Identification of a STAT4 Binding Site in the Interleukin-12 Receptor Required for Signaling*

The specificity of the various STAT SH2 domains for different tyrosine-containing peptides enables cytokines to activate different signaling pathways and to induce distinct patterns of gene expression. We show that STAT4 has a unique peptide specificity and binds to the peptide sequence pYLPSNID (where pY represents phosphotyrosine). This motif is found at tyrosine residue 800 in the b 2 subunit of the interleukin-12 receptor and is required for DNA binding and transcriptional activity of STAT4. Our data demonstrate that transfection of interleukin-12 receptor b 1 and b 2 subunits is sufficient for STAT4 activation but not for STAT1 or STAT3 activation. Mutagenesis

IL-12 1 is a heterodimeric cytokine secreted from antigen presenting cells in response to bacteria and intracellular parasites and thus plays an important role in host defense against bacterial pathogens (1). IL-12 promotes the proliferation of T cells and NK cells and is required for the differentiation of T cells into the Th1 subset of T helper cells. Th1 cells are critical for cell-mediated immune responses, because they secrete IFN-␥, which enhances the activity of cytotoxic T cells and NK cells (1)(2)(3). In addition to these essential functions, Th1-dominated responses are associated with pathologic autoimmune and inflammatory conditions such as rheumatoid arthritis and inflammatory bowel disease (4). Given this potential for immunopathology, it is important to understand the mechanisms that control the Th1 response to develop possible therapeutic interventions.
IL-12 signals through the IL-12 receptor, which is composed of at least two subunits designated ␤1 and ␤2 (5,6). Both IL-12 receptor subunits are members of the hemapoietin receptor superfamily and have strong homology to the gp130 receptor (5). The ␤1 receptor, although a low affinity binder of IL-12, is not capable of transducing an IL-12-mediated signal (6). A second subunit of the IL-12 receptor was subsequently identified that when coexpressed with the ␤1 subunit forms a high affinity receptor for IL-12 and confers IL-12 signaling (6). IL-12R ␤2 expression is differentially regulated in Th1 versus Th2 cells (7). Th1 cells but not Th2 cells express the ␤2 subunit of the IL-12 receptor (8). Following T cell activation, IL-12 and IFN-␥ treatment induces ␤2 expression, whereas IL-4, a cytokine produced by Th2 cells, inhibits ␤2 expression resulting in loss of IL-12 signaling (7,8). Therefore, expression of the ␤2 subunit of the IL-12 receptor is a crucial determinant of Th1 versus Th2 development.
Cytokine binding to its receptor leads to activation of JAK kinases that phosphorylate the receptor on tyrosines located in the intracellular domain. The phosphorylated regions are binding sites for signal transduction molecules called STATs that are rapidly recruited to the receptor and tyrosine phosphorylated by JAK kinases. Tyrosine phosphorylation of STAT proteins induces their dimerization and translocation to the nucleus where they bind to specific DNA sequences and regulate transcription. IL-12 stimulation results in TYK2 and JAK2 phosphorylation and interaction of TYK2 with the ␤1 subunit of the IL-12 receptor and interaction of JAK2 with the ␤2 subunit (9, 10). Unlike STAT4, whose expression is limited to lymphoid and spermatogonia cells, JAK2 and TYK2 are ubiquitously expressed (11,12).
In response to IL-12, STAT4 has been shown to be tyrosine phosphorylated and activated in Th1 lymphocytes and NK cells (9,13). In addition, it has been reported that STAT1, STAT3, and STAT5 can be activated in response to IL-12 (13)(14)(15).
STAT proteins, by way of their SH2 domains, interact with specific phosphotyrosine residues on cytokine receptors (16). Although the phosphotyrosine binding sites of all the other known STAT proteins have been identified, the tyrosine docking site of STAT4 has not previously been reported. The ␤2 subunit of the IL-12 receptor contains three tyrosines in its cytoplasmic domain, whereas the ␤1 subunit contains no tyrosine residues in its cytoplasmic domain (5,6). Therefore, given the importance of tyrosine phosphorylation in cytokine signaling pathways, we focused on the three tyrosines of the ␤2 subunit as possible docking sites for the STAT4 signaling protein in response to IL-12.
