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HMG-I(Y) Phosphorylation Status as a Nuclear Target Regulated through Insulin Receptor Substrate-1 and the I4R Motif of the Interleukin-4 Receptor

1997; Elsevier BV; Volume: 272; Issue: 40 Linguagem: Inglês

10.1074/jbc.272.40.25083

ISSN

1083-351X

Autores

Dingzhi Wang, José Luís Zamorano, Achsah Keegan, Mark Boothby,

Tópico(s)

Pharmacological Effects of Natural Compounds

Resumo

Interleukin (IL)-4 is a cytokine that regulates both the growth and differentiation of hematopoietic cells. Its ligand binding specificity and important signal transduction mechanisms are conferred by the IL-4 receptor α chain (IL-4Rα). The I4R is a tyrosine-containing motif within IL-4Rα that is critical for proliferative responses to IL-4. Although the I4R also contributes to gene regulation, nuclear targets directly regulated by this motif have not been described. It is shown here that the tyrosine at position 497 in the I4R is critical for regulation of the phosphorylation status of a set of nuclear proteins that includes HMG-I(Y), small non-histone chromosomal proteins involved in the control of gene expression in hematopoietic cell lines. Moreover, IL-4 is unable to induce HMG-I(Y) phosphorylation in insulin receptor substrate-1-deficient cells, and the inhibitor wortmannin completely blocks IL-4 regulation of HMG-I(Y) phosphorylation status but not activation of an IL-4 Stat protein. Taken together, these data indicate that HMG-I(Y) is a nuclear target whose phosphorylation status is regulated through the I4R motif via insulin receptor substrate proteins, independent of activation of the Stat pathway. Interleukin (IL)-4 is a cytokine that regulates both the growth and differentiation of hematopoietic cells. Its ligand binding specificity and important signal transduction mechanisms are conferred by the IL-4 receptor α chain (IL-4Rα). The I4R is a tyrosine-containing motif within IL-4Rα that is critical for proliferative responses to IL-4. Although the I4R also contributes to gene regulation, nuclear targets directly regulated by this motif have not been described. It is shown here that the tyrosine at position 497 in the I4R is critical for regulation of the phosphorylation status of a set of nuclear proteins that includes HMG-I(Y), small non-histone chromosomal proteins involved in the control of gene expression in hematopoietic cell lines. Moreover, IL-4 is unable to induce HMG-I(Y) phosphorylation in insulin receptor substrate-1-deficient cells, and the inhibitor wortmannin completely blocks IL-4 regulation of HMG-I(Y) phosphorylation status but not activation of an IL-4 Stat protein. Taken together, these data indicate that HMG-I(Y) is a nuclear target whose phosphorylation status is regulated through the I4R motif via insulin receptor substrate proteins, independent of activation of the Stat pathway. Interleukin (IL) 1The abbreviations used are: IL, interleukin; IL-4R, IL-4 receptor; Stat, signal transduction and activation of transcription; IRS, insulin receptor substrate; Gε, germ line Cε (region of immunoglobulin heavy chain locus); PI 3-K, phosphatidylinositol 3-kinase; hu, human; m, mouse; W.T., wild type; PAGE, polyacrylamide gel electrophoresis; PSL, arbitrary density units.-4 is a cytokine produced by T cells, mast cells, and basophils (1Paul W.E. Blood. 1991; 77: 1570-1589Crossref Google Scholar, 2Jansen J.H. Fibbe W.E. Willemze R. Kluin-Nelemans J.C. Blut. 1990; 60: 269-274Crossref PubMed Scopus (31) Google Scholar). Although originally identified on the basis of its stimulation of B lymphocyte proliferation, the pleiotropic effects of IL-4 are now known to include regulation of proliferation, gene expression, and stable differentiation of both lymphocytic and other hematopoietic cells (2Jansen J.H. Fibbe W.E. Willemze R. Kluin-Nelemans J.C. 