Role of three FKHR phosphorylation sites in insulin inhibition of FKHR action in hepatocytes.
Insulin inhibits insulin-like growth factor binding protein-1 (IGFBP-1) transcription by preventing FKHR protein family members from binding a specific insulin response element in the IGFBP-1 promoter. In most cells, three serine/threonine moieties in FKHR family members are phosphorylated after insulin treatment or protein kinase B/Akt (PKB) transfection, and each of the three phosphorylated PKB sites contributes to insulin- or PKB-mediated inhibition of both the action and the nuclear localization of FKHR family members. In hepatocytes, however, the middle PKB site (PKB2) of FKHR was required for insulin to phosphorylate FKHR and was the only PKB site that participated in insulin inhibition of FKHR action, indicating that insulin utilizes a unique pathway to regulate FKHR action in hepatocytes. In studies presented here, plasmids expressing native or mutant FKHR forms, either with or without N-terminal fusion to green fluorescent protein (GFP), were transiently transfected into HEP G2 cells. All FKHR forms stimulated IGFBP-1 promoter activity, and mutating any of the three FKHR PKB sites impaired the ability of insulin both to inhibit FKHR-stimulated IGFBP-1 promoter activity and to induce FKHR accumulation in cytoplasm. Thus, in hepatocytes as in other cell lines, all three FKHR PKB sites participate in insulin-mediated inhibition of FKHR action and in insulin-mediated accumulation of FKHR in cytoplasm.
Scheimann
A O
AO
Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.
Durham
S K
SK
Suwanichkul
A
A
Snuggs
M B
MB
Powell
D R
DR
D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D049452:Green Fluorescent Proteins; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000235:genetics / Q000502:physiology; D008164:Luminescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D007328:Insulin; D018970:Insulin-Like Growth Factor Binding Protein 1; D008164:Luminescent Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins