©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chain-associated Cytokine Receptors Signal through Distinct Transducing Factors (*)

Alessandra Pernis (1), Sanjay Gupta (1), James Yopp (1) (2), Evan Garfein (1), Helena Kashleva (1), Chris Schindler (1), Paul Rothman (1) (2)(§)

From the (1)Departments of Medicine and (2)Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The IL-2, IL-4, and IL-7 signaling pathways have been shown to utilize shared components. The receptors for these cytokines are composed of ligand-specific binding chains that associate with a shared signaling subunit, the common () chain. In addition, IL-2, IL-4, and IL-7 induce activation of a common set of nonreceptor tyrosine kinases, Jak-1 and Jak-3. We have further investigated the signaling events induced by these cytokines and find that the -associated receptors activate distinct signal transducing factors (STFs). In addition, we show that a 94-kDa STAT-related protein (p94) is activated in response to IL-2 and IL-7, but not IL-4. These data indicate that IL-2, IL-4, and IL-7 activate distinct signaling molecules which might be differentially recruited to the receptor complex by the ligand-specific units of the IL-2, IL-4, and IL-7 receptors.


INTRODUCTION

Cytokines modulate cellular proliferation and differentiation through their interaction with specific cell-surface receptors. A comparison of the genes that encode the cytokine receptors indicates that most are members of a related family, termed the hematopoietin family of receptors(1) . Functional cytokine receptors consist of multicomponent complexes composed of two or more chains. Frequently, one of these chains is responsible for high affinity ligand binding, while the other(s) plays a role in signal transduction(2) . Within the hematopoietin family of receptors, different subfamilies have been identified based on conserved features including the employment of common signaling subunits(3, 4) . For instance, each component of the IL-6 family of receptors (the receptors for IL-6, IL-11, LIF, CNTF, and oncostatin M) associates with gp130(5, 6, 7, 8) . Recently, a new subfamily of cytokine receptors, which includes the IL-2, IL-4, and IL-7 receptors, has been recognized. The ligand binding units of the IL-4 and IL-7 receptors have been found to heterodimerize with the chain of the IL-2 receptor(9, 10, 11, 12) . This common () chain appears to be essential for mitogenic signaling mediated by the IL-2, IL-4, and IL-7 receptors(9, 10, 11, 12, 13) .

The ability of cytokines to rapidly activate genes appears to involve the activation, via tyrosine phosphorylation, of a set of latent cytoplasmic factors which belong to the STAT()(Signal Transducers and Activators of Transcription) family of proteins(14, 15) . Since the hematopoietin receptors lack kinase domains, tyrosine phosphorylation of these factors is hypothesized to be mediated by specific members of the Janus kinase family(16) . These kinases can associate with the hematopoietin receptors and, upon ligand binding, become activated. Phosphorylation of the STATs renders them competent for complex association. These complexes, termed signal transducing factors (STFs), can then undergo nuclear translocation. Once in the nucleus, many of these factors, like the recently characterized IL-4-inducible factor (17-19) (which is composed of a Stat6 homodimer (20),()bind to DNA sequences related to the interferon (IFN) gamma-activation site (GAS). These elements are conserved DNA sequences which mediate transcriptional activation by several STFs, including STF-IFN (a p91Stat1 homodimer) (15) and STF-IL-3(21) .

Interestingly, cytokine receptors which share a common signaling subunit (e.g. IL-6 family) induce activation of a common set of JAK kinases and of identical STFs (e.g. STF-IL-6) upon binding ligand(22) . By analogy with these receptors, employment of a common signaling chain and activation of the same set of JAK kinases (Jak-1 and Jak-3) (23, 24, 25, 26) by the receptors for IL-2, IL-4, and IL-7 would be expected to lead to the activation of the same STF. In this report, we demonstrate that, unlike the IL-6 family, activation of STFs by the members of the family (IL-2, IL-4, and IL-7) is regulated in a more complex manner. IL-2 and IL-7, in fact, induce GAS binding complexes (STF-IL-2 and STF-IL-7, respectively) which are similar by several criteria including GAS binding specificity and antibody reactivity. STF-IL-2 and STF-IL-7, however, are clearly distinct from the STF activated by IL-4 (STF-IL-4). We further demonstrate that IL-2- and IL-7-stimulated extracts contain a 94-kDa tyrosine-phosphorylated protein that is not detected in IL-4-stimulated extracts. This protein, which binds to GAS sequences, appears to be a member of the STAT family of STF component proteins.


