Mutational Analysis of the STAT6 SH2 Domain
1998; Elsevier BV; Volume: 273; Issue: 28 Linguagem: Inglês
10.1074/jbc.273.28.17634
ISSN1083-351X
AutoresThomas Mikita, Carla Daniel, Pengguang Wu, Ulrike Schindler,
Tópico(s)Medicinal Plant Pharmacodynamics Research
ResumoThe SH2 domain of the STAT family of transcription factors is essential for STAT binding to phosphorylated cytoplasmic domains of activated cytokine receptors. Furthermore, the same domain mediates dimerization of activated STAT monomers, a prerequisite for DNA binding by this family of proteins. To identify amino acid residues within the STAT protein that mediate these various interactions, we have carried out an extensive mutational analysis of the Stat6 SH2 domain. Recombinant proteins carrying C-terminal deletions or double alanine substitutions were expressed in mammalian and insect cells and assayed for DNA binding, transcription activation, tyrosine phosphorylation, and the ability to interact with a tyrosine-phosphorylated peptide derived from the interleukin-4 receptor signaling chain. From these studies, we have identified amino acids that are required for both DNA binding and interleukin-4 receptor interaction, as well as residues that when mutated impair only one of the two functions. Our results suggest that the structural homology between the SH2 domain of Stat6 and that of the distantly related Src protein may be higher than predicted on the basis of primary amino acid sequence comparisons. However, the two types of SH2 domains may differ at their C-terminal ends. The SH2 domain of the STAT family of transcription factors is essential for STAT binding to phosphorylated cytoplasmic domains of activated cytokine receptors. Furthermore, the same domain mediates dimerization of activated STAT monomers, a prerequisite for DNA binding by this family of proteins. To identify amino acid residues within the STAT protein that mediate these various interactions, we have carried out an extensive mutational analysis of the Stat6 SH2 domain. Recombinant proteins carrying C-terminal deletions or double alanine substitutions were expressed in mammalian and insect cells and assayed for DNA binding, transcription activation, tyrosine phosphorylation, and the ability to interact with a tyrosine-phosphorylated peptide derived from the interleukin-4 receptor signaling chain. From these studies, we have identified amino acids that are required for both DNA binding and interleukin-4 receptor interaction, as well as residues that when mutated impair only one of the two functions. Our results suggest that the structural homology between the SH2 domain of Stat6 and that of the distantly related Src protein may be higher than predicted on the basis of primary amino acid sequence comparisons. However, the two types of SH2 domains may differ at their C-terminal ends. Tyrosine phosphorylation and specific recognition of these phospho-residues by SH2 1The abbreviations used are: SH2, Src homology domain; aa, amino acids; PCR, polymerase chain reaction; STAT, signal transducer and activator of transcription; IFN, interferon. domain-containing proteins are critical features of many cellular signaling pathways. Hence, this protein-protein interaction domain has been the focus of many studies (1Birge R.B. Hanafusa H. Science. 1993; 262: 1522-1524Crossref PubMed Scopus (66) Google Scholar, 2Pawson T. Gish G.D. Cell. 1992; 71: 359-362Abstract Full Text PDF PubMed Scopus (796) Google Scholar). The best characterized class of SH2 containing proteins is the Src family. Structural analysis of the Src SH2 domain bound to a high affinity phosphopeptide revealed three key features necessary for selective protein recognition (3Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B.J. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Nature. 