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A Gain-of-function Mutation in STAT6

2000; Elsevier BV; Volume: 275; Issue: 19 Linguagem: Inglês

10.1074/jbc.c000129200

1083-351X

Carla Daniel, Anupama Salvekar, Ulrike Schindler,

Helicobacter pylori-related gastroenterology studies

Interleukin-4 (IL-4) is a cytokine that plays a crucial role in the pathophysiology of asthma and allergic diseases. IL-4-induced gene expression is largely mediated through the activation of the latent transcription factor STAT6. We identified a STAT6 mutant (STAT6VT)) that is activated independently of IL-4 stimulation. STAT6VT carries two amino acid changes in the SH2 domain that affect the overall structure and stability of the monomeric and dimeric protein. When overexpressed in mammalian cells, STAT6VT undergoes tyrosine phosphorylation, binds DNA, and activates transcription in the absence of IL-4 stimulation. Using the Jak1- and Jak3-deficient fibroblast line U4A, we demonstrate that phosphorylation is mediated by an IL-4-independent tyrosine kinase that is not able to activate the wild-type STAT6 protein. These results suggest that small changes in STAT6 could result in hyperactivation of the protein and constitutive expression of STAT6-dependent genes. Such a mutation, if found in vivo, could cause genetic predisposition for atopic diseases. Interleukin-4 (IL-4) is a cytokine that plays a crucial role in the pathophysiology of asthma and allergic diseases. IL-4-induced gene expression is largely mediated through the activation of the latent transcription factor STAT6. We identified a STAT6 mutant (STAT6VT)) that is activated independently of IL-4 stimulation. STAT6VT carries two amino acid changes in the SH2 domain that affect the overall structure and stability of the monomeric and dimeric protein. When overexpressed in mammalian cells, STAT6VT undergoes tyrosine phosphorylation, binds DNA, and activates transcription in the absence of IL-4 stimulation. Using the Jak1- and Jak3-deficient fibroblast line U4A, we demonstrate that phosphorylation is mediated by an IL-4-independent tyrosine kinase that is not able to activate the wild-type STAT6 protein. These results suggest that small changes in STAT6 could result in hyperactivation of the protein and constitutive expression of STAT6-dependent genes. Such a mutation, if found in vivo, could cause genetic predisposition for atopic diseases. interleukin 4 T-helper Janus kinase signal transducer and activator of transcription Src homology domain 2 STAT6 V547A/T548A mutation IL-41 is a multifunctional cytokine that stimulates changes in many cell types and is involved in the regulation of immune and inflammatory responses (1.Paul W.E. Seder R.A. Cell. 1994; 76: 241-251Abstract Full Text PDF PubMed Scopus (1700) Google Scholar,2.Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Abstract Full Text PDF PubMed Scopus (1250) Google Scholar). Among the many physiological responses mediated by IL-4 is its ability to promote the differentiation of T-helper (Th) precursors toward the Th2 lineage while inhibiting Th1 development (3.Mosmann T.R. Coffman R.L. Adv. Immunol. 1989; 46: 111-147Crossref PubMed Scopus (1027) Google Scholar, 4.Mosmann T.R. Coffman R.L. Annu. Rev. Immunol. 1989; 7: 145-173Crossref PubMed Scopus (6879) Google Scholar, 5.Abbas A.K. Murphy K.M. Sher A. Nature. 1996; 383: 787-793Crossref PubMed Scopus (3887) Google Scholar). Furthermore, IL-4 stimulation of B-cells triggers immunoglobulin class switching to the IgE isotype (6.Stavnezer J. Adv. Immunol. 1996; 61: 79-146Crossref PubMed Google Scholar). This Ig recombination is thought to be initiated following the transcriptional activation of the germline ε promoter, which leads to the generation of the sterile ε transcript. Numerous studies have shown that the IL-4-induced activation of the ε promoter requires STAT6, a transcription factor activated upon IL-4 stimulation (7.Delphin S. Stavnezer J. J. Exp. Med. 1995; 181: 181-192Crossref PubMed Scopus (189) Google Scholar, 8.Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar, 9.Linehan L.A. Warren W.D. Thompson P.A. Grusby M.J. Berton M.T. J. Immunol. 1998; 161: 302-310PubMed Google Scholar, 10.Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1657) Google Scholar, 11.Akira S. Stem Cells. 1999; 17: 138-146Crossref PubMed Scopus (276) Google Scholar). STAT6 is activated by the Jak/STAT pathway (12.Hou J. Schindler U. Henzel W.J. Ho T.Z. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (730) Google Scholar). Upon IL-4 binding to its cognate receptor, the latent, monomeric STAT6 protein is recruited to the α chain of the IL-4 receptor, where it specifically interacts with two phosphotyrosine-containing regions (12.Hou J. Schindler U. Henzel W.J. Ho T.Z. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (730) Google Scholar, 13.Schindler U. Wu P. Rothe M. Brasseur M. McKnight S.L. Immunity. 1995; 2: 689-697Abstract Full Text PDF PubMed Scopus (233) Google Scholar, 14.Wang H.Y. Paul W.E. Keegan A.D. Immunity. 1996; 4: 113-121Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 15.Ryan J.J. McReynolds L.J. Keegan A. Wang L.-H. Garfein E. Rothman P. Nelms K. Paul W.E. Immunity. 1996; 4: 123-132Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). This interaction requires the integrity of the STAT6 SH2 domain. Subsequently, STAT6 becomes phosphorylated on tyrosine 641 by Jak1 and/or Jak3, two members of the Jak family of tyrosine kinases (8.Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar). The activated protein dimerizes, migrates to the nucleus, binds specificcis-acting elements, and activates transcription of IL-4 responsive genes (16.O'Shea J.J. Immunity. 1997; 7: 1-11Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 17.Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3400) Google Scholar, 18.Mikita T. Kurama M. Schindler U. J. Immunol. 1998; 161: 1822-1828PubMed Google Scholar). One of the best characterized IL-4 responsive elements is located in the germline ε promoter (7.Delphin S. Stavnezer J. J. Exp. Med. 1995; 181: 181-192Crossref PubMed Scopus (189) Google Scholar, 8.Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar). B-cells derived from STAT6 knock-out mice do not produce IgE in response to IL-4 treatment, illustrating that STAT6 is absolutely required for this event (19.Kuperman D. Schofield B. Wills-Karp M. Grusby M.J. J. Exp. Med. 1998; 187: 939-948Crossref PubMed Scopus (406) Google Scholar, 20.Hoey T. Schindler U. Curr. Opin. Genet. Dev. 1998; 8: 582-587Crossref PubMed Scopus (86) Google Scholar, 21.Miyata S. Matsuyama T. Kodama T. Nishioka Y. Kuribayashi K. Takeda K. Akira S. Sugita M. Clin. Exp. Allergy. 1999; 29: 114-123Crossref PubMed Scopus (68) Google Scholar). These observations suggest that STAT6 is an excellent target for therapeutic intervention in cases of allergic rhinitis and allergic asthma, which are often associated with hyper-IgE production. We recently conducted an extensive alanine scan mutagenesis of the STAT6 SH2 domain (22.Mikita T. Daniel C. Wu P. Schindler U. J. Biol. Chem. 1998; 273: 17634-17642Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These studies revealed amino acids that are critical for dimer formation and subsequent DNA binding. Consistent with the model that the STAT SH2 domain is also required for receptor interaction, the same amino acids were shown to be important for binding a tyrosine-phosphorylated peptide derived from the IL-4 receptor signaling chain. Furthermore, STAT6 mutants that were unable to bind DNA were also unable to activate transcription in response to IL-4 stimulation, whereas STAT6 derivatives that could bind DNA were transcriptionally active (22.Mikita T. Daniel C. Wu P. Schindler U. J. Biol. Chem. 1998; 273: 17634-17642Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In this study we have characterized one of these mutants (STAT6VT) in greater detail. STAT6VT differs from the wild-type in that it carries two amino acid changes in the SH2 domain. These mutations alter the confirmation of the protein such that it becomes