A Gain-of-function Mutation in STAT6

Abstract

Interleukin-4 (IL-4) is a cytokine that plays a crucial role in the pathophysiology of asthma and allergic diseases. IL-4-induced gene expression is largely mediated through the activation of the latent transcription factor STAT6. We identified a STAT6 mutant (STAT6VT)) that is activated independently of IL-4 stimulation. STAT6VT carries two amino acid changes in the SH2 domain that affect the overall structure and stability of the monomeric and dimeric protein. When overexpressed in mammalian cells, STAT6VT undergoes tyrosine phosphorylation, binds DNA, and activates transcription in the absence of IL-4 stimulation. Using the Jak1-and Jak3-deficient fibroblast line U4A, we demonstrate that phosphorylation is mediated by an IL-4-independent tyrosine kinase that is not able to activate the wild-type STAT6 protein. These results suggest that small changes in STAT6 could result in hyperactivation of the protein and constitutive expression of STAT6-dependent genes. Such a mutation, if found in vivo, could cause genetic predisposition for atopic diseases.

Induction of the IL-13 receptor α2-chain by IL-4 and IL-13 in human keratinocytes: involvement of STAT6, ERK and p38 MAPK pathways

“…In STAT6VT transfectants, immunoprecipitation experiments revealed a constitutive tyrosine phosphorylation of STAT6 (Figure 7a lower panel), as previously described in other cell lines (Daniel et al, 2000). Subsequently, the transcriptional potential of STAT6VT was assessed using an IL-4 inducible luciferase reporter construct carrying ®ve copies of Figure 6 E ect of pharmacological inhibitors on IL-4/IL-13-induced IL-13Ra2 expression.…”

“…Whether STAT6 is implicated in IL-13Ra2 induction was assessed using a gain-of-function mutant of STAT6 (Daniel et al, 2000). This constitutive active mutant, STAT6VT, was not su cient alone to induce IL-13Ra2 mRNA expression, but strongly potentiated the induction triggered by IL-4 and IL-13.…”

“…The PCR fragment was then cut with XhoI and inserted in frame with the Flag Tag into the XhoI-blunt ended Bglll pCMV-Tag1 (Stratagene, La Jolla, CA, USA). The double mutant STAT6VT was prepared by introducing two alanine residues at amino acid positions 547 and 548 (Daniel et al, 2000) by using the Quick Change site directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The correct nucleotide sequences of PCR products and cloning junctions were veri®ed by DNA sequencing.…”

Complex Regulation of Tartrate-resistant Acid Phosphatase (TRAP) Expression by Interleukin 4 (IL-4)

“…We used the three key transcription factors that are essential for osteoclastic TRAP expression, NFATc1, PU.1, and MITF (8,11), and STAT6VT, a constitutively active form of STAT6 (42). The combination of NFATc1, PU.1, and MITF has been shown to induce TRAP transcription in the absence of RANKL treatment (8,11); STAT6VT can bind to the STAT6-responsive element and regulate transcription without IL-4 treatment (42). Various combinations of these transcription factors were transfected into RAW264.7 cells.…”

IL-10 Inhibits FcεRI Expression in Mouse Mast Cells

“…Though necessary for this signaling event, we have not determined whether Stat6 activation alone is sufficient to decrease IgE receptor expression. To this end, we infected Stat6-deficient BMMC populations with a retrovirus expressing constitutively active Stat6 (Stat6VT) (32) and GFP or with a control retrovirus expressing GFP alone. As shown in Fig.…”