1992; 358: 646-653Crossref PubMed Scopus (575) Google Scholar, 4Waksman G. Shoelson S.E. Pant N. Cowburn D. Kuriyan J. Cell. 1993; 72: 779-790Abstract Full Text PDF PubMed Scopus (656) Google Scholar). First, phosphotyrosine binding is mediated through a mostly polar pocket that contains the conserved GTFLLR motif found in most SH2 domains. Second, a β-sheet structure interacts with the first two amino acids (i + 1 and i + 2) immediately C-terminal to the phosphotyrosine residue. Third, the i + 3 residue is recognized specifically by a second, more hydrophobic pocket (4Waksman G. Shoelson S.E. Pant N. Cowburn D. Kuriyan J. Cell. 1993; 72: 779-790Abstract Full Text PDF PubMed Scopus (656) Google Scholar). Thus, binding selectivity is determined largely by the three amino acids following the phosphotyrosine and specific residues in the interacting SH2 domain (5Bibbins K.B. Boeuf H. Varmus H.E. Mol. Cell. Biol. 1993; 13: 7278-7287Crossref PubMed Scopus (108) Google Scholar, 6Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2391) Google Scholar). In general these features are shared by all SH2 domain-containing proteins for which structural information is available (6Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2391) Google Scholar, 7Marengere L.E.M. Songyang Z. Gish G.D. Schaller M.D. Parsons J.T. Stern M.J. Cantley L.C. Pawson T. Nature. 1994; 369: 502-505Crossref PubMed Scopus (164) Google Scholar, 8Pascal S.M. Singer A.U. Gish G. Yamazaki T. Shoelson S.E. Pawson T. Kay L.E. Forman-Kay J.D. Cell. 1994; 77: 461-472Abstract Full Text PDF PubMed Scopus (230) Google Scholar, 9Songyang Z. Gish G. Mbamalu G. Pawson T. Cantley L.C. J. Biol. Chem. 1995; 270: 26029-26032Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The STAT proteins are the only transcription factors known to contain SH2 domains (10Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1657) Google Scholar). Thus far, seven STAT proteins have been characterized. Some are activated by multiple cytokines or growth factors, whereas others are only activated in response to a specific stimulus (11Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar, 12Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar, 13Lin J.X. Migone T.S. Tsang M. Friedmann M. Weatherbee J.A. Zhou L. Yamauchi A. Bloom E.T. Mietz J. John S. Leonard W.J. Immunity. 1995; 2: 331-339Abstract Full Text PDF PubMed Scopus (678) Google Scholar, 14O'Shea J.J. Immunity. 1997; 7: 1-20Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). Activation of STAT proteins involves cytokine binding to its receptor, which triggers tyrosine phosphorylation of the intracellular receptor domain by an associated Jak kinase (10Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1657) Google Scholar, 15Ihle J.N. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1145) Google Scholar, 16Yan H.M. Krishnan K. Greenlund A.C. Gupta S. Lim J.T.L. Schreiber R.D. Schindler C.W. Krolewski J.J. EMBO J. 1996; 15: 1064-1074Crossref PubMed Scopus (160) Google Scholar). The phosphorylated receptor chain provides a docking site for the latent STAT protein, which resides in the cytoplasm. Once recruited to the receptor, the STAT protein is phosphorylated at a single tyrosine residue by Jak kinase (12Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar). Phosphorylated STAT monomers dimerize, translocate to the nucleus, and modulate transcription through STAT-specific DNA sequence elements (11Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar, 17Ivashkiv L.B. Immunity. 1995; 3: 1-4Abstract Full Text PDF PubMed Scopus (105) Google Scholar). Receptor-associated Jak kinases are relatively nonselective in their ability to phosphorylate individual cytokine receptors and/or STAT proteins (18Heim M.H. Kerr I.M. Stark G.R. Darnell Jr., J.E. Science. 1995; 267: 1347-1349Crossref PubMed Scopus (352) Google Scholar, 19Kohlhuber F. Rogers N.C. Watling D. Feng D. Guschin D. Briscoe J. Witthuhn B.A. Kotenko S.V. Pestka S. Stark G.R. Ihle J.N. Kerr I.M. Mol. Cell. Biol. 1997; 17: 695-706Crossref PubMed Scopus (176) Google Scholar). Furthermore, many STAT proteins, once activated, bind to similar DNA motifs, although not all STAT proteins can activate transcription from the same motif (11Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar, 17Ivashkiv L.B. Immunity. 