Cutting Edge: A Conditionally Active Form of STAT6 Can Mimic Certain Effects of IL-4 Yumiko Kamogawa, Hyun Jun Lee, James A. Johnston, Martin McMahon, Anne O'Garra and Naoko Arai This information is current as of September 29, 2021. J Immunol 1998; 161:1074-1077; ; http://www.jimmunol.org/content/161/3/1074

References This article cites 29 articles, 16 of which you can access for free at: Downloaded from http://www.jimmunol.org/content/161/3/1074.full#ref-list-1

Why The JI? Submit online.

http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: by guest on September 29, 2021 http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ●

Cutting Edge: A Conditionally Active Form of STAT6 Can Mimic Certain Effects of IL-41

Yumiko Kamogawa,* Hyun Jun Lee,* James A. Johnston,* Martin McMahon,* Anne O’Garra,† and Naoko Arai2*

then is tyrosine phosphorylated on residue Y641. The current Binding of IL-4 to its cognate leads to the activation model suggests that tyrosine phosphorylation of STAT6 elicits its Downloaded from of a number of signaling pathways within the cell. Activation of homodimerization and nuclear translocation. STAT6 homodimers the STAT6 by JAK family ty- are then able to bind to specific IFN-␥-activated site (GAS)-like rosine kinases has been shown to be essential for the full re- sequences found in the promoter region of IL-4-inducible genes. sponse of cells to IL-4. To elucidate the role of STAT6 in IL-4 Since the mutation of residue Y641 in STAT6 completely abol- signaling, we have constructed and expressed in cells a condi- ishes DNA-binding activity as well as trans-activation (6), it tionally active form of the protein (STAT6:ER*) by fusing would appear that tyrosine phosphorylation of STAT6 is required http://www.jimmunol.org/ STAT6 to a modified form of the hormone-binding domain of for signal transduction. the . Activation of STAT6:ER* by 4-hy- Gene disruption studies have shown that STAT6 is required for droxytamoxifen leads to specific activation of STAT6-regu- IL-4-mediated biologic functions such as class switching to IgE, lated including the activation of a STAT6 re- induction of IL-4-inducible genes (e.g., CD23, MHC class II, IL-4 porter construct and induction of CD23 in B cell lines. receptor), Th2 cell differentiation, and some effects on lymphoid Interestingly, in contrast to native STAT6, activation of proliferation (7–9). However, it is unclear whether the activation STAT6:ER* occurs in the absence of detectable tyrosine phos- of STAT6 is sufficient for inducing the IL-4-mediated activities or whether there is some dependence on the activation of alternate phorylation of the fusion protein. This type of conditional sys- by guest on September 29, 2021 tem will be helpful in dissecting the mechanisms and specificity signaling molecules as yet undefined. of transcriptional regulation by the STAT family of transcrip- To study the relative importance of STAT6 in IL-4-induced sig- nal transduction and the mechanisms of activation of STAT6, we tion factors. The Journal of Immunology, 1998, 161: 1074– developed a conditionally active form of STAT6 by fusing the 1077. hormone-binding domain of a modified form of the mouse estro- gen receptor (ER*) gene (10–12) to the murine STAT6. Activation ngagement of IL-4 to its receptor leads to heterodimer- of STAT6:ER* led to induced transcription of an IL-4-responsive ization of the receptor (1). Receptor dimerization leads to promoter and in M12.4.1 B lymphoma cells led to induction of activation of the receptor-associated tyrosine kinases, Ja- CD23. This system will be useful in dissecting the role of STAT E 3 nus kinase (JAK) 1 and JAK3, which then phosphorylate distinct in and signaling. tyrosine residues of the receptor (2). This results in the activation of at least two distinct signaling pathways, including STAT6 ac- Materials and Methods tivation (3, 4) and the phosphorylation of the insulin receptor sub- Cell lines and reagents strate 1⁄2 (5). After phosphorylation of the IL-4 receptor, STAT6 BA/F3 cells were cultured in RPMI 1640 (JRH Biosciences, Lenexa, KS) docks onto the receptor through the src homology domain 2 and supplemented with IL-3 (10 ng/ml). M12.4.1 cells, a kind gift from Dr. A. Keegan (American Red Cross, Bethesda, MD), were cultured in RPMI 1640. All tissue culture media were supplemented with 10% FCS, L-glu- Departments of *Cell Signaling and †Immunobiology, DNAX Research Institute of tamine (2 mM), penicillin (100 U/ml), and streptomycin (100 ␮g/ml). Molecular and Cellular Biology, Palo Alto, CA 94304 4-Hydroxytamoxifen (4-HT) (Research Biochemicals Institute, Natick, Received for publication February 18, 1998. Accepted for publication June 1, 1998. MA) was used to activate STAT6 fusion protein. 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 Plasmid DNA with 18 U.S.C. Section 1734 solely to indicate this fact. pMXGSTAT6:ER* (vector pMX (13)) contained STAT6-hbER* fusion 1 DNAX Research Institute of Molecular and Cellular Biology is supported by the (murine STAT6 cDNA (3), hbER* from pBP3:hbER* (12)), and enhanced Schering-Plough Corporation. green fluorescent protein (EGFP) gene that were controlled by SR␣ (14) 2 Address correspondence and reprint requests to Dr. Naoko Arai, Department of Cell and long terminal repeat (LTR) promoter, respectively. Control pMXG Signaling, DNAX Research Institute of Molecular and Cellular Biology, 901 Cali- plasmid contained the EGFP gene controlled by the LTR promoter. fornia Avenue, Palo Alto, CA 94304. E-mail address: [email protected] The reporter construct carrying the luciferase reporter gene (pGLC⑀) ⑀ 3 Abbreviations used in this paper: JAK, ; 4HT, 4-hydroxytamoxifen; was generated by inserting three copies of human C STAT6 binding site ER, estrogen receptor; GAS, IFN-␥-activated site; LTR, long terminal repeat; EGFP, oligonucleotide as direct repeats into the BglII site of pGL2-P (Promega, enhanced green fluorescent protein; EMSA, electrophoretic mobility shift assay. Madison, WI).