MATERIALS AND METHODS

Cell Lines

The M12.4.1 (M12) cells and their culture conditions have been described previously(17) . Ramos 266 cells were grown in Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum (Atlanta Biologicals, Inc.). CTLL (ATCC) cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 50 µM -mercaptoethanol, and recombinant human IL-2 (10 units/ml; Eurocetus). Clone K cells (27) were grown in RPMI 1640 supplemented with 10% fetal calf serum, 50 µM -mercaptoethanol, 1% L-glutamine, and 1% sodium pyruvate on a feeder layer of AC6.21 cells. The derivation of the murine TH1 cell clone, D1.1, and its culture conditions have been described previously(28) .

Cytokine treatments were as follows. M12.4.1 and RAMOS 266 were stimulated, respectively, with recombinant murine IL-4 (400 units/ml; DNAX) or human IL-4 (10 units/ml; Schering-Plough). D1.1 and CTLL cells were stimulated with recombinant human IL-2 (10 units/ml; Eurocetus) or murine IL-4. Clone K cells were stimulated with recombinant murine IL-7 (10 ng/ml; Genzyme). All cytokine treatments were performed for 15 min at 37 °C. Prior to treatment with cytokines, growth factor-dependent cells were starved of these factors for 4 h.

Cell Isolation and Culture

Peripheral blood mononuclear cells (PBM or PBL) were prepared from freshly drawn blood of healthy volunteers by centrifugation on Ficoll-Hypaque (Sigma). The isolation of murine splenocytes has been described previously(29) . Thymocytes were harvested from BALB/c mice. PBM were stimulated for 15 min at 37 °C with the following cytokines: human IL-2 (10 units/ml; Eurocetus), murine IL-7 (10 ng/ml; Genzyme), human IL-4 (10 units/ml; DNAX), murine IL-13 (500 units/ml; DNAX), or human IFN (5 ng/ml; Hoffman-LaRoche). In cycloheximide experiments, PBM were cultured with 10 µg/ml cycloheximide (Sigma) for 1 h prior to the addition of the cytokines. In staurosporine experiments, PBM were cultured in medium containing 500 µM staurosporine (Biomol) for 20 min prior to the addition of the cytokines. Murine splenocytes and thymocytes were stimulated with murine IL-7 (10 ng/ml).

DNA Binding Assays and Cell Extracts

The preparation and employment of DNA oligonucleotide probes for mobility shift assays have been described previously.(30, 31) . The IRF-1 GAS probe employed in these studies was as follows: 5`gatcGATTTCCCGAAAT3`. Double-stranded oligonucleotides used as cold competitors were prepared from single-stranded oligonucleotides (Oligos Etc.) with the following sequences: CAS-GAS (5`gatcGACTTCTTGGAATT3`), Ly6E-GAS (5`gatcATATTCCTGTAAGT3`), and I (-119 to -104) (17) (5`gatctAACTTCCCAAGAACAG3`)(17, 18) .

Peptides from the IL-4 and IFN receptors were obtained from Dr. Douglas Fisher from Pfizer Inc. The peptide sequences were as follows: 1) unphosphorylated IL-4 receptor: CASSGEEGYKPFQDLI(20) , 2) phosphorylated IL-4 receptor: CASSGEEGYKPFQDLI(20) , 3) unphosphorylated IFN receptor: CTSFGYDKPH(32) , 4) phosphorylated IFN receptor: CTSFGYDKPH(32) . Peptides were added in increasing concentrations (30 µM, 100 µM, 300 µM) to whole cell extracts for 30 min at room temperature prior to the addition of the shift probe.

STAT antisera were added (final dilution 1:20) for 30-60 min (at 4 °C), after a standard 20-min (25 °C) incubation of extracts with shift probe. Antiphosphotyrosine antibodies were added to the extracts for 30-60 min (at 4 °C), prior to the addition of the shift probe.