We determined that STAT4 is activated through interaction with the tyrosine at amino acid 800 in the IL-12 receptor ␤2 subunit. STAT4 activation depends on interaction with the peptide sequence pYLPSNID (where pY represents phosphotyrosine), thus providing evidence for the specificity of the STAT4 SH2 domain for a unique binding site in the IL-12 receptor. In addition, we show that neither STAT1 nor STAT3 is activated directly through the IL-12 receptor.

EXPERIMENTAL PROCEDURES
Constructs and Transfections-pRK5-STAT4, a CMV promotor vector, was used for STAT4 expression. A partial clone of IL-12R ␤1 cDNA was isolated from a peripheral blood lymphocyte library and cloned into NaeI and ApaI sites of pcDNA3 (Invitrogen). The N terminus of IL-12R ␤1 was generated using polymerase chain reaction and cloned into BamHI and NaeI sites. An IL-12R ␤2 cDNA was isolated from a peripheral blood lymphocyte library and cloned into the ApaI and NotI sites of pcDNA3. A deletion in this clone was discovered in the 3Ј end; therefore, the C terminus was generated by polymerase chain reaction and cloned into the SalI and ApaI sites of pcDNA3. Mutations changing the tyrosine to phenylalanine at amino acid 678, 767, or 800 in IL-12R ␤2 were generated using QuikChange Site-Directed Mutagenesis (Stratagene). The mutagenesis primers used to generate the mutations were: for ␤2 Y800F, 5Ј-CCCACCCATGATGGCTTCTTACCCTCC-3Ј * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
EMSA-Nuclear extracts were prepared from 293 cells 60 h following transfection by the method of Dignam et al. s (18). EMSAs were performed by using 5 l of nuclear extract, 5 l of binding buffer (100 mM KCl, 25 mM HEPES (pH 7.9), 0.1 mM EDTA, 5 mM MgCl 2 , 200 g/ml bovine serum albumin, 10% glycerol), and 1 g of poly(dI-dC) in a final volume of 15 l. Reactions were incubated at room temperature for 5 min before 1 l of probe was added for 15 min at room temperature. Oligonucleotides containing two high affinity STAT consensus binding sites, (5Ј-TAAAATTTTCCGGGAATTTTTTGAGTTTCCCGGAAAATT-TTG-3Ј and its complement) were annealed and kinased to generate labeled DNA for the EMSA.
Fluorescence Polarization-Tyrosine phosphorylated forms of IL-12R ␤2 peptides Tyr 800 (SHEGpYLPSNID), Tyr 767 (LVDLpYKVLESR), and Tyr 678 (CAKKpYPIAEEK) were synthesized as described previously (19). Peptides were purified as described in Schindler et. al. (20). Fluorescence polarization was measured in an FPM-1 analyzer (Jolly Consulting and Research) as described (20). The sequence of the fluorescent peptide was SFDpYDMPHVL. The binding affinity of STAT4 for the fluorescent peptide is similar to its affinity for Tyr 800 peptide. A fluorescent peptide concentration of 10 nM and a STAT4 concentration of 190 nM were used for STAT4-receptor peptide binding in assay buffer (50 mM NaCl, 10 mM HEPES (pH 7.6), 1 mM EDTA, 0.1% Nonidet P-40, and 2 mM dithithreitol). Full-length STAT4 was expressed with a Cterminal histidine tag in a baculovirus expression system. The protein was purified to approximately 95% homogeneity by nickel affinity chomatography (Qiagen).
Transactivation Reporter Assay-COS cells were plated at 2 ϫ 10 5 / well (35 mm) on 6-well plates. Cells were transfected the next day using Profection Mammalian Transfection System calcium phosphate method (Promega) with 1 g/well of STAT4 expression plasmid, 1.5 g/well of each IL-12R subunit expression plasmid, and 1 g/well of IRF-1 luciferase reporter plasmid. The IRF-1 reporter contains two copies of the ␥-activated sequence (AGCCTGATTTCCCCGAAATGACGGCACG) upstream of herpes simplex virus thymidine kinase (Ϫ50 to ϩ10) in pGL2 luciferase (Promega). 0.25 g/well of CMV ␤-galactosidase plasmid was transfected as a control. After 24 h, cells were treated with IL-12 at 5 ng/ml for 4 -5 h and harvested for luciferase and ␤-galactosidase assays. Luciferase activity was measured using the Promega Luciferase Assay system, and ␤-galactosidase activity was measured using Galacto-light chemiluminescent reporter assay (Tropix Inc.). Luciferase activity was normalized to ␤-galactosidase activity, and the average was taken of three experiments.

