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X. Wu, P.G. Quinn, T.W. Gardner; P70S6K Is Involved in FKHR Translocation in Retinal Neuronal Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4585.
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Purpose: We showed previously that insulin rescues retinal neurons from apoptosis by a PI-3-Kinase dependent mechanism. This study tests the hypothesis that p70S6K regulates FKHR, a transcription factor implicated in cell survival. Methods: Differentiated R28 retinal neuronal cells and explants of normal rat retinas were used. The signal transduction pathways of insulin were investigated by pharmacologic inhibition of PI3-K and p70S6K inhibition with LY294002 and rapamycin, respectively. Cells were also transfected with wild type FKHR and P-site mutant FKHR (T/S/S---A/A/A) constructs and treated with 10 nM insulin. Cell lysates were immunoblotted with antibodies for phospho-specific and total FKHR, and parallel cultures were fixed for immunocytochemistry. Results: Insulin increased FKHR phosphorylation in R28 cells by 9 fold (P<0.001) and in retinal explants by 2.7 fold (P<0.01). LY294002 completely blocked FKHR phosphorylation and rapamycin reduced FKHR phosphorylation by 67% (P<0.01) in R28 cells and 52% (P<0.05) in retinal explants. Insulin induced translocation of wild type but not FKHR (T/S/S) from nucleus to cytoplasm in 90% (P<0.001) of GFP-positive cells; this translocation was completely blocked by LY294002 and reduced by 53% (P<0.001) by rapamycin. Co-transfection of wild type p70S6K and FKHR caused FKHR translocation to cytoplasm in 50% (P<0.001) of GFP-positive cells. Conclusions: Insulin stimulates FKHR phosphorylation and translocation in retinal neuronal cells and S6K1 contributes to FKHR translocation.
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