Whole cell extracts (WCE) were prepared as described previously(31, 33, 34) . Whole cell extracts from Clone K, splenocytes, and thymocytes were prepared using a detergent-free buffer (Buffer C) as described previously(35) .

Immunoprecipitations and Oligoprecipitations

Extracts were immunoprecipitated by p91-ab4 as described previously(31, 33) . The precipitates were fractionated in a 7% SDS-PAGE prior to immunoblotting with an antiphosphotyrosine antibody (4G10, UBI). Bands were detected by ECL (Amersham).

Oligonucleotide precipitation assays were carried out with a 3-unit tandem multimer of the CAS-GAS element. The 11-base pair GAS elements were separated by 10-base pair spacers(21) . This oligomer was biotinylated (Sigma) by a fill-in reaction and linked to streptavidin agarose beads (Sigma) as per recommendations by the manufacturer, at 1-2 µg of DNA per µl of beads. 300 to 1000 µl of extract were incubated with the immobilized oligonucleotides for 2 to 12 h at 4 °C and then eluted with 1 or 2 M NaCl.

Antibodies

Polyclonal rabbit antiserum were generated against various domains of p91 with GST fusion proteins as described previously(33, 36) . p91-ab1 recognizes the carboxyl terminus of p91 (aa-aa), p91-ab2 recognizes the amino-terminal (aa-aa), and p91-ab4 recognizes the SH2 domain of denatured p91 (aa-aa). Stat2 antiserum recognizes p113 (aa-aa)(33, 36) . Polyclonal rabbit antisera against the carboxyl terminus of Stat3 or Stat6 were purchased from Santa Cruz Biotechnology, Inc. The antiphosphotyrosine antibody was purchased from UBI (4G10), and the isotype-matched control OKT4 (ATCC) was a generous gift from M. Yellin.


RESULTS

IL-2 and IL-7 Rapidly Stimulate Signal Transducing Factors

In an effort to determine whether IL-2 and IL-7 can activate specific signal transducing factors (STFs), we stimulated human peripheral blood mononuclear cells (PBMs) with either IL-2, IL-4, IL-7, IL-13, or IFN- for 15 min. We prepared whole cell extracts and assayed them for the induction of DNA binding activity by electrophoretic mobility shift assay (EMSA) with the GAS element from the IRF-1 gene (Fig. 1A). We have previously shown that a wide variety of STFs bind this element(21) . Extracts from untreated PBMs do not contain any DNA binding activities. However, extracts from IL-2- or IL-7-stimulated cells reveal novel complexes, which migrate with similar mobilities. The pattern and mobility of these complexes are clearly different from those of either STF-IL-4 or STF-IFN. As already reported, the DNA binding complex induced by IL-13 migrates with a mobility identical with STF-IL-4(17, 37) . Assaying these extracts by EMSA utilizing a different GAS probe (from the -casein promoter) revealed identical results (data not shown). These data suggest that IL-2 and IL-7 activate unique factors (STF-IL-2 and STF-IL-7, respectively) that are distinct from the Stat6 homodimer induced by IL-4.


Figure 1: A, induction of IRF-1 GAS binding activities in PBM by IL-2, IL-4, IL-7, and IL-13. WCE were prepared from PBM with or without a 15-min stimulation at 37 °C with human IL-2 (10 units/ml), murine IL-7 (10 ng/ml), human IL-4 (10 units/ml), murine IL-13 (500 units/ml), or human IFN (5 ng/ml). B, comparison of IL-2-, IL-4-, and IL-7-inducible GAS binding activities in different cell types. WCE were prepared and stimulated as described under ``Materials and Methods.'' Lanes are as follows: 1, unstimulated PBM; 2, PBM stimulated with human IL-2 (10 units/ml); 3, PBM stimulated with murine IL-7 (10 ng/ml); 4, PBM stimulated with human IL-4 (10 units/ml); 5, unstimulated D1.1; 6, D1.1 stimulated with human IL-2 (10 units/ml); 7, D1.1 stimulated with murine IL-7 (10 ng/ml); 8, D1.1 stimulated with murine IL-4 (400 units/ml); 9, unstimulated CTLL; 10, CTLL stimulated with human IL-2 (10 units/ml); 11, unstimulated Clone K; 12, Clone K stimulated with murine IL-7 (10 ng/ml). The WCE were examined by EMSA using an IRF-1 GAS probe.



