Interleukin-4 induces two distinct GAS-binding complexes containing STAT6: evidence for DNA binding of STAT6 monomer.
Treatment of tonsillar mononuclear cells with interleukin-4 (IL-4) rapidly induced the formation of two distinct complexes upon reaction of the cell extracts with the IL-4-responsive element (IL-4RE) of the CD23b promoter in the electrophoretic mobility sift assays (EMSA). The two complexes were detected with a similar activation kinetics upon IL-4 stimulation. They were both immunoreactive with antibodies to signal transducer and activator of transcription (STAT) 6. The upper complex, however, appeared more stable in the competitor oligomer binding assays and more resistant in the anti-phosphotyrosine antibody inhibition assays than the lower complex. Western blot analysis revealed a single peptide of 105 kDa reacting with anti-STAT6 antibodies, which ruled out the possibility for multiple isoforms of STAT6 in these cells. Subsequently, Southwestern analysis demonstrated that monomeric STAT6 in the IL-4-treated nuclear extract can bind the labeled IL-4RE in an activation-dependent manner. Our data strongly suggests that in addition to dimeric STAT6, monomeric STAT6, albeit with a lower affinity, can bind the IL-4RE/gamma activation site (GAS) upon IL-4-induced activation. Also, the upper and lower bands observed in EMSA are likely to represent dimeric and monomeric STAT6 bound to the IL-4RE oligomer, respectively. The functional implication of the STAT6 monomer binding to GAS in the IL-4-induced gene activation is discussed.
Department of Biological Science and Institute for Basic Science, College of Natural Science, SungKyunKwan University, Suwon, Korea. celee@yurim.skku.ac.kr
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CE
Lee
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HJ
Park
D015139:Blotting, Southern; D015153:Blotting, Western; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015847:Interleukin-4 / Q000494:pharmacology*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D014066:Palatine Tonsil / Q000166:cytology; D011401:Promoter Regions, Genetic / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D017455:Receptors, IgE / Q000235:genetics / Q000378:metabolism; D050800:STAT6 Transcription Factor; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D017455:Receptors, IgE; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D015534:Trans-Activators; D015847:Interleukin-4