1995; 3: 1-4Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 20Horvath C.M. Wen Z. Darnell Jr., J.E. Genes Dev. 1995; 9: 984-994Crossref PubMed Scopus (452) Google Scholar, 21Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar, 22Schindler U. Wu P. Rothe M. Brasseur M. McKnight S.L. Immunity. 1995; 2: 689-697Abstract Full Text PDF PubMed Scopus (233) Google Scholar, 23Seidel H.M. Milocco L.H. Lamb P. Darnell Jr., J.E. Stein R.B. Rosen J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3041-3045Crossref PubMed Scopus (383) Google Scholar). Selectivity in gene activation upon stimulation with different cytokines appears to be achieved at the STAT-receptor interaction (11Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar, 18Heim M.H. Kerr I.M. Stark G.R. Darnell Jr., J.E. Science. 1995; 267: 1347-1349Crossref PubMed Scopus (352) Google Scholar, 24Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell Jr., J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1352Crossref PubMed Scopus (869) Google Scholar). This interaction is mediated by the STAT-SH2 domain, which also dictates the specificity in STAT:STAT dimer formation (25Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (731) Google Scholar, 26Li X. Leung S. Qureshi S. Darnell Jr., J.E. Stark G.R. J. Biol. Chem. 1996; 271: 5790-5794Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 27Shuai K. Horvath C.M. Huang L.H.T. Qureshi S.A. Cowburn D. Darnell Jr., J.E. Cell. 1994; 76: 821-828Abstract Full Text PDF PubMed Scopus (687) Google Scholar). The selectivity of Stat1 for the IFN-γ receptor can be transferred to Stat2 via the SH2 domain, suggesting that the STAT-SH2 domain is a modular structure as are other SH2 domains (2Pawson T. Gish G.D. Cell. 1992; 71: 359-362Abstract Full Text PDF PubMed Scopus (796) Google Scholar, 18Heim M.H. Kerr I.M. Stark G.R. Darnell Jr., J.E. Science. 1995; 267: 1347-1349Crossref PubMed Scopus (352) Google Scholar, 28Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1444) Google Scholar). Strikingly, STAT-SH2 domains share little sequence similarity with other SH2 domains (Fig. 1). Most of the similarity is restricted to the N-terminal half which contains the conserved GTFLLR motif (4Waksman G. Shoelson S.E. Pant N. Cowburn D. Kuriyan J. Cell. 1993; 72: 779-790Abstract Full Text PDF PubMed Scopus (656) Google Scholar). As with other SH2 domains, mutation of the invariant arginine leads to loss of phosphotyrosine recognition in STATs (5Bibbins K.B. Boeuf H. Varmus H.E. Mol. Cell. Biol. 1993; 13: 7278-7287Crossref PubMed Scopus (108) Google Scholar, 21Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar, 29Mayer B.J. Jackson P.K. Van Etten R.A. Baltimore D. Mol. Cell. Biol. 1992; 12: 609-618Crossref PubMed Scopus (238) Google Scholar, 30Qureshi S.A. Leung S. Kerr I.A. Stark G.R. Darnell Jr., J.E. Mol. Cell. Biol. 1996; 16: 288-293Crossref PubMed Scopus (149) Google Scholar). Specific peptide recognition is mediated by residues in the C-terminal half of the SH2 domain (4Waksman G. Shoelson S.E. Pant N. Cowburn D. Kuriyan J. Cell. 1993; 72: 779-790Abstract Full Text PDF PubMed Scopus (656) Google Scholar, 6Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2391) Google Scholar) where little homology exists between the STAT-SH2 and other SH2 domains (Fig. 1). In addition, no structural information is available for any of the STAT-SH2 domains. Given the involvement of this domain in selective protein-protein interactions, we sought to define aspects of this domain that participate in achieving the fidelity of the Jak/STAT signaling pathway. We are interested primarily in the IL-4-inducible protein, Stat6. To determine the C terminus of the Stat6 SH2 domain, we generated a series of C-terminal deletion mutants. To identify residues critical for Stat6 function, we carried out a systematic mutational analysis of the SH2 domain by changing two amino acids at a time in the context of the