Requirements for interleukin-4-induced gene expression and functional characterization of Stat6

Interleukin-4 (IL-4) stimulation leads to the activation of the signal transducer and activator of transcription 6 (Stat6). In this study, we present data relating to the functional properties of Stat6. Human embryonic kidney 293 cells were shown to be deficient of Stat6 yet express all other components of the IL-4 signaling cascade. This cell line was used for transient-transfection studies of wild-type and mutant Stat6 proteins. The wild-type protein was shown to activate a reporter construct carrying multiple copies of the IL-4 response element derived from the human immunoglobulin heavy-chain germ line epsilon promoter. Similarly, a truncated protein lacking 41 amino acids of the N terminus was fully active. However, removal of the C-terminal 186 amino acids completely abolished transcription activation. Amino acid substitutions were introduced into the putative DNA binding domain (VVI at positions 411 to 413), the SH2 domain (R-562), or the tyrosine (Y-641) which presumably becomes phosphorylated upon activation. All three of these Stat6 mutants were unable to activate transcription in 293 cells. Wild-type and mutant Stat6 derivatives were also expressed in insect cells, and purified proteins were analyzed in vitro for the ability to interact with both DNA and tyrosine-phosphorylated peptides derived from the IL-4 receptor alpha chain. Mutations within the DNA binding domain, the SH2 domain, or tyrosine 641 completely abolished DNA binding. In contrast, only the SH2 mutant failed to interact with tyrosine-phosphorylated peptides. The transdominant effects of all Stat6 derivatives were analyzed by using HepG2 cells, which express endogenous Stat6 protein. Differential effects were observed with various mutants, supporting the current model of the Jak/STAT activation cycle.

[1]  T. Hoey,et al.  Cooperative DNA Binding and Sequence-Selective Recognition Conferred by the STAT Amino-Terminal Domain , 1996, Science.

[2]  S. McKnight,et al.  Differentiation of T‐helper lymphocytes: selective regulation by members of the STAT family of transcription factors , 1996, Genes to cells : devoted to molecular & cellular mechanisms.

[3]  W. Paul,et al.  Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted State6 gene , 1996, Nature.

[4]  S. Akira,et al.  Essential role of Stat6 in IL-4 signalling , 1996, Nature.

[5]  Allard Kaptein,et al.  Dominant Negative Stat3 Mutant Inhibits Interleukin-6-induced Jak-STAT Signal Transduction (*) , 1996, The Journal of Biological Chemistry.

[6]  M. Kaplan,et al.  Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. , 1996, Immunity.

[7]  D. Levy,et al.  Targeted Disruption of the Mouse Stat1 Gene Results in Compromised Innate Immunity to Viral Disease , 1996, Cell.

[8]  J. Ihle STATs: Signal Transducers and Activators of Transcription , 1996, Cell.

[9]  R. Schreiber,et al.  Targeted Disruption of the Stat1 Gene in Mice Reveals Unexpected Physiologic Specificity in the JAK–STAT Signaling Pathway , 1996, Cell.

[10]  K. Izuhara,et al.  Signal Transduction Pathway of Interleukin-4 and Interleukin-13 in Human B Cells Derived from X-linked Severe Combined Immunodeficiency Patients (*) , 1996, The Journal of Biological Chemistry.

[11]  J. Darnell,et al.  Function of Stat2 protein in transcriptional activation by alpha interferon , 1996, Molecular and cellular biology.

[12]  B. Groner,et al.  Cloning and expression of Stat5 and an additional homologue (Stat5b) involved in prolactin signal transduction in mouse mammary tissue. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[13]  M. White,et al.  Characterization of the Interleukin-4 Nuclear Activated Factor/STAT and Its Activation Independent of the Insulin Receptor Substrate Proteins (*) , 1995, The Journal of Biological Chemistry.

[14]  L. Ivashkiv Cytokines and STATs: how can signals achieve specificity? , 1995, Immunity.

[15]  S. McKnight,et al.  Components of a Stat recognition code: evidence for two layers of molecular selectivity. , 1995, Immunity.