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These varied biological functions of IL-4 are regulated through one or more receptor complexes that include a high affinity binding chain, IL-4Rα, a member of the hematopoietin receptor gene superfamily (11Mosley B. Beckmann M.P. March C.J. Idzerda R.L. Gimpel S.D. VandenBos T. Friend D. Alpert A. Anderson D. Jackson J. Wignall J.M. Smith C. Gallis B. Sims J.E. Urdal D. Widmer M.B. Cosman D. Park L.S. Cell. 1989; 59: 335-348Abstract Full Text PDF PubMed Scopus (487) Google Scholar, 12Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Silvennoinen O. Annu. Rev. Immunol. 1995; 13: 369-398Crossref PubMed Scopus (550) Google Scholar, 13Callard R.E. Matthews D.J. Hibbert L. Immunol. Today. 1996; 17: 108-110Abstract Full Text PDF PubMed Scopus (167) Google Scholar). The IL-4Rα chain pairs with an accessory chain, γc, while other forms of IL-4 receptor may use a different accessory chain (13Callard R.E. Matthews D.J. Hibbert L. Immunol. 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Conserved phosphotyrosine residues at positions 575, 2Amino acid residues are numbered according to their positions in the human IL-4Rα chain (with +1 as the initiator methionine at the N terminus of the signal peptide). 603, and 631 are recognized by the SH2 domains of the latent cytoplasmic transcription factor Stat6, a member of the SignalTransduction and Activation ofTranscription (Stat) family, and docking of Stat6 monomers leads to their phosphorylation, dimerization, and nuclear translocation (19Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (731) Google Scholar, 20Quelle F.W. Shimoda K. Thierfelder W. Fischer C. Kim A. Ruben S.M. Cleveland J.L. Pierce J.H. Keegan A. Nelms K. Paul W.E. Ihle J.N. Mol. Cell. Biol. 1995; 15: 3336-3343Crossref PubMed Scopus (304) Google Scholar, 21Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5062) Google Scholar, 22Ryan J.J. 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Nature. 1996; 380: 630-633Crossref PubMed Scopus (1117) Google Scholar, 25Kaplan M.H. Schindler U. Smiley S.T. Gruesby M.J. Immunity. 1996; 4: 313-319Abstract Full Text Full Text PDF PubMed Scopus (1341) Google Scholar). However, certain mRNAs may be IL-4-inducible in Stat6-deficient mouse hematopoietic cells (64Kaplan M.H. Grusby M.J. J. Allerg. Clin. Immunol. 1997; 99 (abstr.): S484Google Scholar), thus raising further questions about Stat6-independent signal transduction. The most membrane-proximal conserved tyrosine, Tyr-1, is at position 497 of the primary sequence of human IL-4Rα. In contrast to the distal residues thought most responsible for Stat6 recruitment, phosphorylation of the Tyr-1 residue leads to recruitment of adaptor proteins termed insulin receptor substrate (IRS)-1 and IRS-2 (originally called 4PS), and this phosphorylation is critical for the ability of IL-4 to stimulate proliferation of the myeloid progenitor cell line 32D (26Wang L. Myers Jr., M.G. Sun X. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (371) Google Scholar, 27Wang L. Keegan A.D. Paul W.E. Heidaran M.A. Gutkind J.S. Pierce J.H. EMBO J. 1992; 11: 4899-4908Crossref PubMed Scopus (159) Google Scholar, 28Keegan A.D. Nelms K. White M. Wang L.-M. Pierce J. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (288) Google Scholar). Based on a shared sequence motif and homology with insulin receptor signal transduction, the region spanning IL-4Rα Tyr-1 has been termed the I4R motif (28Keegan A.D. Nelms K. White M. Wang L.-M. Pierce J. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (288) Google Scholar). Although the I4R motif clearly regulates proliferative responses, its contribution to the regulation of nuclear proteins and gene transcription is less well understood. Emerging data indicate that the I4R motif can contribute to modulation of transcriptional activity through Stat6-independent mechanism(s) (29Wang H.Y. Paul W.E. Keegan A.D. Immunity. 1995; 4: 113-121Abstract Full Text Full Text PDF Scopus (76) Google Scholar). 3J. Youn, H. Y. Wang, M. Boothby, and A. D. Keegan, unpublished observations. However, the set of nuclear proteins whose phosphorylation status is regulated through the I4R and the dependence of their regulation on Stat6 remain unclear. One potential target for Stat6-independent regulation is the non-histone chromosomal protein HMG-I(Y). HMG-I(Y) is a nuclear protein involved in the signal-induced regulation of multiple genes, including virus activation of interferon β, IL-1 activation of E-selectin, induction of IL-2Rα chain and repression of IL-4 transcription in activated T cells, and regulation of the germ line immunoglobulin epsilon (Gε) promoter (30Thanos D. Maniatis T. 