Since PBM represent a complex population of cells and the activation of STF-IL-2, STF-IL-4, and STF-IL-7 may be occurring in different cell types, we were concerned that some of the differences in GAS binding activity might be cell-type-specific. Therefore, we assayed clonal cell populations for their ability to induce GAS binding activities after stimulation with IL-2, IL-4, or IL-7 (Fig. 1B). Extracts from cells of a murine T cell clone (D1.1) stimulated with IL-2, or with IL-7, exhibited complexes which migrated with a mobility identical with that of the IL-2- and IL-7-inducible factors detected in human PBM. In contrast, as already observed in PBM, extracts of D1.1 cells cultured with IL-4 displayed a complex whose mobility was clearly distinct from that of STF-IL-2 or STF-IL-7. Interestingly, the murine STF-IL-4 displayed a different ratio of individual bands as well as a slightly different mobility from its human counterpart. Since different tissues have been shown to contain different Stat6 mRNA species(20) , it is possible that different forms of Stat6 protein are present in different cell types. Thus, the multiple bands induced by IL-4 in PBM may be related to different forms of Stat6 found in this heterogeneous cell population. Extracts from another IL-2-stimulated murine T cell line, CTLL, also revealed the appearance of a factor that on EMSA migrates with a mobility identical with that of the IL-2-inducible activities in PBMs and in D1.1. The IL-2-inducible complex in D1.1, in addition, contains a faster migrating band which is not consistently detected. The nature of this band is unclear. Interestingly, extracts of an IL-7-stimulated pre-B cell line (Clone K) exhibit a GAS binding activity with slightly slower mobility than the other IL-2- or IL-7-treated extracts. By several criteria, including competition by unlabeled GAS-related oligonucleotides and antibody supershifting, the IL-7-inducible complex detected in Clone K behaves exactly like the STF-IL7 present in PBMs and D1.1 (data not shown). Taken together, these results illustrate that, both in murine and human cells, IL-2 and IL-7 induce similar GAS binding activities which are distinct from STF-IL-4. However, STF-IL-2 and STF-IL-7 from different cell types can exhibit slight variations in both mobility and pattern of bands. This might be secondary either to the presence of additional cell-type-specific factors or to different forms of the same factor(s).

STF-IL-2 and STF-IL-7 Are Activated by Tyrosine Phosphorylation

In order to better characterize the GAS binding activities induced by IL-2 and IL-7, we studied whether the activation of STF-IL-2 and STF-IL-7 follows the pattern delineated for other STFs. Previous studies have demonstrated that the component proteins of STFs reside in the cytoplasm prior to activation. After receptor-cytokine interaction, and in the absence of new protein synthesis, these factors are quickly activated. Once activated, they rapidly translocate to the nucleus(17, 38) . In order to assess the requirement for new protein synthesis in the induction of STF-IL-2, and STF-IL-7, we pretreated cells with cycloheximide for 1 h prior to cytokine stimulation. As in the case of STF-IL-4 and other STFs characterized to date, the appearance of STF-IL-2 and STF-IL-7 is resistant to pretreatment with cycloheximide (Fig. 2A), demonstrating that the protein components of these complexes are present prior to activation by cytokines.


Figure 2: Mode of activation of STF-IL-2 and STF-IL-7. A, effects of cycloheximide or staurosporine on STF-IL-2 and STF-IL-7 induction. Extracts from PBM stimulated with IL-2 or IL-7 alone or after pretreatment with cycloheximide (C) (10 µg/ml) for 1 h or with staurosporine (S) (500 µM) for 20 min were assayed as described in Fig. 1. B, STF-IL-2 and STF-IL-7 contain phosphotyrosyl residues. Extracts were incubated with either an antiphosphotyrosine antibody (4G10) or a control antibody (OKT4) for 60 min at 4 °C prior to analysis by EMSA. 3 µg of antibodies were added for each reaction except for the IL-7-stimulated extracts to which 4 µg of antibody were added.



