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.
Mikita
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T
Tularik, Inc., South San Francisco, California 94080, USA.
Campbell
D
D
Wu
P
P
Williamson
K
K
Schindler
U
U
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D015870:Gene Expression / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D015847:Interleukin-4 / Q000494:pharmacology*; D007668:Kidney; D008156:Luciferases / Q000096:biosynthesis; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010748:Phosphopeptides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D050800:STAT6 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D015534:Trans-Activators; D015847:Interleukin-4; D008156:Luciferases