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00

● The Journal of Immunology 1075

Retrovirus infection Three micrograms of DNA were transfected into the packaging cell line Phoenix (provided by Dr. G. Nolan, Stanford University, Stanford, CA) using lipofectamine (Life Technologies, Gaithersburg, MD). After 2 days of transfection, viral supernatants were used for infection of M12.4.1 and BA/F3 cells. Luciferase assay BA/F3 cells were transfected with 10 ␮g of reporter plasmid pGLC⑀ and 1 ␮g of reference plasmid pRSV-LacZ by electroporation. After culturing for 24 h in phenol red-free RPMI 1640 (Life Technologies) medium, the cells were stimulated with either 1 ␮M 4HT or 100 U/ml IL-4 for 24 h at 37°C. The cells were harvested and assayed for luciferase activities using a Luciferase Assay Kit (Promega, Madison, WI). Immunoprecipitation and Western blotting Cell lysates were immunoprecipitated with anti-mouse STAT6 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) as previously described (15). West- ern blotting was performed with either anti-STAT6 Ab, anti-ER Ab (Santa Cruz), or monoclonal anti-phosphotyrosine Ab (Upstate Biotechnology, FIGURE 1. Expression of STAT6:ER* fusion protein in established Lake Placid, NY) using the ECL detection system (Amersham, Amersham, BA/F3 (BS6ER*) and M12.4.1 (MS6ER*) cell lines. Cell lysates were Downloaded from Buckinghamshire, U.K.). prepared from BS6ER* and MS6ER* cells as well as control BGFP and MGFP cells and immunoprecipitated with anti-STAT6 Ab. Western blots Electrophoretic mobility shift assay for STAT6 were prepared and probed with either a polyclonal rabbit anti-mouse Nuclear extracts and electrophoretic mobility shift assay (EMSA) were STAT6 Ab (Santa Cruz Biotechnology) or a rabbit anti-human (cross-react prepared as previously described (16), except that binding buffer included with mouse ER) ER Ab (Santa Cruz Biotechnology). The bands corre- 1 ␮M 4HT. In the supershift assay, nuclear extracts were incubated with 1 sponding to STAT6:ER* (ϳ160 kDa) and the endogenous STAT6 (ϳ100 ␮g of each Ab at room temperature for 10 min before adding the probe. kDa) were indicated. The structure of plasmids used is presented schemat- http://www.jimmunol.org/ ically in the lower panel. Results Construction and expression of conditionally active STAT6:ER* To derive a conditionally active form of STAT6, we made a ret- binding activity is strongly and rapidly (within 1 h, data not rovirus construct, pMXGSTAT6:ER*, encoding the EGFP marker shown) induced after addition of 4HT to these cells. They also and, a chimeric protein of mouse STAT6 and a modified form of indicate that STAT6:ER* does not respond to IL-4 in the absence the hormone-binding domain of ER* that fails to respond to ␤-es- of 4HT. Next, to test the trans-activation of the STAT6:ER* protein,