phosphorylated, binds DNA, and activates transcription independent of IL-4 stimulation. This hyperactivation of STAT6, if found in vivo, could lead to increased IgE production. Hence, it will be interesting to see whether such a mutation occurs in patients suffering from allergic rhinitis and/or allergic asthma. HEK293 cells were cultured in Dulbecco's modified Eagle's medium/F12 (Meditech, Herndon, VA) supplemented with 10% fetal bovine serum. The human fibrosarcoma U4A cells (a gift from Dr. George Stark) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Human IL-4 (4.1 × 107 unit/mg) was purchased from R&D Systems (Minneapolis, MN). The IL-4 inducible luciferase reporter construct carries four copies of the IL-4 response element (C/EBP-N4 site) found in the human germline ε promoter. Preparation of this construct has been described previously (8.Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar). The mammalian expression plasmids for Jak1, the wild-type STAT6 protein as well as the mutant STAT6 derivatives, carrying two amino acid changes in the SH2 domain, have been described elsewhere (22.Mikita T. Daniel C. Wu P. Schindler U. J. Biol. Chem. 1998; 273: 17634-17642Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The triple mutant STAT6VT/Y was prepared using polymerase chain reaction to introduce two alanine residues at amino acid positions 547/548 and a phenylalanine at amino acid position 641. The fragment containing the indicated mutations was then used to replace the BglII/SacI fragment of TPU 389 (22.Mikita T. Daniel C. Wu P. Schindler U. J. Biol. Chem. 1998; 273: 17634-17642Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The presence of the mutations was confirmed by DNA sequence analysis. The baculovirus expression plasmids, wild-type STAT6 and STAT6VT, have also been described (8.Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar, 22.Mikita T. Daniel C. Wu P. Schindler U. J. Biol. Chem. 1998; 273: 17634-17642Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). HEK293 or U4A cells were transfected using calcium phosphate precipitation (Promega, Madison, WI). A control plasmid carrying the β-galactosidase gene under the cytomegalovirus promoter was co-transfected in each experiment. The cells were stimulated with IL-4 (10 ng/ml) 6 h before harvesting. Luciferase and β-galactosidase activities were determined 2 days post-transfection using the Promega assay system. Probes corresponding to the STAT6 binding site (N4) have been described previously (8.Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar). Nuclear extracts were prepared, and mobility shift assays were performed as described (12.Hou J. Schindler U. Henzel W.J. Ho T.Z. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (730) Google Scholar, 24.Osborn L. Kunkel S. Nable G.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1373) Google Scholar). Peptide binding studies using the tyrosine-phosphorylated IL-4 receptor peptide, ASSGEEGPY*KPFQDLI, have been described elsewhere (12.Hou J. Schindler U. Henzel W.J. Ho T.Z. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (730) Google Scholar, 24.Osborn L. Kunkel S. Nable G.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1373) Google Scholar). The same nuclear extracts were used for immunoprecipitations with an anti-STAT6-specific polyclonal antibody. The precipitated material was electrophoretically separated and subjected to anti-phosphotyrosine Western blots using the 4G10 monoclonal anitbody (Upstate Biotechnology). Wild-type STAT6 and STAT6VT were expressed in insect cells and purified to homogeneity as described previously (13.Schindler U. Wu P. Rothe M. Brasseur M. McKnight S.L. Immunity. 1995; 2: 689-697Abstract Full Text PDF PubMed Scopus (233) Google Scholar). 30 μg of each protein were exposed to 3 μg of activated trypsin (Sigma) protease. The reaction was incubated at 37 °C in 1× digestion buffer (20 mm Tris, pH 7.2, 1 mm dithiothreitol). 