Phosphorylation and nuclear exclusion of the forkhead transcription factorFKHR after epidermal growth factor treatment in human breast cancer cells

Akt, when activated by IGF/insulin, can phosphorylate forkhead transcriptio n factors. We undertook this study to determine whether epidermal growth fa ctor (EGF) treatment could produce a signaling cascade resulting in phospho rylation of the forkhead transcription factor FKHR in a breast cancer cell line, MDA-MB-231, After establishing ErbB1, cbl, PI3 kinase and Akt were ac tivated in EGF treated MDA-MB-231, we determined by immunoblot with FKHR an tiserum that the electrophoretic mobility of FKHR was retarded after EGF tr eatment. This mobility retardation was reversible by treatment with alkalin e phosphatase, and immunoblot with phospho-Ser(256) FKHR antibody further c onfirmed phosphorylation on an Akt consensus site after EGF treatment. EGF stimulated FKHR phosphorylation was blocked by the PI3 kinase inhibitor LY2 94002, and the ErbB1 inhibitor AG1478, FKHR immunoblotting after purificati on of nuclear and cytoplasmic proteins showed that EGF induced a simultaneo us increase of FKHR in the cytoplasm and decrease in the nucleus. This find ing was confirmed by immunofluorescence staining. Treatment of cells with p harmacological inhibitors of PI3 kinase or ErbB1 blocked this effect. Thus, these results demonstrate the phosphorylation and nuclear exclusion of FKH R after EGF treatment by a PI3 kinase dependent mechanism, and represent th e first report of growth factor regulation of endogenous FKHR localization.