tradiol but retains responsiveness to estrogen analogues such as by guest on September 29, 2021 4-HT and ICI 182,780 (11). Retrovirus infection was followed by pGLC⑀ was transfected into BS6ER* and BGFP by electropora- several rounds of sorting for EGFP-positive cells in a mouse pre-B tion. In BS6ER* cells, the reporter gene was efficiently transcribed cell line (BA/F3) and a mouse B lymphoma line (M12.4.1) to yield in the presence of 4HT. Importantly, the luciferase activity induced stable cell lines, BS6ER* and MS6ER*, respectively. As shown in by 4HT was similar to that induced by IL-4. As expected, 4HT did Figure 1, both BS6ER* and MS6ER* expressed comparable not induce luciferase activity in control BGFP cells, whereas IL-4 amounts of STAT6:ER* fusion protein (160 kDa) and endogenous did (Fig. 2B). These data indicate that STAT6:ER* activates C⑀- STAT6 (100 kDa). The control pMXG infecting BA/F3 and driven transcription in a 4HT-dependent fashion. M12.4.1 cell lines (named BGFP and MGFP, respectively) ex- Tyrosine phosphorylation is not required for activation of pressed only endogenous STAT6. STAT6:ER* The fusion protein STAT6:ER* binds to the STAT6-specific It has been shown that tyrosine phosphorylation of STAT proteins GAS-like element and activates transcription in response to is essential for their activation (18). Thus, we investigated whether 4-hydroxytamoxifen STAT6:ER* activation by 4-HT was accompanied by tyrosine phos- To examine 4HT-dependent activation of the STAT6:ER* protein, phorylation by Western blot (Fig. 3). Treatment of cells with IL-4 we first performed EMSA on nuclear extracts from MS6ER*, us- led to tyrosine phosphorylation of both STAT6 and STAT6:ER*, ing a probe containing GAS-like sequence specific for STAT6 which was readily detected after 15 min of cytokine treatment and from the human C⑀ promoter (17). As shown in Figure 2A, treat- was sustained for up to 24 h after the addition of IL-4. By contrast, ment of cells with murine IL-4 induced formation of complex (I), activation of STAT6:ER* by the addition of 4HT did not result in which appeared in nuclear extracts from both MS6ER* and MGFP any detectable phosphorylation in spite of the expression of cells. By contrast, activation of STAT6:ER* by 4HT-induced com- STAT6:ER* protein. High levels of STAT6:ER* protein accumu- plex (II) which had reduced mobility compared with complex (I). lation were observed after 6 to 24 h of 4HT addition, possibly due Complex (II) appeared only in nuclear extracts from MS6ER* to the selective stabilization of the protein as observed in ⌬Raf:ER cells but not in those from MGFP cells. Furthermore, complex (II) fusion proteins after prolonged addition of 4HT (19). was supershifted by both an anti-STAT6 Ab and an anti-ER Ab, indicating that complex (II) contained STAT6:ER*. In accordance 4HT induced up-regulation of CD23 expression in M12.4.1 cells with the above observations, IL-4-induced complex (I) was super- infected by pMXGST6:ER* shifted only by the anti-STAT6 Ab in MS6ER* cells. A minor part It has previously been shown that treatment of the M12.4.1 cell of complex (I) was also supershifted by adding the anti-ER Ab in lines with IL-4 leads to an elevation of CD23 expression (20). We MS6ER* cells, suggesting that complex (I) consists of either a examined whether STAT6:ER* could mimic the effects of IL-4 in STAT6-STAT6 homodimer or a small amount of STAT6-STAT6: the regulation of CD23 expression. Cells expressing STAT6:ER* ER* heterodimer. These data indicate that STAT6:ER* DNA- as well as the control obtained by infecting pMXGST6:ER* and 1076 CUTTING EDGE