The activation of STFs has been shown to be dependent on the Janus family of tyrosine kinases for a number of cytokines(39, 40, 41, 42) . For example, the activation of STF-IFN is dependent on the presence of Jak-1 and Jak-2(43, 44) . The induction of STF-IL-4 also requires tyrosine phosphorylation(17, 18) . The observation that STF-IL-2 and STF-IL-7 contain STAT-related proteins (see below) prompted us to investigate whether these factors are also dependent on the activity of tyrosine kinases. When cells are pretreated with the kinase inhibitor staurosporine, which is known to block the Janus kinases, the induction of STF-IL-2 and STF-IL-7 is inhibited (Fig. 2A). This finding suggests that phosphorylation is critical for the activation of these factors.

If these factors are activated by tyrosine phosphorylation, then the IL-2- and IL-7-induced GAS binding activities should contain phosphotyrosyl residues. To examine this possibility, extracts from stimulated cells were incubated with an antiphosphotyrosine antibody or with a control antibody prior to analysis by EMSA (Fig. 2B). In a pattern similar to STF-IL-4 and other STFs(17, 45) , addition of an antiphosphotyrosine antibody, but not of equivalent amounts of an isotype-matched control antibody, prevented the appearance of these STFs. These results provide further evidence that STF-IL-2 and STF-IL-7 contain phosphotyrosyl residues which are essential for their binding to specific DNA elements. These results are consistent with the paradigm of activation of these latent cytoplasmic factors that has been delineated for the IFNs.

IL-2 and IL-7 Stimulate Signal Transducing Factors That Are Distinct from STF-IL-4

Although STF-IFN and STF-IL-4 bind common DNA elements (e.g. GAS from the IRF-1 and -casein (CAS) genes), these unique factors can be distinguished by their differential binding affinity for unique GAS-like elements (e.g. GAS from the Ly6E gene and the I element from the Ig germ line promoter)(17) . In order to further define the relationship between the factors induced by IL-2, IL-4, and IL-7, we assessed the ability of the CAS-GAS, Ly6E-GAS, and I (high affinity STF-IL-4 binding site) elements to compete for binding of these STFs (Fig. 3A). The binding of STF-IL-2 and STF-IL-7 to IRF-1 GAS is competed by a similar pattern of GAS elements, i.e. with high affinity by the CAS-GAS, but only weakly by the Ly6E-GAS or by I. This pattern differs from the one exhibited by STF-IL-4, which is strongly competed with the CAS-GAS and the I element, but not the Ly6E-GAS. The competition pattern exhibited by STF-IL-2 and STF-IL-7 is also distinct from that shown by STF-IFN, which is strongly competed by the Ly6E-GAS(17) . These findings provide additional evidence that IL-2 and IL-7 induce similar factors, which differ from the factor induced by IL-4.


Figure 3: Comparison of STF-IL-2, STF-IL-4, and STF-IL-7 by oligonucleotide or IL-4 receptor phosphopeptide competition, as well as antisera reactivity. Cells were stimulated, and extracts were prepared and examined as in Fig. 1. A, oligonucleotide competition assays were performed on WCE from IL-2-, IL-4-, or IL-7-stimulated D1.1 either in the absence (lanes 1, 5, and 9) or in the presence of a 100-fold molar excess of cold GAS oligonucleotides added to the shift reaction as indicated (lanes 2-4, 6-8, and 10-12). Competitors included CAS-GAS (lanes 2, 6, and 10), I (lanes 3, 7, and 11), and Ly6E-GAS (lanes 4, 8, and 12). B, antibody interference mobility shift assays were carried out by the addition of either an antiserum raised against a conserved domain of Stat1 (p91-ab4) (lanes 2, 5, and 8) (17) or preimmune serum (lanes 3, 6, and 9) to WCE derived from IL-2-, IL-4-, or IL-7-stimulated PBM. Antisera were added at a final dilution of 1:20 for 30 min at 4 °C to extracts which had been incubated with the IRF-1 GAS probe for 20 min. at 25 °C. C, antibody interference mobility shift assays were carried out, as described above, by the addition of either an antiserum raised against the carboxyl terminus of Stat6 (lanes 2, 5, 8, and 11) (17) or preimmune serum (lanes 3, 6, 9, and 12) to WCE derived from untreated or from IL-2-, IL-4-, or IL-7-stimulated PBM. D, IL-4 receptor peptide competition assay were performed by adding either a phosphorylated (lanes 2-4) or an unphosphorylated (lanes 5-7) form of this peptide to WCE from human PBL which had been stimulated with either IL-2 (panel a), IL-7 (panel b), or IL-4 (panel c). The final concentrations of peptide added were 30 µM (lanes 2 and 5), 100 µM (lanes 3 and 6), and 300 µM (lanes 4 and 7).