STAT4 Protein Has the Highest Affinity for pYLPSNID Sequence in IL-12
Receptor-It seemed likely that STAT4 phosphorylation in response to IL-12 is dependent on interaction with one of the three tyrosines in the cytoplasmic domain of the IL-12R ␤2 subunit, because the ␤1 subunit cytoplasmic domain contains no tyrosines. We tested the specificity of the STAT4 SH2 domain for phosphopeptides corresponding to tyrosinecontaining regions of the IL-12R ␤2 subunit by fluorescence polarization competition binding assays. The sequence of the fluorescent peptide was SFDpYDMPHVL. This is a mutated version of the STAT1 binding site derived from the IFN-␥ receptor. STAT4 binds to the mutant peptide approximately 8-fold better than it does to the wild-type sequence, SFpYDK-PHVL. The peptide containing tyrosine 800 with the sequence pYLPSNID had an IC 50 of 2.9 M, whereas the other peptides pYKVLESR (Tyr 678 ) and pYPIAEEK (Tyr 767 ) had IC 50 values of greater than 100 M, equivalent to nonspecific background binding (Fig. 1A). These results indicate that STAT4 has the highest affinity for the peptide sequence that contains tyrosine 800 in the IL-12R ␤2 subunit, suggesting that this is the in vivo docking site for STAT4.
The Tyrosine at Amino Acid 800 in the IL-12 Receptor Is Required for STAT4 Activation-We reconstituted IL-12 receptor signaling in 293 cells to determine whether the ␤1 and ␤2 subunits of the IL-12 receptor are sufficient for STAT4 activation in response to IL-12. Both subunits of the IL-12 receptor and STAT4 were transfected into 293 cells, and STAT4 activation was tested by EMSA using nuclear extracts isolated from the transfected cells after addition of IL-12. IFN-␣ stimulation has also been shown to activate STAT4 (21) and was used as a control for activation of STAT4 in these experiments. STAT4 was activated to bind DNA following IL-12 addition (Fig. 1B,  lane 3) and IFN-␣ addition (Fig. 1B, lane 10) but was not activated in the absence of IL-12 addition (Fig. 1B, lane 2). The ␤1 and ␤2 subunits of the IL-12 receptor were sufficient for activation of STAT4 by IL-12 in 293 cells. Both of the IL-12 receptor subunits were required for activation of STAT4 be-  2, 3, and 7), and STAT4 (lanes 2-11) and after 72 h were treated with no cytokine (lanes 1 and 2), IL-12 (lanes 3-9), or IFN-␣ (lanes 10 and 11) at 10 ng/ml for 4 h. Nuclear extracts were prepared and analyzed for STAT4 binding activity by EMSA using a high affinity double STAT consensus site as probe. Specificity of probe interaction was shown by adding anti-STAT4 antibody (Ab) to nuclear extract and probe mix (lanes 7-9 and 11), which specifically blocked the interaction of STAT4 protein with the probe. The arrow indicates the protein-DNA complex.

STAT4 Binding Site in the IL-12 Receptor 1876
cause neither one alone resulted in STAT4 DNA binding (data not shown).
IL-12R ␤2 subunit mutants that each contain one of the tyrosines in the cytoplasmic domain changed to phenylalanine were then analyzed for their ability to recruit STAT4. IL-12 ␤2 receptor mutants at both tyrosine 678 and tyrosine 767 were capable of activating STAT4, whereas the mutant at tyrosine 800 was not able to activate STAT4 (Fig. 1B, lanes 4 -6). When anti-STAT4 antiserum was added to the binding reactions, the STAT4 DNA binding complex was removed, indicating that STAT4 protein was responsible for binding to the probe (Fig.  1B, lanes 7-9 and 11). This evidence, along with the in vitro peptide binding data, indicates that the STAT4 SH2 domain binds to a specific phosphorylated tyrosine in the IL-12R ␤2 subunit that contains the sequence pYLPSNID.
