Foxo1 nuclear‐cytoplasmic movement in living skeletal muscle

Abstract

The transcription factor Foxo1 is integral to the regulation of expression of proteins which promote muscle atrophy. Phosphorylation of Foxo1 causes its translocation to the cytoplasm and thus prevents Foxo1-DNA binding and consequent transcription of genes that cause muscle atrophy and cell death. Thus, phosphorylation of Foxo1 leads to cell survival and muscle hypertrophy. Maintenance of Foxo1 phosphorylation and its resulting cytoplasmic retention could be used to suppress muscle atrophy and thereby shift the atrophy/hypertrophy balance in favor of hypertrophy. Akt and serum- and glucocorticoid-inducible kinase (SGK) are important regulators of the phosphorylation status of Foxo1. These pathways have been well characterized and the effects on Foxo1 localization have been reported. However, the mechanisms which regulate nuclear influx and nuclear efflux have not been separately evaluated. Here, we determine the effects of Akt kinase activity specifically on nuclear influx of Foxo1. To accomplish th/is goal, we quantified nuclear and cytoplasmic levels of adenovirally expressed Foxo1-GFP in cultured flexor digitorum brevis. The nuclear influx during treatment with kinase inhibitor Akt IV alone and in combination with the nuclear efflux inhibitor lemptomycin B provides insight into the activity of Akt as a Foxo1 kinase. Surprisingly, Akt inhibition reveals Akt to have little effect on the rate of nuclear influx of Foxo1. To evaluate translocation of endogenous Foxo1 in a similar manner immunocytochemistry and western blotting techniques were used. These results indicate that Akt either phosphorylates Foxo1 primarily in the nucleus and not in the cytoplasm, or that there is a mechanism for cytoplasmic phosphorylation of Foxo1 other than via Akt. Supported by training grants T32 AR0075592 and T32 HL072751 and research grant R01 AR056477.