full-length protein. Recombinant mutant proteins were tested for DNA binding, tyrosine phosphorylation, and transcription activation. Proteins were also tested for their ability to interact with a tyrosine-phosphorylated peptide derived from the IL-4 receptor. Mutants that were unable to bind DNA but did interact with the receptor-derived peptide could partially inhibit IL-4-induced gene expression when overexpressed in cells that contain endogenous Stat6. Our analysis provides insight into the structure and function of the Stat6 SH2 domain. BJAB cells were grown in RPMI 1640(1×) (Hyclone) supplemented with 10% fetal calf serum (PAA Laboratories), 2 mml-glutamine (Life Technologies, Inc.), and 5 × 10−5mβ-mercaptoethanol. Stably transfected BJAB cell lines were grown in the same media supplemented with 1 mg/ml G418 (Sigma). BJAB cells were stably transfected with the use of electroporation as described by Tewari and Dixit (31Tewari M. Dixit V.M. J. Biol. Chem. 1995; 270: 3255-3260Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar). Human embryonic kidney 293 cells and HepG2 cells were grown in Dulbecco's modified Eagle's medium (Mediatech) containing 10% fetal calf serum. Transfections in embryonic kidney 293 cells and HepG2 cells were carried out as described (21Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar). Luciferase and β-galactosidase activity was determined 48 h following transfection using the Promega assay systems. The cells were stimulated with 5 ng/ml IL-4 6 h before harvesting. Expression constructs for full-length Stat6 (TPU272) and Stat6ΔC (TPU285) carrying a stop codon at position 662 have been described previously (21Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar, 22Schindler U. Wu P. Rothe M. Brasseur M. McKnight S.L. Immunity. 1995; 2: 689-697Abstract Full Text PDF PubMed Scopus (233) Google Scholar). All C-terminal deletion mutants (aa 602, TPU601; aa 620, TPU600; aa 633, TPU599; aa 636, TPU598; aa 641, TPU597; aa 645, TPU596; aa 650, TPU595) were generated by replacing the XmaI/SacI fragment of TPU285 with DNA fragments that carried stop codons at the appropriate residues. The fragments were generated with the polymerase chain reaction (PCR). Double amino acid substitutions in the Stat6-SH2 domain were generated by substituting theXmaI/SacI fragment of TPU595 with mutated DNA fragments that were prepared by PCR. The integrity of the resulting clones was determined by DNA sequence analysis. All proteins carried nine histidine residues and a pentapeptide substrate (RRASV) for protein kinase A at their C terminus. With the exception of three mutants (WS, FS, and LY), all proteins were expressed in Hi-5 cells and purified using Ni2+ affinity chromatography (22Schindler U. Wu P. Rothe M. Brasseur M. McKnight S.L. Immunity. 1995; 2: 689-697Abstract Full Text PDF PubMed Scopus (233) Google Scholar). The expression construct for full-length Stat6 (TPU389) differs from the previously described construct TPU388 (20Horvath C.M. Wen Z. Darnell Jr., J.E. Genes Dev. 1995; 9: 984-994Crossref PubMed Scopus (452) Google Scholar) in that a flag epitope tag was introduced at the C terminus. All C-terminal deletion constructs (aa 602, TPU608; aa 620, TPU607; aa 636, TPU605; aa 641, TPU604; aa 645, TPU603; aa 650, TPU602) were generated by replacing the BglII/SpeI fragment of TPU389 with a DNA fragment carrying the indicated 3′ deletion. Double alanine mutants were prepared by replacing the BglII/SacI fragment of TPU389 with a DNA fragment that carried the indicated mutation. In each case, the fragments were generated by PCR, and the integrity of the clones was determined by DNA sequence analysis. Mutants WS, FS, and LY failed to express in insect cells and were consequently purified from stably transfected BJAB cells. Proteins expressed in mammalian cells contained C-terminal flag epitope tags, which allowed purification on anti-flag M2 affinity resin (Kodak). BJAB (108) cells were resuspended in lysis buffer (30 ml; 0.2m NaCl, 30 mm Hepes, pH 7.6, 