IL-12-dependent transcriptional activation through STAT4 was also tested by co-transfecting the IL-12 receptor subunits, STAT4, and an IRF-1 luciferase reporter gene into cells and measuring luciferase assay following treatment with IL-12. STAT1 can induce the IRF-1 gene (22), and STAT4 has the same DNA binding specificity as STAT1 (23). Therefore, given that IL-12 stimulation has also been shown to induce IRF-1 gene expression in human peripheral blood lymphocytes, 2 we used the IRF-1 STAT binding site for our reporter assays. We determined that in the transient transfection assays STAT4 transcriptional activation of the IRF-1 gene was dependent on IL-12 and was induced 4-fold in IL-12 treated transfected cells ( Fig. 2A). The IL-12 receptor mutants ␤2-Y678F and ␤2-Y767F were also able to induce transcriptional activation of the IRF-1 gene to levels equivalent to wild-type IL-12 ␤2 receptor ( Fig.  2A). However, STAT4-mediated transcriptional activation with the ␤2-Y800F mutant of IL-12R ␤2 was not detected. Expression of each of the mutant ␤2 subunits was confirmed by Western blot (Fig. 2B). Protein levels of the IL-12R ␤1 subunit and STAT4 were also shown to be equivalent in each transfection (data not shown). We conclude that the tyrosine at amino acid 800 in IL-12R ␤2 is necessary for efficient activation of STAT4 for both DNA binding and transcriptional activation.

IL-12R␤1 and ␤2 Are Not Sufficient for STAT1 or STAT3
Activation-It has previously been reported that IL-12 is able to induce tyrosine phosphorylation and DNA binding of STAT3 and STAT1 in addition to STAT4 (13,14). We tested the ability of IL-12 to activate STAT1 and STAT3 through the IL-12 receptor in our 293 transfection system. Following transfection of 293 cells with the IL-12 receptor subunits and STAT4, STAT1, or STAT3, nuclear extracts were prepared and analyzed for DNA binding activity by EMSA using the STAT consensus probe. Although we detected STAT4 DNA binding following activation with IL-12, we were unable to detect DNA binding by STAT1 or STAT3 (Fig. 3), even though their expression levels were equivalent to STAT4 expression (data not shown). STAT1 was capable of being activated for DNA binding by IFN-␥ as seen in lane 7 of Fig. 3. Our results indicate that in this system, STAT1 and STAT3, unlike STAT4, cannot be activated by IL-12 through the IL-12 receptor. In addition, we also tested the ability of STAT1 or STAT3 to be activated when STAT4 is co-transfected into 293 cells. When extracts from IL-12 stimulated 293 cells co-transfected with the IL-12R subunits and STAT4 plus STAT1 or STAT4 plus STAT3 were analyzed by EMSA, supershift, and co-immunoprecipitation experiments, only STAT4 homodimer complexes were detected (data not shown). These data suggest that neither STAT1 nor STAT3 is activated by IL-12 through an interaction with STAT4. DISCUSSION IL-12 stimulation results in activation of STAT4 through interaction with the IL-12 receptor (13,24). In response to IL-12 stimulation, STAT4 is tyrosine phosphorylated and is competent for DNA binding and transcriptional activation of target genes. STAT4 function is necessary for mediating the IL-12 response and is crucial for Th1 development and efficient IFN-␥ production as shown by STAT4 knock-out studies (25,26).