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The phosphorylation status of HMG-I(Y) is regulated in response to IL-4, and the phosphorylated form of HMG-I(Y) exhibits a lower affinity for Gε promoter DNA in vitro, suggesting that phosphorylation decreases the repressor effect of HMG-I(Y) at the Gε promoter in B lymphocytes (37Wang D.-Z. Ray P. Boothby M. J. Biol. Chem. 1995; 270: 22924-22932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Since HMG-I(Y) phosphorylation status is regulated through a pathway that appeared independent from Stat6 activation but sensitive to the immunosuppressive agent rapamycin (37Wang D.-Z. Ray P. Boothby M. J. Biol. Chem. 1995; 270: 22924-22932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 38Wang D.-Z. Cherrington A.L. Famakin-Mosuro B.M. Boothby M. Int. Immunol. 1996; 8: 977-989Crossref PubMed Scopus (21) Google Scholar), we formulated the hypothesis that this phosphorylation is regulated through the I4R motif rather than the membrane-distal phosphotyrosine residues implicated in Stat6 activation. To investigate this hypothesis, the ability of wild-type and mutant forms of the IL-4 receptor to regulate HMG-I(Y) phosphorylation was measured. Because the I4R motif is linked to IRS-1 recruitment and activation of the lipid kinase PI 3-kinase, we also investigated the requirement for these signal transduction elements in IL-4-inducible HMG-I(Y) phosphorylation. Stable transfectants of the IL-3-dependent myeloid progenitor line 32D, bearing wild-type or mutant human IL-4Rα chains with or without transfected rat IRS-1, have been described previously (26Wang L. Myers Jr., M.G. Sun X. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (371) Google Scholar, 28Keegan A.D. Nelms K. White M. Wang L.-M. Pierce J. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (288) Google Scholar). Specific clones used in this study were as follows: wild-type hIL-4R (W.T.; clones 8-2B4 and 8-2B4A6), full-length hIL-4R with Y497F (clones 8-5B6 and 8-5D3), and truncations whose end points are at residue 657 (d657) or 557 (d557) (5-3WY0 and 5-3WY2; 6-3W3 and 6-3W13, respectively). These cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 50 units ml−1 penicillin, 50 units ml−1 streptomycin, 3 mml-glutamine, 100 μm 2-mercaptoethanol (RP/10F is complete RPMI 1640 media with 10% fetal bovine serum), and 5% conditioned medium from WEHI-3 cells as a source of IL-3. To prepare conditioned medium, WEHI-3 cells were grown to confluence in Iscove's medium supplemented with 10% fetal bovine serum, 50 units ml−1 penicillin, 50 units ml−1 streptomycin, and 3 mml-glutamine. After culture for 4 days in stationary phase, cell debris were removed by centrifugation and sterile filtration. For metabolic labeling experiments, 32D cells (106 cells ml−1) were removed from WEHI-3-conditioned medium and then cultured for 20 h in RP/10F. M12 B lymphoma cells transfected with a truncated huIL-2Rβ and the Chim-1 chimera of huIL-2Rβ-huIL4R (residues 457–557) have been described previously (29Wang H.Y. Paul W.E. Keegan A.D. Immunity. 1995; 4: 113-121Abstract Full Text Full Text PDF Scopus (76) Google Scholar). Mouse splenocytes were prepared as described (5Boothby M. Gravallese E. Liou H.-C. Glimcher L.H. Science. 