[16]  R. Schreiber,et al.  Stat recruitment by tyrosine-phosphorylated cytokine receptors: an ordered reversible affinity-driven process. , 1995, Immunity.

[17]  S. Szabo,et al.  Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4 , 1995, The Journal of experimental medicine.

[18]  J. Darnell,et al.  Tyrosine-phosphorylated Stat1 and Stat2 plus a 48-kDa protein all contact DNA in forming interferon-stimulated-gene factor 3. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[19]  J. Darnell,et al.  A STAT protein domain that determines DNA sequence recognition suggests a novel DNA-binding domain. , 1995, Genes & development.

[20]  S. McKnight,et al.  Identification and purification of human Stat proteins activated in response to interleukin-2. , 1995, Immunity.

[21]  W. Leonard,et al.  The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. , 1995, Immunity.

[22]  J. Rosen,et al.  Nuclear factor I and mammary gland factor (STAT5) play a critical role in regulating rat whey acidic protein gene expression in transgenic mice , 1995, Molecular and cellular biology.

[23]  J. Darnell,et al.  Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Darnell,et al.  Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors , 1995, Science.

[25]  J. Stavnezer,et al.  Characterization of an interleukin 4 (IL-4) responsive region in the immunoglobulin heavy chain germline epsilon promoter: regulation by NF- IL-4, a C/EBP family member and NF-kappa B/p50 , 1995, The Journal of experimental medicine.

[26]  J. Darnell,et al.  Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. , 1995, Annual review of biochemistry.

[27]  J. Stavnezer,et al.  Activation of the Ig germ-line gamma 1 promoter. Involvement of C/enhancer-binding protein transcription factors and their possible interaction with an NF-IL-4 site. , 1994, Journal of immunology.

[28]  S. McKnight,et al.  An interleukin-4-induced transcription factor: IL-4 Stat. , 1994, Science.

[29]  B. Groner,et al.  Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. , 1994, The EMBO journal.

[30]  J. Darnell,et al.  Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. , 1994, Science.

[31]  E. Rieber,et al.  Human interleukin‐13 activates the interleukin‐4‐dependent transcription factor NF‐IL4 sharing a DNA binding motif with an interferon‐γ‐induced nuclear binding factor , 1994, FEBS letters.

[32]  J. Darnell,et al.  Stat3 and Stat4: members of the family of signal transducers and activators of transcription. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[33]  B. Groner,et al.  Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. , 1994, The EMBO journal.

[34]  B. Viviano,et al.  Ligand‐induced IFN gamma receptor tyrosine phosphorylation couples the receptor to its signal transduction system (p91). , 1994, The EMBO journal.

[35]  J. Darnell,et al.  Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. , 1994, Science.

[36]  J. Darnell,et al.  Interferon activation of the transcription factor Stat91 involves dimerization through SH2-phosphotyrosyl peptide interactions , 1994, Cell.

[37]  W. Paul,et al.  Lymphocyte responses and cytokines , 1994, Cell.

[38]  Andrew Ziemiecki,et al.  Polypeptide signalling to the nucleus through tyrosine phosphorylation of Jak and Stat proteins , 1993, Nature.

[39]  N. Reich,et al.  Requirement of tyrosine phosphorylation for rapid activation of a DNA binding factor by IL-4. , 1993, Science.

[40]  J. Darnell,et al.  A single phosphotyrosine residue of Stat91 required for gene activation by interferon-gamma. , 1993, Science.

[41]  J. Kuriyan,et al.  Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: Crystal structures of the complexed and peptide-free forms , 1993, Cell.

[42]  O. Hagenbüchle,et al.  A rapid method for the isolation of DNA-binding proteins from purified nuclei of tissues and cells in culture. , 1992, Nucleic acids research.

[43]  T Pawson,et al.  SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. , 1991, Science.

[44]  M. Ptashne,et al.  A vector for expressing GAL4(1-147) fusions in mammalian cells. , 1989, Nucleic acids research.

[45]  G. Nabel,et al.  Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. , 1989, Proceedings of the National Academy of Sciences of the United States of America.