10% glycerol, 0.1% Nonidet P-40, 1 mm EDTA), incubated on ice for 10 min, and sonicated and clarified by centrifugation at 10,000 ×g for 10 min. The supernatant was incubated with anti-flag M2 (1 ml) resin for 1 h at 4 °C. Unbound material was washed from the resin with lysis buffer (50 ml). Bound protein was eluted with flag peptide (400 ng/ml). Biotinylated peptides (2.5 mmol: IL-4R peptide, ASSGEEGPYKPFQDLI, or IFN-γ peptide, GGGGGFGYPDKPHVL) were coupled to 25 μl of packed streptavidin-agarose beads (Sigma) in 0.5 ml of binding buffer (0.1 m NaCl, 30 mmHepes, pH 7.6, 10% glycerol, 0.1% Nonidet P-40, 1 mmEDTA) for 30 min at 4 °C. Unbound peptide was removed by washing the resin 4 times with binding buffer (1 ml). Purified protein (20 μg) was incubated with peptide-coupled streptavidin beads (25 μl in a final volume of 500 μl binding buffer) for 90 min at 4 °C. Unbound protein was removed by washing the resin 4 times with binding buffer (1 ml) for 10 min each. Bound protein was eluted with 50 μl of SDS sample buffer, and 5 μl were subjected to Western analysis with antibodies directed against Stat6. An aliquot of the starting material (25 μl) was separated on an SDS-polyacrylamide gel, and proteins were visualized by Coomassie R-250 staining to ensure that the input was equivalent for each binding reaction. Purified Stat6 was activated in vitrowith Jak1 kinase (both Stat6 and Jak1 were expressed and purified from insect cells (13Lin J.X. Migone T.S. Tsang M. Friedmann M. Weatherbee J.A. Zhou L. Yamauchi A. Bloom E.T. Mietz J. John S. Leonard W.J. Immunity. 1995; 2: 331-339Abstract Full Text PDF PubMed Scopus (678) Google Scholar)). Phosphorylation conditions were 10 mmHepes, pH 7.4, 50 mm NaCl, 50 mmMgCl2, 50 μm ATP, 0.1 mmNa3VO4, 0.5 μg of Jak1, and 1 μg of Stat6 in a 50-μl reaction volume. Reactions were incubated at room temperature for 30 min. Typically, 1 μl of the reaction was used for mobility shift assays. Nuclear extract preparations and mobility shift assays have been described previously (21Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar). BJAB cells (1.5 × 10 6) stably expressing wild-type Stat6 or mutant derivatives or 293 cells (2 × 106) transiently transfected with Stat6 C-terminal deletion mutants were either treated or not treated with IL-4 for 15 min. Cells were lysed in immune precipitation buffer (50 mm Hepes, pH 7.9, 200 mm NaCl, 1 mm EDTA, 1 mmdithiothreitol, 10% glycerol, and 0.5% Nonidet P-40), and recombinant STAT proteins were immune-precipitated with anti-flag M2-coupled beads. The beads were washed 5 times with immune precipitation buffer, and bound proteins were eluted in SDS sample buffer (50 μl). Proteins were subjected to Western blots with anti-phosphotyrosine antibody (4G10, Upstate Biotechnology) and anti-flag M2 antibody (Kodak). A sequence alignment of the Stat6 and Src SH2 domains is shown in Fig. 1. The conserved tryptophan residue constitutes the N-terminal border of the Src SH2 domain. The GTFLLR motif, which is involved in phosphotyrosine recognition, represents the most conserved region (28Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1444) Google Scholar). Some sequence identity exists N-terminal to the GTFLLR motif, whereas very little similarity is observed in the C-terminal half. Thus it is impossible to determine the C-terminal border of the Stat6 SH2 domain on the basis of sequence similarity. We showed previously that a truncated version of Stat6 (amino acids (aa) 1–661) that lacks the C-terminal 186 aa binds with wild-type affinity to a tyrosine-phosphorylated peptide derived from the IL-4 receptor (21Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar). This mutant retains the tyrosine residue (aa 641) critical for phosphorylation and dimerization; hence, the protein is able to bind DNA when activated in cells. In order to define more precisely the C-terminal border of the Stat6 SH2 domain, we generated a series of C-terminal deletion mutants by inserting a flag epitope tag and a stop codon at various positions