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, w e present data relating to the functional properties of Stat6, Human e mbryonic kidney 293 cells were shown to be deficient of Stat6 yet expr ess all other components of the IL-4 signaling cascade, This cell line was used for transient-transfection studies of wild-type and mutant S tat6 proteins, The wild-type protein was shown to activate a reporter construct carrying multiple copies of the IL-4 response element derive d from the human immunoglobulin heavy-chain germ line epsilon promoter , Similarly, a truncated protein lacking 41 amino acids of the N termi nus was fully active, However, removal of the C-terminal 186 amino aci ds completely abolished transcription activation, Amino acid substitut ions were introduced into the putative DNA binding domain (VVI at posi tions 411 to 413), the SH2 domain (R-562), or the tyrosine (Y-641) whi ch presumably becomes phosphorylated upon activation. All three of the se Stat6 mutants were unable to activate transcription in 293 cells, W ild-type and mutant Stat6 derivatives were also expressed in insect ce lls, and purified proteins were analyzed in vitro for the ability to i nteract with both DNA and tyrosine-phosphorylated peptides derived fro m the IL-4 receptor alpha chain. Mutations within the DNA binding doma in, the SH2 domain, or tyrosine 641 completely abolished DNA binding, In contrast, only the SH2 mutant failed to interact with tyrosine-phos phorylated peptides, The transdominant effects of all Stat6 derivative s were analyzed by using HepG2 cells, which express endogenous Stat6 p rotein, Differential effects were observed with various mutants, suppo rting the current model of the Jak/STAT activation cycle.