It has recently been shown that several STFs contain p91 (Stat1) or other p91-related (STAT family) proteins(30, 33, 38, 41, 45, 46, 47) . In order to determine whether STAT family members participate in the formation of STF-IL-2 and STF-IL-7, we assayed these GAS binding activities for reactivity to a panel of antibodies raised against various conserved domains of the human Stat1 protein. We found that an antiserum (p91-ab4) which recognizes the denatured, but not the native, Stat1 strongly supershifts both STF-IL-2 and STF-IL-7 as assayed by EMSA using an IRF-1 GAS probe (Fig. 3B). Although this antiserum can inhibit STF-IL-4 binding in certain human cell lines (21), it did not interfere with PBM-derived STF-IL-4 binding (Fig. 3B) or with D1.1-derived STF-IL-4 binding (data not shown). In contrast, an antiserum directed against the carboxyl terminus of Stat6 was able to supershift the STF-IL-4 complex but did not affect either STF-IL-2 or STF-IL-7 GAS binding activity (Fig. 3C). Other antisera directed against Stat1 (p91-ab1 and p91-ab2) or against Stat3 had no effects on either STF-IL-2 or STF-IL-7, while Stat2 (p113) antiserum only very weakly supershifted these GAS binding activities (data not shown). The ability of a Stat1 antiserum to supershift STF-IL-2 and STF-IL-7 suggests that these complexes contain a STAT-related protein. In addition, the finding that a Stat6 antiserum reacts with STF-IL-4 but not with STF-IL-2 or STF-IL-7 implies that STF-IL-2 and STF-IL-7 are not composed of Stat6 homodimers. Taken together, these data point to the fact that the component proteins of STF-IL-2/STF-IL-7 appear to be distinct from those of the IL-4-inducible complex.

In order to better define whether STF-IL-2 and STF-IL-7 are distinct from STF-IL-4, we took advantage of the recent finding that the ability of purified Stat6 to bind GAS elements is inhibited by phosphopeptides derived from the IL-4 receptor, but not by an IFN- receptor-derived phosphopeptide known to inhibit Stat1 activation(20) . This inhibition is believed to be due to the fact that SH2-phosphotyrosine interactions are essential for the recruitment of STATs to specific cytokine receptors as well as for the dimerization of STAT proteins(20, 32) . We, therefore, ran a series of experiments to determine whether one of the IL-4 receptor-derived phosphopeptides would affect GAS binding by STF-IL-2 and STF-IL-7. Consistent with previous reports, the addition of the tyrosine-phosphorylated, but not the unphosphorylated, peptide blocked DNA binding by STF-IL-4 in a dose-dependent manner (Fig. 3D). In contrast, GAS binding by either STF-IL-2 or STF-IL-7 was inhibited only weakly by the highest concentration of the IL-4 receptor-derived phosphopeptide (300 µM) (Fig. 3D). A slight inhibitory effect on STF-IL-2 and STF-IL-7 DNA binding was, however, also seen by the addition of a similar concentration of an IFN- receptor phosphopeptide, which is known to inhibit Stat1 GAS binding (data not shown). Since binding of STATs to cytokine receptors is believed to be mediated by specific SH2-phosphotyrosine interactions, these results suggest that the STF-IL-2STF-IL-7 complexes will be recruited to sites on the receptor chains distinct from those involved in STF-IL-4 binding.