between aa 602 and 650 (Fig. 1). The proteins were expressed in embryonic kidney 293 cells and assayed for DNA binding upon IL-4 stimulation. The 293 cells lack endogenous Stat6 protein but express all other components of the IL-4 signaling pathway. Therefore, any IL-4-inducible DNA binding activity is due to the recombinant Stat6 protein (21Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar). Fig. 2 A shows the DNA binding activity of nuclear extracts prepared from IL-4-treated 293 cells transiently expressing the truncated Stat6 proteins. Only the wild-type protein and the mutant ending with aa 650 bound DNA. Mutants ending with aa 645, 641, and 636 did not bind DNA, although the proteins were expressed at the same level as the wild-type (Fig. 2 B). Proteins ending with aa 620 and 602 were not expressed in mammalian cells, suggesting that these truncated proteins are unstable (data not shown). These results indicate that only the mutant ending at aa 650 is, upon phosphorylation, able to dimerize, translocate to the nucleus, and bind DNA. Mutant proteins (ending with aa 645 and 641) retain the critical tyrosine residue but were unable to bind DNA. Hence, we investigated whether these truncated proteins became tyrosine-phosphorylated upon cytokine stimulation. Proteins were immune-precipitated from IL-4-treated 293 cells and probed with anti-phosphotyrosine antibodies (Fig. 2 C). Only the DNA-binding positive derivative ending with aa 650 was tyrosine-phosphorylated. The absence of phosphorylation with mutants 645, 641, and 635 showed that no nonspecific tyrosine phosphorylation occurs in response to IL-4. Furthermore, the data suggest that the 9 aa following the critical tyrosine (aa 641) are required for proper cytokine-induced Stat6 activation. This lack of phosphorylation observed in mammalian cells might reflect an unproductive interaction between the truncated Stat6 proteins and the Jak kinase when both are bound to the IL-4 receptor. To resolve this issue, we investigated whether these same mutants could bind DNA when activated in vitro. The proteins were expressed in insect cells, purified to homogeneity, and tyrosine-phosphorylatedin vitro with recombinant Jak1. Again, a deletion mutant ending at aa 650 was able to bind DNA, whereas Stat6 proteins truncated at 641 and 636 were inactive (Fig. 2 D). However, while deletion to aa 645 blocked Stat6 phosphorylation in IL-4-treated 293 cells, this mutant was activated in vitro to bind DNA. Thus, the 9 aa following tyrosine 641 are essential for Stat6 activationin vivo, whereas only four of these residues are required for in vitro activation. We next assessed the C-terminal boundary of the SH2 domain with respect to peptide binding. We previously identified two tyrosine-containing peptides derived from the IL-4 receptor signaling chain that, when phosphorylated (YPKAFS and YPKPFQ), associate specifically with Stat6 (25Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (731) Google Scholar). Similarly, a tyrosine-phosphorylated peptide derived from the interferon-γ receptor bound specifically to Stat1 but not Stat6 (22Schindler U. Wu P. Rothe M. Brasseur M. McKnight S.L. Immunity. 1995; 2: 689-697Abstract Full Text PDF PubMed Scopus (233) Google Scholar, 32Greenlund A. Morales M.O. Viviano B.L. Yan H. Krolewski J. Schreiber R.D. Immunity. 1995; 2: 677-687Abstract Full Text PDF PubMed Scopus (252) Google Scholar). Thus, the specificity of the STAT-receptor interaction can be accurately mimicked with the use of peptides derived from individual receptor chains. We investigated whether truncated Stat6 proteins purified from insect cells could bind specifically to the IL-4 receptor-derived peptide YPKPFQ (S, Fig. 2 E). A phosphorylated peptide derived from the interferon-γ receptor served as a negative control (N, Fig. 2 E). Biotinylated peptides were attached to streptavidin beads and incubated with purified Stat6 proteins. After binding the beads were washed, and the eluted proteins were analyzed by Western blot. All C-terminal