Receptor binding by STAT proteins has been shown to be specific and dictated by the SH2 domain of the STAT proteins (reviewed in Refs. 16, 27, and 28). STAT interaction with cytokine receptors depends on the phosphotyrosine residue in the receptor and the amino acid residues C-terminal to the phosphotyrosine that mediate specificity. In this paper, we demonstrate that STAT4 is activated by interacting with the IL-12 2 T. Hoey, unpublished results.  and 3), STAT1 (lanes 5 and 6), and STAT3 (lanes 8 and 9) and after 72 h were treated with no cytokine (lanes 2, 5, and 8) or  IL-12 (lanes 3, 6, and 9). As a positive control, 293 cells were transfected with STAT4 alone and treated with IFN-␣ at 10 ng/ml for 5 h (lane 4) or transfected with STAT1 alone and treated with IFN-␥ at 10 ng/ml for 5 h (lane 7). Lane 1 is the mock-transfected control. Nuclear extracts were prepared and analyzed for STAT binding activity by EMSA using a high affinity double STAT consensus site as probe. The arrows indicate the protein-DNA complexes. STAT4 binds as a tetramer (upper arrow), and STAT1 binds as both a tetramer and a dimer (lower arrow). STAT4 Binding Site in the IL-12 Receptor 1877 receptor and that the interaction of the STAT4 SH2 domain is specific for the peptide sequence pYLPSNID, the peptide at tyrosine 800 in the IL-12R ␤2 subunit. This tyrosine at amino acid 800 in the IL-12R ␤2 subunit is required for STAT4 DNA binding activity and transcriptional activation of the IRF-1 gene. STAT4 is not capable of binding the other peptide sequences at tyrosine residues 678 or 767 of the IL-12 ␤2 subunit, and these peptide sequences are not required for the activation of STAT4. The IL-12R peptide sequence pYLPSNID appears to be a specific protein binding site for STAT4. Other STATs, including STAT1, which has a closely related SH2 domain, cannot recognize the IL-12R pYLPSNID peptide. 3 STAT1 and STAT3 are activated by a number of cytokines (28,29). Additionally, it has been reported that STAT1 and STAT3 are activated in response to IL-12. Jacobson et al. (13) reported that IL-12 induced weak tyrosine phosphorylation of STAT3 and DNA binding activity of STAT3 in addition to STAT4. They showed that STAT3 was part of a IL-12-induced DNA complex that included STAT4, suggesting that STAT3 and STAT4 form heterodimers. Yu et al. (14) demonstrated DNA-protein complexes containing STAT4, STAT1␣, and STAT3 following treatment of NK cells with IL-12. These reports, however, did not address the issue of whether STAT1, STAT3, or STAT4 directly interacts with the IL-12 receptor. Interestingly, in our experiments, neither STAT1 nor STAT3 was activated by IL-12 through the transfected IL-12 receptor subunits under conditions in which STAT4 was phosphorylated. Perhaps STAT1 and STAT3 could be recruited to the IL-12 receptor through another factor that functions as an adaptor protein and is not present in 293 cells.
For certain SH2-containing proteins, the ϩ1 and ϩ3 positions in phosphotyrosine peptides have been shown to confer binding specificity (30). All the known docking sites for STAT3, for instance, have a glutamine residue at the ϩ3 position relative to tyrosine. None of the three tyrosine peptides in the IL-12 ␤2 receptor contain a glutamine in the ϩ3 position, nor do they share amino acid sequence similarities to the STAT1 docking site in the IFN-␥ receptor, pYDKPH (31,32). This is consistent with our results that STAT1 and STAT3 are not activated directly by contact with the IL-12 receptor.
The specificity of the various STAT protein SH2 domains for different tyrosine-containing peptides results in activation of specific cytokine signaling pathways and the induction of distinct gene expression patterns. We conclude that STAT4 has a unique peptide binding specificity among the STAT family and binds to the peptide sequence pYLPSNID. The unique specificity of STAT4 for its tyrosine-containing peptide sequence helps explain the distinct effects of IL-12 signaling.
STAT binding peptides derived from cytokine receptors have the ability to disrupt STAT dimers and inhibit STAT tyrosine phosphorylation in vivo (19,31,33). It may be possible to develop therapeutically useful compounds that, analogous to the receptor-derived peptides, will block the STAT-receptor interaction, thus preventing STAT activation and subsequent signaling.
The ability to reconstitute IL-12 signaling by transfecting the ␤1 and ␤2 subunits of the IL-12 receptor will facilitate the analysis of IL-12 receptor structure and function. In addition to activation of the JAKs resulting in STAT tyrosine phosphorylation, IL-12 also stimulates STAT4 serine phosphorylation possibly by mitogen-activated protein kinase or Lck (21,34,35). It will be interesting to determine which regions of the receptor are required for triggering these signaling pathways.