10 μl aliquots were removed every 20 min for analysis by SDS-polyacrylamide gel electrophoresis. For each reaction, a negative control was performed without protease. Previously, we generated a series of STAT6 mutants that differ from the wild type in that they carry two amino acid substitutions in the SH2 domain (22.Mikita T. Daniel C. Wu P. Schindler U. J. Biol. Chem. 1998; 273: 17634-17642Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In the present study, we have compared the transcription potential of these mutants in the presence and absence of cytokine. The proteins were co-expressed in HEK293 cells with an IL-4 inducible luciferase reporter carrying four copies of the STAT6-dependent IL-4 response element derived from the human germline ε promoter. HEK293 cells lack endogenous STAT6, but they are able to activate an IL-4 responsive promoter upon ectopic expression of STAT6, suggesting that this is the only component missing in the IL-4/STAT6 signaling cascade (8.Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar). As observed previously, all STAT6 derivatives that were known to bind DNA also activated transcription (Fig. 1). With one exception, all proteins were active only in the presence of IL-4 and were not active without cytokine stimulation. Strikingly, the substitution of two residues at positions 547 (V) and 548 (T) in the SH2 domain resulted in a STAT6 mutant that was transcriptionally active even in the absence of IL-4 stimulation (VT, Fig. 1). In addition, this protein was about three times more active than wild-type STAT6 in the induced state. These data suggested that mutations of these two amino acids (VT) dramatically affect the IL-4-dependent regulation of STAT6. To explore the mechanism underlying the constitutive activity of STAT6VT, we asked whether the protein is able to dimerize and bind DNA without IL-4 stimulation. STAT6 or STAT6VT were expressed in HEK293 cells, and nuclear extracts were prepared from IL-4-treated or untreated cells and subjected to DNA binding studies using the IL-4 response element. Fig.2 shows that the DNA binding activity of wild-type STAT6 can be seen only upon IL-4 stimulation (lane 2), but not under unstimulated conditions (lane 1). In contrast, STAT6VT was able to bind the IL-4 response element even in the absence of cytokine (lane 5). Some increase in binding was observed after IL-4 treatment (lane 6). These results show that the double amino acid substitution yields a protein that binds DNA and activates transcription in an IL-4-independent manner. Phosphorylation of Tyr-641 is critical for STAT6 dimerization and DNA binding (8.Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar). To explore whether Tyr-641 phosphorylation is required in the context of the V547A/T548A mutation, we substituted tyrosine 641 with phenylalanine (STAT6VT/Y) and analyzed the protein using DNA binding. Fig. 2 A shows that the tyrosine mutation abolished DNA binding activity even in the presence of IL-4 stimulation (lanes 7 and 8). STAT6Y carrying the Tyr-641 mutation in the context of the wild-type was used as control (lanes 3 and 4). We also investigated the phosphorylation status of these proteins in the absence and presence of IL-4 stimulation using immunoprecipitations followed by anti-phosphotyrosine immunoblotting. Fig. 2 Bshows that STAT6VT is tyrosine-phosphorylated in the absence of IL-4 treatment (lane 5). Consistent with the DNA binding studies, some increase in tyrosine phosphorylation was seen upon IL-4 stimulation (lane 6). In contrast, no phosphorylation of the wild-type protein was seen in the uninduced state, whereas strong phosphorylation was seen following IL-4 treatment (lanes 1and 2). Phosphorylation was completely abolished when Tyr-641 was replaced by phenylalanine in the context of the wild-type (lanes 3 and 4) or the mutant protein (lanes 7 and 8). We also asked whether these proteins were able to activate transcription when expressed in HEK293 cells (Fig. 2 C). Both STAT6Y and STAT6VT/Y were unable to activate transcription even after IL-4 stimulation. In contrast, STAT6 and STAT6VT were fully active. These studies clearly show that Tyr-641 is absolutely essential for STAT6 function even in the context of the V547A/T548A mutation. STAT6VT is not able to utilize an alternative tyrosine residue for dimerization nor to bind DNA or activate transcription in the absence of tyrosine phosphorylation. IL-4 binding to its receptor triggers the phosphorylation of tyrosines in the intracellular domain of the IL-4 receptor α chain, which allow the specific recruitment of STAT6 (8.Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar, 12.Hou J. Schindler U. Henzel W.J. Ho T.Z. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (730) Google Scholar, 13.Schindler U. Wu P. Rothe M. Brasseur M. McKnight S.L. Immunity. 1995; 2: 689-697Abstract Full Text PDF PubMed Scopus (233) Google Scholar). So far, we have demonstrated that phosphorylation of STAT6VT is required but also that this event is independent of IL-4 binding to its receptor. Hence, one could argue that the gain-of-function phenotype seen with STAT6VT is due to a structural change mediated by the two amino acid substitutions. To address the possibility of conformational change, we compared the stability of STAT6 and STAT6VT by limited proteolysis (Fig.3 A). Recombinant proteins were expressed in insect cells, purified to homogeneity (lane 1), and exposed to trypsin protease for increasing incubation periods (lanes 2-9). The results clearly demonstrate that STAT6VT is more resistant to protease digestion. Even after prolonged exposure to the protease, STAT6VT did not generate the complex digestion pattern obtained for wild-type STAT6. This finding suggests that the two amino acid changes in the SH2 domain alter the conformation of the protein. Tyrosine-phosphorylated peptides derived from the IL-4 receptor α chain inhibit STAT6 DNA binding because they interact with the SH2 domain, which is required for dimer formation (12.Hou J. Schindler U. Henzel W.J. Ho T.Z. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (730) Google Scholar, 13.Schindler U. Wu P. Rothe M. Brasseur M. McKnight S.L. Immunity. 1995; 2: 689-697Abstract Full Text PDF PubMed Scopus (233) Google Scholar). The transcriptional activity of STAT6VT is the same in the induced and uninduced state even though its phosphorylation status and DNA binding activity can still be increased upon IL-4 stimulation. Hence, we wished to determine whether the V547A/T548A mutation stabilizes the dimer upon DNA binding. Proteins expressed in HEK293 cells were subjected to DNA binding in the presence or absence of tyrosine-phosphorylated receptor peptide (Fig. 3 B). In agreement to previous results (13.Schindler U. Wu P. Rothe M. Brasseur M. McKnight S.L. Immunity. 1995; 2: 689-697Abstract Full Text PDF PubMed Scopus (233) Google Scholar), the peptide inhibited DNA binding of the wild-type STAT6 protein in a dose-dependent manner (lanes 2-4). Interestingly, the peptide had no effect on DNA binding of the mutant STAT6VT protein (lanes 7-9). Using fluorescence polarization experiments (13.Schindler U. Wu P. Rothe M. Brasseur M. McKnight S.L. Immunity. 1995; 2: 689-697Abstract Full Text PDF PubMed Scopus (233) Google Scholar) we demonstrated that the monomeric wild-type and mutant proteins bound this peptide with the same affinity (K d ∼ 0.3 μm). The failure of IL-4 receptor-derived peptides to displace the STAT6VT dimer as it is seen for the wild-type protein strongly suggests that the STAT6VT dimer is more stable. These results help explain the high transcriptional activity in unstimulated cells (Fig. 1). The data indicate that receptor ligation is not required for the activation of STAT6VT. However, tyrosine phosphorylation at residue Tyr-641 is absolutely essential. HEK293 cells do not express Jak3, demonstrating that this kinase is not necessary for STAT6 phosphorylation. We next determined whether the function of Jak1, which is associated with the IL-4 receptor α chain, is required for STAT6VT activation. In these studies we characterized the behavior of wild-type STAT6 and STAT6VT in U4A cells, a Jak1-deficient fibroblast cell line (25.McKendry R. John J. Flavell D. Muller M. Kerr I.M. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11455-11459Crossref PubMed Scopus (229) Google Scholar, 26.Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5061) Google Scholar). The parental line 2fTGH served as positive control. First, we compared the transcription activation potentials of STAT6 and STAT6VT in both 2fTGH and U4A cells. Fig. 4 A shows that IL-4-induced luciferase activity could be detected in 2fTGH but not in U4A. Overexpression of wild-type STAT6 increased the activity in 2fTGH about 2-fold but had no effect in U4A cells, supporting the notion that Jak1 is absolutely essential for STAT6 function. In contrast, strong luciferase activity was observed in 2fTGH and U4A cells upon overexpression of STAT6VT. Again, transcription activation was seen in the absence of cytokine stimulation. For reasons we do not understand, STAT6VT was even more active in U4A cells than in the parental line, 2fTGH. However, the data shows that STAT6VT is constitutively active even in the absence of Jak1 and Jak3. Next we wished to determine the DNA binding activity of both proteins when expressed in U4A or 2fTGH cells. Both cell lines were transfected with constructs encoding either wild-type STAT6 or STAT6VT. Nuclear extracts were prepared and assayed for their ability to associate with a STAT6-specific DNA binding element (Fig. 4 B). In agreement with our activation studies and earlier data, wild-type STAT6 protein could not be activated in U4A cells because of the lack of Jak1 (top panel, lane 4) (26.Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5061) Google Scholar, 27.Haque S.J. Wu Q. Kammer W. Friedrich K. Smith J.M. Kerr I.M. Stark G.R. Williams B.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8563-8568Crossref PubMed Scopus (65) Google Scholar). However, the protein was active upon IL-4 stimulation in the parental line 2fTGH (bottom panel, lane 4). Furthermore, when Jak1 was co-expressed, IL-4-induced activation of wild-type STAT6 was reconstituted in U4A cells (top panel, lane 10). In contrast, STAT6VT was active in both the parental line 2fTGH as well as the mutant U4A line independent of whether Jak1 was co-transfected (lanes 6 and 12). As observed in HEK293 cells, activation of STAT6VT did not require cytokine stimulation. Taken together, these findings clearly demonstrate that STAT6VT is phosphorylated by a kinase that does not need to be activated by IL-4 stimulation. We have identified two amino acids in the STAT6 SH2 domain that, when mutated, uncouple the activity of the STAT6 protein from its regulatory cytokine, IL-4. Strikingly, this mutant, STAT6VT, is tyrosine-phosphorylated and is able to bind DNA and activate transcription in the absence of IL-4. Using limited proteolysis, we show that STAT6VT expressed in insect cells is more resistant to protease digestion than the wild-type protein. This observation does not reflect the difference in tyrosine phosphorylation, because STAT6VT is not differentially phosphorylated in insect cells when compared with the wild-type protein (data not shown). Hence, we argue that the two amino acid changes stabilize STAT6VT so that it is less susceptible to protease digestion. In mammalian cells, this change in conformation seems to give rise to a protein that is phosphorylated by an IL-4-independent tyrosine kinase. These results are supported by the observation that Jak1- and Jak3-deficient cells are able to phosphorylate STAT6VT but fail to phosphorylate the wild-type protein. Furthermore, STAT6VT forms a more stable protein:DNA complex than the wild-type protein. These two mechanisms, phosphorylation in the absence of IL-4 and increased DNA binding stability, help to explain the constitutive transcription activity seen with STAT6VT. Obviously, these studies do not rule out the possibility that wild-type STAT6 is also phosphorylated by this IL-4-independent kinase. In this scenario, STAT6 may be rapidly dephosphorylated in the absence of IL-4 stimulation. This phosphatase may be unable to recognize STAT6VT because of the two amino acid changes. Our results do not allow us to discriminate between these two potential mechanisms. Constitutively active versions of STAT1 and STAT5 have been identified (28.Shuai K. Liao J. Song M.M. Mol. Cell. Biol. 1996; 16: 4932-4941Crossref PubMed Scopus (132) Google Scholar, 29.Onishi M. Nosaka T. Misawa K. Mui A.L.