1988; 242: 1559-1562Crossref PubMed Scopus (52) Google Scholar). Cells were rinsed twice with serum- and phosphate-free RPMI 1640 medium and then resuspended in RPMI 1640 medium supplemented with 50 units ml−1 penicillin, 50 units ml−1streptomycin, 3 mml-glutamine, 100 μm 2-mercaptoethanol, and 10% dialyzed fetal bovine serum (Life Technologies, Inc.). Cells were cultured for 4 h at 37 °C with 40 μCi ml−1[32P]orthophosphate (NEN Life Science Products) and then stimulated 4 h with huIL-2 (100 units ml−1; for M12 cells only), huIL-4 (10 ng ml−1), or mIL-4 (10 ng ml−1). For experiments with pharmacologic inhibitors, cells were separately incubated with rapamycin (100 nm), genistein (75 μm), or wortmannin (100 nm), or the solvent (a 1:1 v/v mixture of dimethyl sulfoxide and ethanol) at 1:1000 for 20 h and 2 h prior to stimulation with IL-4. Purified recombinant human IL-4 (huIL-4) was obtained as generous gifts from M. Widmer (Immunex, Seattle, WA) and J. Devries (DNAX, Palo Alto, CA). Purified recombinant mouse IL-4 (mIL-4) was purchased from BioSource International (Camarillo, CA), and IL-4 conditioned medium was prepared from a myeloma cell line transduced with a mIL-4 expression vector as described previously (39Karasuyama H. Melchers F. Eur. J. Immunol. 1988; 18: 97-104Crossref PubMed Scopus (1081) Google Scholar). Purified recombinant huIL-2 was obtained from the Biological Response Modifiers Program (Frederick, MD). Rapamycin was a generous gift of S. Sehgal (Wyeth-Ayerst, Princeton, NJ), and was stored at −80 °C in absolute ethanol. Sodium orthovanadate and sodium fluoride were purchased from Sigma, and fresh aqueous solutions were prepared prior to each experiment. Wortmannin and genistein were purchased from Sigma and Life Technologies, Inc., respectively, dissolved in dimethyl sulfoxide, and stored at −20 °C. Protein concentrations were determined by the Bradford method (Bio-Rad). After labeling as described above, cells were harvested, rinsed twice with ice-cold phosphate-buffered saline, and lysed with 0.5% Nonidet P-40 in 10 mm Tris-Cl, pH 7.5, 10 mm NaCl, 3 mm MgCl2, 0.1 mm EGTA, 0.5 mm dithiothreitol (RSB), supplemented with 20 μm leupeptin, 10 μg ml−1 aprotinin, 1 mm phenylmethylsulfonyl fluoride, 0.5 mm sodium orthovanadate, and 1 mm sodium fluoride. Nuclei were pelleted after lysis of the cells, and nuclear extracts were prepared as described previously (37Wang D.-Z. Ray P. Boothby M. J. Biol. Chem. 1995; 270: 22924-22932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) except for supplementation with protease and phosphatase inhibitors as above. Resolution of basic nuclear proteins by two-dimensional electrophoresis on acid-urea gels followed by SDS-PAGE was performed as described previously (37Wang D.-Z. Ray P. Boothby M. J. Biol. Chem. 1995; 270: 22924-22932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 40Kardalinou E. Zhelev N. Hazzalin C.A. Mahadevan L.C. Mol. Cell. Biol. 1994; 14: 1066-1074Crossref PubMed Google Scholar). For selected experiments, the two-dimensional gels were silver-stained to detect relative protein levels, then exposed to autoradiographic film and phosphorimaging plates, whereas in other experiments aliquots of the nuclear proteins were silver-stained separately. To quantify incorporation of 32P into phosphorylated HMG-I(Y) using a Fuji BAS 1000 phosphorimager, the net density units (“PSLs”) incorporated into HMG-I(Y) in each gel were adjusted by subtracting plate background. This incorporation into HMG-I(Y) was normalized by dividing HMG-I(Y)-specific PSLs by the net PSLs in an internal reference spot that was consistently resolved from other labeled proteins. The efficiency of the huIL-4R transfected into a given 32D clone relative to the mouse IL-4R was defined as follows: ({(normalized PSLs in HMG-I(Y)}huIL-4-treated − {normalized PSLs in HMG-I(Y)}basal) ÷ ({normalized PSLs in HMG-I(Y)}mouse IL-4-treated − {normalized PSLs in HMG-I(Y)}basal). For gel shift analyses, 32D cells were cultured in RPMI 1640 medium with 10% fetal bovine serum in the absence of IL-3 for 0 or 2 h and then stimulated with IL-4 for 0.5 h. Electrophoretic mobility shift analyses were performed using a double-stranded oligonucleotide representing the Stat6 binding site spanning nucleotides −122 to −104 of the Gε promoter after preparation of whole cell or nuclear extracts, as described previously (38Wang D.-Z. Cherrington A.L. Famakin-Mosuro B.M. Boothby M. Int. Immunol. 1996; 8: 977-989Crossref PubMed Scopus (21) Google Scholar, 41Schindler C. Shuai K. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (727) Google Scholar). Analyses of phosphotyrosine-containing proteins were performed as described previously (28Keegan A.D. Nelms K. White M. Wang L.-M. Pierce J. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (288) Google Scholar). After pretreatment with genistein as appropriate, cells deprived of serum for 2 h at 37 °C were cultured in the presence or absence of huIL-4 (5 ng ml−1) for 5 min at 22 °C. The reaction was terminated by dilution in ice-cold PBS containing 100 μm Na3VO4. Cells were lysed in 50 mm Hepes, pH 7.5, 0.15 m NaCl, 0.5% Nonidet P-40, with 50 mm NaF, 10 mmNaPPi, and protease inhibitors (28Keegan A.D. Nelms K. White M. Wang L.-M. Pierce J. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (288) Google Scholar), followed by immunoprecipitation of proteins in the soluble fraction using a polyclonal rabbit antiserum against rat IRS-1 (generous gift of L. M. Wang and J. Pierce, LCMB, National Institutes of Health). Precipitates were washed in lysis buffer, dissolved in SDS sample buffer, and separated by SDS-PAGE. Resolved proteins were transferred to polyvinylidene difluoride membranes and probed with the 4G10 monoclonal antiphosphotyrosine antibody or rabbit anti-IRS-1. Bound antibodies were detected using enhanced chemiluminescence (Amersham Corp.). At least two spatially distinct motifs have been identified within the cytoplasmic domain of the IL-4 receptor α chain as follows: an I4R region originally linked to control of proliferation but not gene expression or Stat6 phosphorylation (20Quelle F.W. Shimoda K. Thierfelder W. Fischer C. Kim A. Ruben S.M. Cleveland J.L. Pierce J.H. Keegan A. Nelms K. Paul W.E. Ihle J.N. Mol. Cell. Biol. 1995; 15: 3336-3343Crossref PubMed Scopus (304) Google Scholar, 28Keegan A.D. Nelms K. White M. Wang L.-M. Pierce J. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (288) Google Scholar), and dominant Stat6 docking sites distal to amino acid 557 (19Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (731) Google Scholar, 22Ryan J.J. McReynolds L.J. Keegan A.D. 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, 42Pernis A. Witthuhn B. Keegan A.D. Nelms K. Garfein E. Ihle J.N. Paul W.E. Pierce J.H. Rothman P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7971-7975Crossref PubMed Scopus (82) Google Scholar). More recent evidence indicates that lymphocyte proliferation is impaired by Stat6 gene disruption (23Takeda K. Tanaka T. Shi W. Matsumoto M. Minami M. Kashiwamura S.-I. Nakanishi K. Yoshida N. Kishimoto T. Kira S. Nature. 1996; 380: 627-630Crossref PubMed Scopus (1285) Google Scholar, 24Shimoda K. van Deursen J. Sangster M.Y. Sarawar S.R. Carson R.T. Tripp R.A. Chu C. Quelle F.W. Nosaka T. Vignali D.A.A. Doherty P.C. Grosveld G. Paul W.E. Ihle J.N. Nature. 1996; 380: 630-633Crossref PubMed Scopus (1117) Google Scholar, 25Kaplan M.H. Schindler U. Smiley S.T. Gruesby M.J. Immunity. 1996; 4: 313-319Abstract Full Text Full Text PDF PubMed Scopus (1341) Google Scholar), whereas HMG-I(Y) phosphorylation status has been correlated with proliferation of cells (43Giancotti V. Pani B. Andrea P.D. Berlingieri M.T. Di Fiore P.P. Fusco A. Vecchio G. Philp R. Crane-Robinson C. Nicolas R.H. Wright C.A. Goodwin G.H. EMBO J. 1987; 6: 1981-1987Crossref PubMed Scopus (150) Google Scholar, 44Reeves R. Langan T.A. Nissen M.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1671-1675Crossref PubMed Scopus (115) Google Scholar). These observations are consistent with a role for either or both of the IL-4Rα motifs in regulation of HMG-I(Y) phosphorylation status. To determine which of these IL-4Rα motifs, the I4R or Stat6 docking motifs, mediates IL-4 regulation of HMG-I(Y) phosphorylation, the ability of wild-type and mutant forms of the human IL-4Rα (huIL-4R) to confer regulated HMG-I(Y) phosphorylation was analyzed using transfectants derived from the myeloid progenitor line 32D. To measure IL-4 regulation of HMG-I(Y) phosphorylation, metabolic labeling experiments and two-dimensional acid-urea/SDS-PAGE analyses were performed using a panel of 32D transfectants all of which also expressed rat IRS-1. Because of the known potential for clone-to-clone