FIGURE 3. STAT6:ER* are not tyrosine phosphorylated during activa- tion. MS6ER* cells (2 ϫ 107) were either unstimulated or stimulated with either 4HT (1 ␮M) or IL-4 (100 U/ml) for indicated times. The cell lysates were prepared and immunoprecipitated with anti-STAT6 Ab. The precip- itated proteins were separated by SDS-PAGE. Western blotting was per- formed with either an anti-phosphotyrosine or an anti-STAT6 Ab. Posi- tions of endogenous STAT6 and STAT6: ER* were indicated.

form of the hormone-binding domain of the mouse ER. Activation Downloaded from of STAT6:ER* with 4HT leads to the rapid induction of specific DNA-binding activity and activation of a STAT6-specific reporter gene. When expressed stably in cells, activation of STAT6:ER* mimics some of the effects of IL-4 in that it induces the cell surface expression of CD23. We observed that the rate and kinetics of CD23 induction by STAT6:ER* were similar to those observed in http://www.jimmunol.org/ response to IL-4. In addition, we confirmed that STAT6:ER* and native STAT6 share the same specificity for DNA binding and promoter activation (data not shown). Importantly, however, acti- vation of STAT6:ER* occurred in the absence of tyrosine phos- phorylation of the fusion protein. These data indicate that we can activate STAT6 protein in cells in the absence of engagement, allowing the anal- ysis of STAT6-specific events in the absence of other “parallel”