-F. Gorman D. McMahon M. Miyajima A Ktamura T. Mol. Cell. Biol. 1998; 18: 3871-3879Crossref PubMed Scopus (349) Google Scholar). In the case of STAT1, it was shown that a mutation of amino acid 31 (Arg to Ala) inhibits the dephosphorylation of STAT1. Furthermore, a deletion of the N terminus resulted in a protein that was constitutively phosphorylated (28.Shuai K. Liao J. Song M.M. Mol. Cell. Biol. 1996; 16: 4932-4941Crossref PubMed Scopus (132) Google Scholar). A similar mechanism does not apply for STAT6 because deletion of the N terminus has no effect on the activation/phosphorylation properties of STAT6 (8.Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar). Two amino acids have been identified in STAT5 that, when mutated simultaneously, yield a constitutively active STAT5 protein (29.Onishi M. Nosaka T. Misawa K. Mui A.L.-F. Gorman D. McMahon M. Miyajima A Ktamura T. Mol. Cell. Biol. 1998; 18: 3871-3879Crossref PubMed Scopus (349) Google Scholar). One of these resides is upstream of the DNA binding domain and the other is located in the C-terminal activation domain. Also for STAT6VT, both amino acid changes were required to observed the full constitutive phenotype. Hence, it appears that different mutations can yield STAT proteins that are either phosphorylated in a ligand-independent manner or that render them less susceptible to dephosphorylation. Inappropriate activation of STAT proteins has been described for some pathological conditions. For example, STAT3 and STAT5 are constitutively active in HTLV-1 transformed T-cells (30.Migone T.S. Lin J.X. Cereseto A. Mulloy J.C. O'Shea J.J. Franchini G. Leonard W.J. Science. 1995; 269: 79-81Crossref PubMed Scopus (507) Google Scholar). STAT3 has also been shown to be activated in a cytokine-independent manner by Src, resulting in cell transformation (31.Campbell G.S., Yu, C.L. Jove R. Carter-Su C. J. Biol. Chem. 1997; 272: 2591-2594Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 32.Turkson J. Bowman T. Garcia R. Caldenhoven E. De Groot R.P. Jove R. Mol. Cell. Biol. 1998; 18: 2545-2552Crossref PubMed Scopus (594) Google Scholar, 33.Bromberg J.F. Horvath C.M. Besser D. Lathem W.W. Darnell Jr., J.E. Mol. Cell. Biol. 1998; 18: 2553-2558Crossref PubMed Scopus (571) Google Scholar). Furthermore, constitutively active STAT3 is found in bone marrow mononuclear cells from patients with multiple myelomas and is thought to contribute to pathogenesis by preventing apoptosis (34.Catlett-Falcone R. Landowski T.H. Oshiro M.M. Turkson J. Levitzki A. Savino R. Ciliberto G. Moscinski L. Fernandez-Luna J.L. Nunez G. Dalton W.S. Jove R. 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Our finding that the mutation of two key residues within the STAT6 SH2 domain results in a protein that is constitutively active provides the first reported evidence of a hyperactive form of STAT6. Asthma is familial, and many genetic loci predispose individuals to the disease (38.Daniels S.E. Bhattacharrya S. James A. Leaves N.I. Young A. Hill M.R. Faux J.A. Ryan G.F. le Souef P.N. Lathrop G.M. Musk A.W. Cookson W.O. Nature. 1996; 383: 247-250Crossref PubMed Scopus (712) Google Scholar). Furthermore, asthma as well as allergy are multifactorial in that they are influenced by many genetic and environmental factors. IL-4 is a critical cytokine in the development of atopy, the IgE-mediated syndrome of allergic asthma and rhinitis (39.Marsh D.G. Neely J.D. Breazeale D.R. Ghosh B. Freidhoff L.R. Ehrlich-Kautzky E. Schou C. Krishnaswamy G. Beaty T.H. Science. 1994; 264: 1152-1156Crossref PubMed Scopus (953) Google Scholar). 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