Estimating the Rate of FOXO1 Phosphorylation and Dephosphorylation Using Live Cell Imaging

Abstract

FoxO1 is a signaling transcription factor regulated by the growth factor/PI3K/AKT pathway. Phosphorylation of FOXO1 by the serine/threonine kinase AKT, sequesters FOXO1 in the cytoplasm by blocking the interaction of FOXO1’s nuclear localization signal (NLS) with nuclear transport receptors and promoting FOXO1 binding to the cytoplasmic 14-3-3 proteins. Dephosphorylation of FOXO1 by the phosphatase PP2A restores NLS function and leads to accumulation of FOXO1 in the nucleus. Here, we use fluorescently labeled FOXO1 to characterize its nuclear trafficking dynamics under conditions of AKT and PP2A inhibition in order to describe the relative cytoplasmic dephosphorylation rate by PP2A and relative nuclear phosphorylation rate by AKT on FOXO1 respectively. Measured results affirm previous data that indicates AKT is less active in the nucleus than the cytoplasm and suggests that FOXO1 may undergo rapid shuttling into and out of the nucleus even during AKT activation.