STF-IL-2 and STF-IL-7 Contain a Novel 94-kDa STAT-related Protein

The ability of p91-ab4 to supershift STF-IL-2 and STF-IL-7 GAS binding activities permitted us to investigate the nature of the component(s) this antiserum recognizes. Extracts prepared from cells stimulated with IL-2, IL-4, and IL-7 were immunoprecipitated with p91-ab4. The precipitants were separated on SDS-PAGE and immunoblotted with an antiphosphotyrosine antibody. We found that both IL-2 and IL-7 induce the tyrosine phosphorylation of a 94-kDa protein (Fig. 4A). Since this protein (p94) is not activated by IL-4, these results provide additional evidence that the similarities between STF-IL-2 and STF-IL-7 demonstrated by EMSA (see above) are likely to be due to the ability of IL-2 and IL-7 to activate common components. Furthermore, these studies show that activation of STF-IL-2 and STF-IL-7 involves tyrosine phosphorylation.


Figure 4: A 94-kDa tyrosine-phosphorylated protein can be immunoprecipitated using an anti-Stat1 antibody from IL-2- and IL-7-, but not from IL-4-, stimulated cells. Extracts were prepared from cells as described in Fig. 1. Extracts were then examined by immunoprecipitation using p91-ab4. A, IL-2-stimulated CTLL, IL-7-stimulated Clone K, PBM, IL-2-stimulated PBM, IL-7-stimulated PBM, IL-4-stimulated PBM. B, CTLL, IL-2-stimulated CTLL, IL-4-stimulated CTLL, M12 (murine B cell line), IL-4-stimulated M12, RAMOS 266 (human B cell line), IL-4-stimulated RAMOS 266, Clone K, and IL-7-stimulated Clone K.



In order to ascertain whether p94 can be activated by IL-4, we immunoprecipitated extracts from various cell lines stimulated with IL-4 and assayed for tyrosine phosphorylation of p94 (Fig. 4B). p94 is not tyrosine-phosphorylated in response to IL-4 in either a human (RAMOS 266) or a murine (M12) B cell line which contain STF-IL-4 binding activity(17, 21) . In addition, CTLL cells, which activate p94 in response to IL-2 (Fig. 4, A and B), do not tyrosine-phosphorylate p94 in response to IL-4 (Fig. 4B). These data confirm our previous results that STF-IL-2 and STF-IL-7 contain a common, if not identical, component which is not a constituent of STF-IL-4.

To determine whether p94 has GAS binding activity, as has been observed with other STAT proteins(17, 38, 46, 48) , we precipitated extracts from IL-2- and IL-7-stimulated cells using an agarose-linked GAS element. These precipitants were immunoblotted with an antiphosphotyrosine antibody. The precipitants from both IL-2-stimulated CTLL and IL-7-stimulated Clone K contained the same 94-kDa tyrosine-phosphorylated protein detected by immunoprecipitations using p91-ab4 (Fig. 5). In addition, both murine splenocytes and thymocytes also activate a protein of identical size in response to IL-7 (Fig. 5). These results indicate that the 94-kDa protein (p94) recognized by p91-ab4 is part of the complex that binds to the GAS element that we have used to characterize STF-IL-2 and STF-IL-7.


Figure 5: A 94-kDa tyrosine-phosphorylated protein can be identified in IL-2- and IL-7-stimulated extracts by GAS oligonucleotide affinity precipitations. Extracts were precipitated using agarose-immobilized CAS-GAS multimers except for the last two lanes which represents CTLL cells immunoprecipitated with p91-ab4. The protein components were then separated on 7% SDS-PAGE and analyzed by Western blotting using an antiphosphotyrosine antibody. Lanes are as follows: splenocytes, IL-7-stimulated splenocytes, thymocytes, IL-7-stimulated thymocytes, Clone K, IL-7-stimulated Clone K, CTLL, IL-2-stimulated CTLL, CTLL immunoprecipitated using p91-ab4, IL-2-stimulated CTLL immunoprecipitated using p91-ab4.




