Role of p90 ribosomal S6-kinase-1 in oltipraz-induced specific phosphorylation of CCAAT/enhancer binding protein-beta for GSTA2 gene transactivation.
Oltipraz, which has been extensively studied as a cancer chemopreventive agent, promotes phosphatidylinositol 3-kinase-mediated activation of CCAAT/enhancer binding protein-beta (C/EBPbeta). Activated p90 ribosomal S6-kinase-1 (RSK1) phosphorylates major transcription factors, including C/EBPbeta. This study examined whether oltipraz induces phosphorylation of C/EBPbeta at specific residues, and if so, whether RSK1 regulates C/EBPbeta phosphorylation by oltipraz for the GSTA2 gene transactivation. Subcellular fractionation and immunoblot analyses revealed that oltipraz treatment increased the level of C/EBPbeta phosphorylated at Ser(105) in the cytoplasm, which translocated to the nucleus for DNA binding in rat H4IIE cells. Immunoprecipitation-immunoblot, chromatin-immunoprecipitation, and specific mutation analyses revealed that Ser(105)-phosphorylated C/EBPbeta recruited the cAMP response element-binding protein binding protein for histone acetylation and transactivation of the GSTA2 gene. The role of RSK1 in Ser(105)-phosphorylation of C/EBPbeta by oltipraz and its gene transactivation was evidenced by transfection experiments with dominant-negative mutants of RSK1. In mouse Hepa1c1c, human HepG2 cells, and rat primary hepatocytes, oltipraz induced phosphorylation of C/EBPbeta at Thr(217), Thr(266), and Ser(105), respectively, via RSK1. The experiment using small-interference RNA of RSK1 confirmed the essential role of RSK1 in the gene expression. Inhibition of PI3-kinase activity prevented oltipraz-inducible Ser(105)-phosphorylation of rat C/EBPbeta. Oltipraz treatment led to increases in the catalytic activity and nuclear translocation of RSK1, which was abrogated by PI3-kinase inhibition. In summary, oltipraz induces the phosphorylation of rat C/EBPbeta at Ser(105) (functionally analogous Thr(217/266) in mouse and human forms) in hepatocytes, which results in cAMP response element-binding protein-binding protein (CBP) recruitment for the GSTA2 gene transactivation, and the specific C/EBPbeta phosphorylation is mediated by RSK1 downstream of PI3-kinase.
Lee
Seung Jin
SJ
National Research Laboratory, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea.
Kim
Sang Geon
SG
D000595:Amino Acid Sequence; D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011719:Pyrazines / Q000494:pharmacology*; D034741:RNA, Small Interfering; D051381:Rats; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013871:Thiones; D013876:Thiophenes; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection
D022782:CCAAT-Enhancer-Binding Protein-beta; D005982:Glutathione Transferase; D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases; D011719:Pyrazines; D034741:RNA, Small Interfering; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D012694:Serine; D013871:Thiones; D013876:Thiophenes; C026209:oltipraz; C119995:glutathione S-transferase alpha