deletion mutants (with the exception of mutant 602 which did not express) bound selectively to peptides with affinities equal to that of wild-type Stat6 (Fig. 2 E). Consistent with the peptide binding results is the observation that the mutants exhibited a dominant negative phenotype when overexpressed in an IL-4-responsive cell line (data not shown). Similar results were obtained for Stat6 mutants that retained receptor/peptide binding activity but lacked the ability to bind DNA (21Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar). From the above results we tentatively designated aa 620 as the most C-terminal aa of the Stat6-SH2 domain. To identify more precisely amino acid residues critical for function of the Stat6 SH2 domain, we carried out an extensive mutational analysis. We substituted 2 aa at a time with alanines in the context of the full-length protein. Mutagenesis began at the conserved Trp-533 and ended at Pro-620. The mutants were expressed in lymphoid (BJAB) and nonlymphoid cells (293 cells), and their DNA binding properties and tyrosine phosphorylation status were determined. Fig. 3 shows the DNA binding activity of these mutants in nuclear extracts prepared from transiently transfected 293 cells following IL-4 treatment. Identical results were obtained with stably transfected BJAB cell extracts (data not shown). Many of the double alanine substitutions did not impair DNA binding. Because dimerization is a prerequisite for DNA binding, a positive DNA binding signal indicates that the SH2 domain of these mutants is capable of mediating both receptor binding and dimerization. However, certain substitutions reduced or completely abolished Stat6 DNA binding. Some of these loss-of-function substitutions target residues that are conserved between the SH2 domains of Stat6 and Src. For example, all changes in the conserved GTFLLR motif completely abolished DNA binding, consistent with the observation that this region is required for phosphotyrosine binding. Other double amino acid changes in conserved residues (WS, LI, and LY) also abolished DNA binding, whereas changes in the conserved LLLN sequence had no effect. Substitutions in certain regions without homology to Src (SK, DS, IT, IA, EN, IQ, PF, IR, RI, and RD) interfered with DNA binding. To determine the effect of these mutations on transcription, we overexpressed the proteins in 293 cells in the presence of a luciferase reporter carrying four copies of IL-4 response elements derived from the germ line epsilon promoter. Previously we showed that this reporter is not active in 293 cells in the presence or absence of IL-4 since 293 cells do not contain endogenous Stat6 (21Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (229) Google Scholar) (Fig. 3, A andB). However, the reporter can be activated through overexpression of recombinant Stat6 and in the presence of IL-4 (wt, Fig. 3 B). By using this assay system, we analyzed all SH2 mutants for their ability to activate transcription. A comparison between Fig. 3, A and B, shows that all proteins that are able to bind DNA also activate transcription, whereas mutants that do not bind DNA are not transcriptionally active. In general the mutants were slightly less active than the wild-type protein which may reflect differences in the expression levels. For reasons we do not understand, mutant VT was significantly more active than the wild-type protein. The loss-of-function mutants were analyzed further to determine where in the Stat6 activation cycle they cease to function. Various reasons could account for the lack of DNA binding seen with some of our mutants. One possibility is that these mutants are not phosphorylated in the cell following IL-4 treatment, because they either failed to bind the receptor or bound the receptor-Jak complex in an unproductive manner. In contrast, mutants that do get phosphorylated completed the receptor binding and activation steps but failed subsequently, most likely beca
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