DISCUSSION

Our studies demonstrate that, although the IL-2, IL-4, and IL-7 signaling pathways share common components, only IL-2 and IL-7 activate closely related STFs. By several criteria including mobility on EMSA, competition profile by GAS-related sequences as well as by IL-4 receptor phosphopeptides, and reactivity to Stat1 and Stat6 antisera, the Stat6 homodimer induced by IL-4 behaves clearly differently from STF-IL-2 and STF-IL-7. In addition, only IL-2 and IL-7, but not IL-4, induce the rapid tyrosine phosphorylation of a 94-kDa protein (p94). The fact that this p94 protein possesses GAS binding activity (which has also been shown for Stat1, Stat3, Stat4, and Stat6(22, 38, 46, 49) ) along with its cross-reactivity with some, but not all, Stat1 antisera suggests that it is a member of the STAT family of proteins.

The receptors for the cytokines we have studied are composed of multicomponent complexes consisting of the common chain in addition to ligand-specific chain(s). Our data, which demonstrate that there are distinct complexes induced by different chain-associated receptors, in spite of their association with the same set of JAK kinases, suggest that factors other than the kinases themselves, perhaps the ligand-binding chain, play a critical role in defining the specificity of the transduced signal. This notion is supported by the observation that a phosphorylated peptide derived from the IL-4 receptor ligand-specific chain is able to block GAS binding by STF-IL-4 more effectively than that by STF-IL-2 or STF-IL-7. Hence, complex interactions between the individual components of the IL-2, IL-4, and IL-7 receptors appear to dictate the recruitment and activation of specific signal transducing factors by receptor-associated kinases.

The data presented above indicate that the STFs activated by IL-2 and IL-7, but not IL-4, contain a 94-kDa protein (p94) that is related to Stat1. Interestingly, on EMSA, the mobility displayed by STF-IL-2 and STF-IL-7 is identical with that exhibited by the IL-3-inducible complex (data not shown), which appears to be composed of a Stat5 homodimer (50). In addition, preliminary results reveal that p94 reacts with a monoclonal antibody directed against Stat5. These findings raise the possibility that the STAT-related protein activated by IL-2 and IL-7 is indeed Stat5 or a Stat5-related protein. Activation of p94 is detected in a variety of IL-7 responsive cell types. We have shown that p94 is activated in response to IL-7 in thymocytes, splenocytes, and an IL-7-dependent pre-B cell line (Clone K). Interestingly, the mobility of STF-IL-7 in Clone K is slightly slower than the one detected in murine splenocytes and thymocytes (data not shown), raising the possibility that additional components of STF-IL-7 exist that are specific for different stages of differentiation and/or cell type. In addition, it is possible that STF-IL-2 and STF-IL-7 may contain proteins that complex with p94 and are unique for each STF. Further characterization and cloning of these factors will allow a more careful analysis of the component proteins of these complexes.

Cytokine-induced STFs have been shown to bind to unique enhancer elements in the promoter of target genes and are responsible for the activation of early response genes(14, 15) . The fact that IL-2 and IL-7 induce similar STFs may indicate that the early transcriptional events induced by these two cytokines are identical. Our data are consistent with prior work which shows that the pattern of tyrosine-phosphorylated proteins induced by IL-2 overlaps with that stimulated by IL-7(51) , but differs from that induced by IL-4(52) . Distinctions between the IL-2 and the IL-4 signaling pathways have also been noted in the activation of p21 which is activated in response to IL-2, but not IL-4(53) . The early divergence of the signaling pathways among these cytokines might explain their ability to perform their distinctive biologic functions.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL 21006-17 (to C. S.), AI 33450-02 (to P. R.), and T 32CA09503 (to A. P.), the James S. McDonnell Foundation (to C. S.), Warner Lambert Grant 6-49556 (to C. S.), the Pew Scholars Program (to P. R.), and the Stephen I. Morse Fellowship (to A. P.). 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 and reprint requests should be addressed: Columbia University, Black Bldg. 1029, 630 West 168th St., New York, NY 10032.

The abbreviations used are: STAT, signal transducers and activators of transcription; STF, signal transducing factor; GAS, activation site; IFN, interferon; PBM, peripheral blood mononuclear cells; WCE, whole cell extracts; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay.

A. Pernis, unpublished results.


ACKNOWLEDGEMENTS

We thank Robert Coffman and Jan de Vries of DNAX Research Institute for recombinant IL-4 and IL-13 and Doug Fisher of Pfizer Corp. for cytokine receptor peptides. We also thank Kathryn Calame and Ned Braunstein for their critical reading of this manuscript.


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