Phosphorylation and nuclear exclusion of the forkhead transcription factor FKHR after epidermal growth factor treatment in human breast cancer cells.
Akt, when activated by IGF/insulin, can phosphorylate forkhead transcription factors. We undertook this study to determine whether epidermal growth factor (EGF) treatment could produce a signaling cascade resulting in phosphorylation of the forkhead transcription factor FKHR in a breast cancer cell line, MDA-MB-231. After establishing ErbB1, cbl, PI3 kinase and Akt were activated in EGF treated MDA-MB-231, we determined by immunoblot with FKHR antiserum that the electrophoretic mobility of FKHR was retarded after EGF treatment. This mobility retardation was reversible by treatment with alkaline phosphatase, and immunoblot with phospho-Ser256 FKHR antibody further confirmed phosphorylation on an Akt consensus site after EGF treatment. EGF stimulated FKHR phosphorylation was blocked by the PI3 kinase inhibitor LY294002, and the ErbB1 inhibitor AG1478. FKHR immunoblotting after purification of nuclear and cytoplasmic proteins showed that EGF induced a simultaneous increase of FKHR in the cytoplasm and decrease in the nucleus. This finding was confirmed by immunofluorescence staining. Treatment of cells with pharmacological inhibitors of PI3 kinase or ErbB1 blocked this effect. Thus, these results demonstrate the phosphorylation and nuclear exclusion of FKHR after EGF treatment by a PI3 kinase dependent mechanism, and represent the first report of growth factor regulation of endogenous FKHR localization.
Pubmed ID: 11030146 RIS Download
Alkaline Phosphatase | Biological Transport | Breast Neoplasms | Cell Nucleus | Chromones | Cytoplasm | DNA-Binding Proteins | Epidermal Growth Factor | Female | Forkhead Transcription Factors | Gene Expression Regulation, Neoplastic | Humans | Macromolecular Substances | Morpholines | Neoplasm Proteins | Phosphatidylinositol 3-Kinases | Phosphorylation | Protein Processing, Post-Translational | Protein-Serine-Threonine Kinases | Proto-Oncogene Proteins | Proto-Oncogene Proteins c-akt | Proto-Oncogene Proteins c-cbl | Quinazolines | Receptor, Epidermal Growth Factor | Signal Transduction | Transcription Factors | Tumor Cells, Cultured | Tyrphostins | Ubiquitin-Protein Ligases