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

Abstract

Stat6 transcription factor is a critical mediator of IL-4-specific gene responses. Tyrosine phosphorylation is required for nuclear localization and DNA binding of Stat6. The authors investigated whether Stat6-dependent transcriptional responses are regulated through IL-4-induced serine/threonine phosphorylation. In Ramos B cells, the serine/threonine kinase inhibitor H7 inhibited 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 phosphorylation and DNA binding of Stat6. To analyze whether the H7-sensitive pathway was regulating Stat6-activated transcription, we used reporter constructs 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 maps revealed that IL-4 induced phosphorylation of Stat6 on serine and tyrosine residues in Ramos cells and in 32D cells lacking endogenous IRS proteins. However, H7 treatment did not inhibit the phosphorylation of Stat6. Instead, H7 inhibited the IL-4-induced phosphorylation of RNA polymerase II. These results indicate that Stat6-induced transcription is dependent on phosphorylation events mediated by H7-sensitive kinase(s) but that it also involves serine phosphorylation of Stat6 by an H7-insensitive kinase independent of the IRS pathway.

Lipin-1 contributes to IL-4 mediated macrophage polarization

“…Flow cytometric evaluation of the surface expression of IL-4 receptor showed no difference between wild type and lipin-1 m KO BMDM (Fig 2A). Ligand binding of the IL-4 receptor-α (IL4Rα) triggers tyrosine phosphorylation at the cytoplasmic tail to facilitate recruitment and subsequent tyrosine phosphorylation of STAT6 by JAK1/JAK3 pathway [22]. Wildtype and lipin-1 m KO BMDMs were stimulated with IL-4 for 30 minutes and protein was collected.…”

“…IL-4 binding to the IL-4R leads to phosphorylation and activation of STAT6. STAT6 binds to DNA promoters that leads to recruitment of PPARγ:RXR transcription factors to promote gene expression in macrophages [2; 22]. PPARγ is highly expressed during macrophage differentiation and its expression is a prominent feature of IL-4 stimulated macrophages [27].…”

Identification of p100 as a coactivator for STAT6 that bridges STAT6 with RNA polymerase II

“…The TAD of STAT6 is rich in Pro, Ser, Thr, Leu and Glu residues, but lacks acidic residues commonly found in TADs. Serine phosphorylation of STAT6 (Pesu et al, 2000) may also increase its negative net charge and regulate the interaction with co-regulators. p100 has been linked to a signaling pathway from Pim-1 Ser/Thr kinase to c-Myb transcription factor (Leverson et al, 1998).…”

“…To characterize the mechanism of p100-mediated augmentation of STAT6 activity, we analyzed whether p100 would affect the immediate signaling events of STAT6. These experiments were carried out in COS-7 cells that do not express detectable levels of STAT6, but express a functional IL-4R (Moriggl et al, 1997;Pesu et al, 2000).…”

Regulation of inflammation by interleukin-4: a review of “alternatives”

“…STAT6 often forms cooperative interactions with other transcription factors and coactivators, such as NF-B or p300/CEBP. Other post-translational modifications of STAT6 that impact its transcriptional function include acetylation [99], methylation [100], and serine phosphorylation [101][102][103][104]. The STAT6 pathway plays a central role in gene regulation and allergic responses regulated by IL-4 or IL-13, including T cell proliferation and survival and Th2 differentiation [105].…”