signal transduction pathways. This conditional system can be by guest on September 29, 2021 FIGURE 2. A, STAT6:ER* binds to STAT6-specific GAS-like se- readily exploited to identify STAT6-regulated genes in a variety of quences containing oligonucleotide. MEGFP or MS6ER* cells were cell systems. treated either with 4HT (1 ␮M) for 13 h or with IL-4 (100 U/ml) for 15 The hormone-binding domain of steroid receptors has proven min. Nuclear extracts were prepared, and EMSA was performed using the remarkably versatile in the establishment of conditional forms of human C⑀ GAS-like sequences (5Ј-GATCAAGACCTTTCCCAAGAA ATCTATC-3Ј) containing probe in the presence or absence of indicated Ab. The positions of the IL-4-induced DNA-binding complex (I), the 4HT- induced binding complex (II), and the supershifted complexes (*) are in- dicated. B, STAT6:ER* trans-activates the reporter construct driven by STAT6-binding sites. BS6ER* and a control line, BGFP, were transiently transfected with a reporter plasmid pGLC⑀ (10 ␮g). The cells were split into three groups and either unstimulated (none) or stimulated either with 100 U/ml IL-4 or with 1 ␮M 4HT for 24 h. Then they were harvested and assayed for luciferase activities. The luciferase activity without stimulation was assigned a value of 1.0; the promoter activity was expressed as relative fold increase after normalization for ␤-galactosidase activity of pRSV- LacZ. Data represent the mean Ϯ SD from three independent experiments. pMXG, respectively, were treated with either IL-4 or 4HT for 16 h. The activation of STAT6:ER* by 4HT or IL-4 was evaluated by the level of cell surface CD23 expression assessed by flow cytom- etry (Fig. 4). It was apparent that CD23 induction was observed only in STAT6:ER expressing populations (Fig. 4f) but not in cells infected with the control virus (Fig. 4c) after addition of 4HT, although IL-4 caused increased CD23 expression in all cell pop- FIGURE 4. Conditional up-regulation of CD23 expression by STAT6: ulations (Fig. 4, b, c, e, and f). These data indicate that STAT6: ER*. M12.4.1 cells were infected with virus containing either control ER* can mimic IL-4 function to induce CD23 expression. pMXG (top, a--c) or pMXGSTAT6:ER* (bottom, d–f). Two days after primary retroviral infection, the cells were unstimulated (black line) or Discussion stimulated with either 1 ␮M 4HT (green line) or 100 U/ml IL-4 (pink line) for 16 h, and cell surface expression of CD23 was analyzed by FACS We describe the construction and utility of a conditionally active within either the EGFP-negative population (R1: EGFP(Ϫ)(b, e)orthe form of STAT6 derived by fusing mouse STAT6 to a modified EGFP-positive population (R2: EGFP(ϩ)(c, f). The Journal of Immunology 1077 transcription factors (E1A, c-, c-Fos, CEBP, v-Rel) (21–25) 5. Chen, X. H., B. K. Patel, L. M. Wang, M. Frankel, N. Ellmore, R. A. Flavell, and signaling molecules (Raf, c-Abl) (26, 27). It has been clearly W. J. LaRochelle, and J. H. Pierce. 1997. Jak1 expression is required for medi- ating interleukin-4-induced tyrosine phosphorylation of insulin receptor substrate demonstrated that the isolated C-terminal hormone-binding do- and Stat6 signaling molecules. J. Biol. Chem. 272:6556. main of ER dimerizes and undergoes conformational changes as 6. Mikita, T., D. Campbell, P. Wu, K. Williamson, and U. Schindler. 1996. Re- observed in the whole molecule after binding (28, 29). Al- quirements for interleukin-4-induced gene expression and functional character- ization of Stat6. Mol. Cell. Biol. 16:5811. though the exact mechanism of the observed conditionality re- 7. Takeda, K., T. Tanaka, W. Shi, M. Matsumoto, M. Minami, S. Kashiwamura, mains unclear, it is likely that hormone-regulated dimerization K. Nakanishi, N. Yoshida, T. Kishimoto, and S. Akira. 1996. Essential role of may be important. In this regard, hormone-induced dimerization Stat6 in IL-4 signalling. Nature 380:627. 8. Shimoda, K., D. J. van Deursen, M. Y. Sangster, S. R. Sarawar, R. T. Carson, may mimic the functional consequences of STAT6 tyrosine phos- R. A. Tripp, C. Chu, F. W. Quelle, T. Nosaka, D. A. Vignali, P. C. Doherty, phorylation believed to be essential for dimerization, which is a G. Grosveld, W. E. Paul, and J. N. Ihle. 1996. Lack of IL-4-induced Th2 response prerequisite for transcriptional regulation (6). Moreover, since and IgE class switching in mice with disrupted Stat6 gene. Nature 380:630. 9. Kaplan, M. H., U. Schindler, S. T. Smiley, and M. J. Grusby. 1996. Stat6 is STAT6:ER* activation occurred in the absence of tyrosine phos- required for mediating responses to IL-4 and for development of Th2 cells. Im- phorylation, it seems likely that tyrosine phosphorylation is re- munity 4:313. quired only for dimerization and not for activation of transcription 10. Danielian, P. S., R. White, S. A. Hoare, S. E. Fawell, and M. G. Parker. 1993. Identification of residues in the estrogen receptor that confer differential sensi- per se. Furthermore, the low level of reporter activity in the ab- tivity to estrogen and hydroxytamoxifen. Mol. Endocrinol. 7:232. sence of 4HT indicates that the STAT6:ER* fusion protein is not 11. Littlewood, T. D., D. C. Hancock, P. S. Danielian, M. G. Parker, and G. I. Evan. leaky. 1995. A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins. Nucleic Acids Res. 23:1686. Both tyrosine phosphorylation and DNA binding of STAT6:

12. Pritchard, C. A., M. L. Samuels, E. Bosch, and M. McMahon. 1995. Condition- Downloaded from ER* resisted the effects of IL-4. However, in the presence of 4HT, ally oncogenic forms of the A-Raf and B-Raf protein kinases display different both tyrosine phosphorylation and DNA binding of STAT6:ER* biological and biochemical properties in National Institutes of Health 3T3 cells. Mol. Cell. Biol. 15:6430. were strongly potentiated in response to costimulation with IL-4. 13. Onishi, M., S. Kinoshita, Y. Morikawa, A. Shibuya, J. Phillips, L. L. Lanier, These data suggest that the ER* domain renders the STAT6:ER* D. M. Gorman, G. P. Nolan, A. Miyajima, and T. Kitamura. 1996. Applications fusion resistant to the activation of IL-4 receptor by ligand en- of retrovirus-mediated expression cloning. Exp. Hematol. 24:324. 14. Takebe, Y., M. Seiki, J. Fujisawa, P. Hoy, K. Yokota, K. Arai, M. Yoshida, and gagement. Neither the inactive form of STAT6:ER* nor the phos- N. Arai. 1988. SR alpha promoter: an efficient and versatile mammalian cDNA http://www.jimmunol.org/ phorylated form of STAT6:ER* displayed a dominant-negative expression system composed of the simian virus 40 early promoter and the R-U5 effect, since activation of cells in combination of IL-4 and 4HT segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol. 8:466. caused additive effects on endogenous CD23 expression as well as 15. Migone, T. S., J. X. Lin, A. Cereseto, J. C. Mulloy, J. J. O’Shea, G. Franchini, trans-activation of the reporter gene (data not shown). and W. J. Leonard. 1995. Constitutively activated Jak-STAT pathway in T cells Taken together, these data indicate the feasibility of using the transformed with HTLV-I. Science 269:79. 16. Masuda, E. S., J. Liu, R. Imamura, S. I. Imai, K. I. Arai, and N. Arai. 1997. hormone-binding domain of the ER to regulate the function of Control of NFATx1 nuclear translocation by a calcineurin-regulated inhibitory STAT family transcription factors. Activation of STAT6:ER* domain. Mol. Cell. Biol. 17:2066. clearly mimicked one of the effects of IL-4 treatment of B lym- 17. Rothman, P., S. C. Li, B. Gorham, L. Glimcher, F. Alt, and M. Boothby. 1991. Identification of a conserved lipopolysaccharide-plus-interleukin-4-responsive el- phoma cells. We are presently identifying additional STAT6-reg- ement located at the promoter of germ line ⑀ transcripts. Mol. Cell. Biol. 11:5551. by guest on September 29, 2021 ulated genes and attempting to identify their role in the effects of 18. Darnell, J. E., Jr. 1997. STATs and gene regulation. Science 277:1630. IL-4. Finally, given the degree of structural similarity of the mem- 19. Woods, D., D. Parry, H. Cherwinski, E. Bosch, E. Lees, and M. McMahon. 1997. bers of the STAT family of transcription factors, it seems likely Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1. Mol. Cell. Biol. 17:5598. that this approach will have broad utility in the analysis of STAT- 20. Kikutani, H., M. Suemura, H. Owaki, H. Nakamura, R. Sato, K. Yamasaki, mediated regulation of gene expression. E. L. Barsumian, R. R. Hardy, and T. Kishimoto. 