases that are capable of phosphorylating the various Stats.


FOOTNOTES

*
This work was supported by Grant P30 CA21765 from the National Cancer Institute Cancer Center Support (CORE), by Grant PO1 HL53749 from the National Heart, Lung, and Blood Institute (to J. N. I.), by Grant RO1 DK/HL42932 from the National Institute of Diabetes and Digestive and Kidney Diseases (to J. N. I.), by Grant RO1-HD00700 (to S. C.) from the National Institute of Child Health and Development, and by funds from the American Lebanese Syrian Associated Charities. 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: Dept. of Biochemistry, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Tel.: 901-522-0422; Fax: 901-525-8025.

(^1)
The abbreviations used are: IFN, interferon(s); EGFR, epidermal growth factor receptor; PAGE, polyacrylamide gel electrophoresis; GAS, interferon-gamma-activated sequence.


ACKNOWLEDGEMENTS

We gratefully acknowledge the technical support provided by Linda Synder and Melissa Norwood in these studies. We also thank H. Yan and J. Krolewski for providing the Tyk2 baculovirus expression construct.


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