and inter-experimental variation, we employed a widely used strategy to control for these technical issues. Human and mouse IL-4 exhibit species-specific binding to their respective receptors. Thus, the effect of human IL-4 receptors expressed on a given 32D clone can be compared with the effect of the endogenous mouse IL-4 receptors, and huIL-4R expressed on different clones also can be compared. As shown in Fig. 1 A, IL-4 increases the phosphorylation of a set of the basic nuclear proteins which enter acid-urea gels. Of particular note, human IL-4 induced an increase in HMG-I(Y) (arrows) phosphorylation comparable to that observed with mouse IL-4. Since no significant changes in the amount of HMG-I(Y) and other nuclear proteins were observed on silver-stained gels (Fig. 1 B), this increase in labeled protein represents an increase in the specific activity of HMG-I(Y). Function of the I4R motif has previously been shown to require a tyrosine at position 497 (Tyr-1) (28Keegan A.D. Nelms K. White M. Wang L.-M. Pierce J. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (288) Google Scholar). Accordingly, to test if I4R function is required for IL-4 to regulate HMG-I(Y) phosphorylation, parallel labeling experiments were performed using cells transfected with a full-length human IL-4Rα bearing the Tyr → Phe substitution that blocks IRS-1 recruitment and inactivates the I4R motif (28Keegan A.D. Nelms K. White M. Wang L.-M. Pierce J. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (288) Google Scholar). This Y497F (Y1F) substitution completely eliminated the ability of huIL-4 to induce increases in HMG-I(Y) phosphorylation in each of the two clones tested (8-5B6 and 8-5D3). In sharp contrast, the endogenous wild-type mouse IL-4R in these clones was fully competent to increase HMG-I(Y) phosphorylation (Fig. 1 A, right-hand panels; Fig.1 C). This finding indicates that the failure to increase HMG-I(Y) labeling is specific for the mutant human receptor and contradicts the alternative possibility that the huIL-4R Y497F mutant leads to clones in which the basal rate of HMG-I(Y) phosphorylation cannot be increased by IL-4 signaling. Quantitation of the results of these experiments showed that the wild-type human receptor was as potent as the mouse receptor, whereas the Y497F (Y1F) mutation dramatically inhibited this function of the IL-4Rα chain (Fig.1 C). The phosphorylation status of other basic nuclear proteins resolved in these two-dimensional gels also was sensitive to IL-4 and dependent on I4R function. Although the identity of these other proteins has not been determined, 4Spots co-migrating with the core histones (H2a, H2b, H3, and H4) demonstrated no significant phosphorylation, whereas histone H1 migrated in a cluster of highly labeled proteins exhibiting an IL-4-inducible increase in phosphorylation and could not be quantified separately. we nonetheless can conclude that an intact I4R is essential for regulation of IL-4-inducible increases in HMG-I(Y) phosphorylation. The above data provide evidence that an intact I4R is essential for regulation of HMG-I(Y) phosphorylation by IL-4. However, it was possible that the quantitative level of Stat6 activation might also contribute to regulation of HMG-I(Y) phosphorylation (37Wang D.-Z. Ray P. Boothby M. J. Biol. Chem. 1995; 270: 22924-22932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Human IL-4 receptors that terminate at amino acid residue 557 (d557), between Tyr-1 and Tyr-2, have a greatly reduced competence to induce Stat6 (Fig. 2 A). In contrast, receptors truncated at residue 657 (d657) are fully competent to induce Stat6 (20Quelle F.W. Shimoda K. Thierfelder W. Fischer C. Kim A. Ruben S.M. Cleveland J.L. Pierce J.H. Keegan A. Nelms K. Paul W.E. Ihle J.N. Mol. Cell. Biol. 1995; 15: 3336-3343Crossref PubMed Scopus (304) Google Scholar, 22Ryan J.J. McReynolds L.J. Keegan A.D. 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, 42Pernis A. Witthuhn B. Keegan A.D. Nelms K. Garfein E. Ihle J.N. Paul W.E. Pierce J.H. Rothman P. Proc. Nat

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