1986. Fc⑀ receptor, a specific differentiation marker transiently expressed on mature B cells before isotype switching. J. Exp. Med. 164:1455. Acknowledgments 21. Picard, D., S. J. Salser, and K. R. Yamamoto. 1988. A movable and regulable inactivation function within the steroid binding domain of the glucocorticoid We thank Dr. J. Ihle for the gift of the STAT6 cDNA, Dr. G. Nolan for receptor. Cell 54:1073. retrovirus packaging cell line, Dr. T. Kitamura for a retroviral vector pMX, 22. Eilers, M., D. Picard, K. R. Yamamoto, and J. M. Bishop. 1989. Chimaeras of Dr. M. Howard for the BA/F3 cell line, and Dr. S. Menon for mouse IL-3 myc oncoprotein and steroid receptors cause hormone-dependent transformation and IL-4. We thank J. Cupp, E. Callas, D. Polakoff, and of cells. Nature 340:66. E. Murphy for cell sorting and D. Liggett for for oligonucleotide synthesis 23. Superti, F. G., G. Bergers, D. Picard, and M. Busslinger. 1991. Hormone-depen- dent transcriptional regulation and cellular transformation by Fos-steroid receptor and Drs. T. Migone, E. Masuda, A. Mui, D. Robinson, Y. Amasaki, fusion proteins. Proc. Natl. Acad. Sci. USA 88:5114. R. Imamura, D. Wylie, and K. Arai for critical comments and reading of 24. Umek, R. M., A. D. Friedman, and S. L. McKnight. 1991. CCAAT-enhancer the manuscript. binding protein: a component of a differentiation switch. Science 251:288. 25. Capobianco, A. J., and T. D. Gilmore. 1993. A conditional mutant of vRel con- taining sequences from the human estrogen receptor. Virology 193:160. References 26. Samuels, M. L., M. J. Weber, J. M. Bishop, and M. McMahon. 1993. Conditional 1. Keegan, A. D., K. Nelms, L. M. Wang, J. H. Pierce. and W. E. Paul. 1994. transformation of cells and rapid activation of the mitogen-activated protein ki- receptor: signaling mechanisms. Immunol. Today 15:423. nase cascade by an estradiol-dependent human raf-1 protein kinase. Mol. Cell. 2. Schindler, C., and J. E. Darnell, Jr. 1995. Transcriptional responses to polypep- Biol. 13:6241. tide ligands: the JAK-STAT pathway. Annu. Rev. Biochem. 64:621. 27. Jackson, P., D. Baltimore, and D. Picard. 1993. Hormone-conditional transfor- 3. Quelle, F. W., K. Shimoda, W. Thierfelder, C. Fischer, A. Kim, S. M. Ruben, mation by fusion proteins of c-Abl and its transforming variants. EMBO J. 12: J. L. Cleveland, J. H. Pierce, A. D. Keegan, K. Nelms, W. Paul, and J. Ihle. 1995. 2809. Cloning of murine STAT6 and human STAT6, STAT proteins that are tyrosine 28. Parker, M. G., N. Arbuckle, S. Dauvois, P. Danielian, and R. White. 1993. Struc- phosphorylated in responses to IL-4 and IL-3 but are not required for mitogen- ture and function of the estrogen receptor. Ann NY Acad. Sci. 684:119. esis. Mol. Cell. Biol. 15:3336. 29. Brandt, M. E., and L. E. Vickery. 1997. Cooperativity and dimerization of re- 4. Hou, J., U. Schindler, W. J. Henzel, T. C. Ho, M. Brasseur, and S. L. McKnight. combinant human estrogen receptor hormone-binding domain. J. Biol. Chem. 1994. An interleukin-4-induced transcription factor: IL-4 Stat. Science 265:1701. 272:4843.