Interleukin-4-induced transcriptional activation by Stat6 involves multiple serine/threonine kinase pathways and serine phosphorylation of Stat6

Stat6 transcription factor is a critical mediator of IL-4-specific gene res ponses. Tyrosine phosphorylation is required for nuclear localization and D NA binding of Stat6, The authors investigated whether State-dependent trans criptional responses are regulated through IL-4-induced serine/threonine ph osphorylation, In Ramos B cells, the serine/threonine kinase inhibitor H7 i nhibited IL-4-induced expression of CD23. Treatment with H7 did not affect IL-4R-mediated immediate signaling events such as tyrosine phosphorylation of Jak1, Jak3, insulin receptor substrate (IRS)-1 and IRS-2, or tyrosine ph osphorylation and DNA binding of Stat6, To analyze whether the H7-sensitive pathway was regulating Stat6-activated transcription, we used reporter con structs containing different IL-4 responsive elements. H7 abrogated Stat6-, as well as Stat5-, mediated reporter gene activation and partially reduced C/EBP-dependent reporter activity. By contrast, IL-4-induced transcription was not affected by wortmannin, an inhibitor of the phosphatidyl-inositol 3'-kinase pathway, Phospho-amino acid analysis and tryptic phosphopeptide m aps revealed that IL-4 induced phosphorylation of Stat6 on serine and tyros ine residues in Ramos cells and in 32D cells lacking endogenous IRS protein s. However, H7 treatment did not inhibit the phosphorylation of Stat6, Inst ead, H7 inhibited the IL-4-induced phosphorylation of RNA polymerase II. Th ese results indicate that Stat6-induced transcription Is dependent on phosp horylation events mediated by H7-sensitive kinase(s) but that it also invol ves serine phosphorylation of Stat6 by an H7-insensitive kinase independent of the IRS pathway, (Blood, 2000;95:494-502) (C) 2000 by The American Soci ety of Hematology.