Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms

Abstract

FOXO1, a Forkhead transcription factor, is an important target of insulin and growth factor action. Phosphorylation of Thr-24, Ser-256 and Ser-319 promotes nuclear exclusion of FOXO1, yet the mechanisms regulating nuclear/cytoplasmic shuttling of FOXO1 are poorly understood. Previous studies have identified an NLS (nuclear localization signal) in the C-terminal basic region of the DBD (DNA-binding domain), and a leucine-rich, leptomycin-B sensitive NES (nuclear export signal) located further downstream. Here, we find that other elements in the DBD also contribute to nuclear localization, and that multiple mechanisms contribute to nuclear exclusion of FOXO1. Phosphorylation of Ser-319 and a cluster of nearby residues (Ser-322, Ser-325 and Ser-329) functions co-operatively with the nearby NES to promote nuclear exclusion. The N-terminal region of FOXO1 (amino acids 1-149) also is sufficient to promote nuclear exclusion, and does so through multiple mechanisms. Amino acids 1-50 are sufficient to promote nuclear exclusion of green fluorescent protein fusion proteins, and the phosphorylation of Thr-24 is required for this effect. A leucine-rich, leptomycin B-sensitive export signal is also present nearby. Phosphorylated FOXO1 binds 14-3-3 proteins, and co-precipitation studies with tagged proteins indicate that 14-3-3 binding involves co-operative interactions with both Thr-24 and Ser-256. Ser-256 is located in the C-terminal region of the DBD, where 14-3-3 proteins may interfere both with DNA-binding and with nuclear-localization functions. Together, these studies demonstrate that multiple elements contribute to nuclear/cytoplasmic shuttling of FOXO1, and that phosphorylation and 14-3-3 binding regulate the cellular distribution and function of FOXO1 through multiple mechanisms. The presence of these redundant mechanisms supports the concept that the regulation of FOXO1 function plays a critical role in insulin and growth factor action.

shRNA against CD44 inhibits cell proliferation, invasion and migration, and promotes apoptosis of colon carcinoma cells

“…shRNA CD44 inhibited Ser241 phosphorylation of PDK1. This inhibition of the PDK1/Akt molecule is an important signal for cell survival (27). GSK3β is the target of PDK1/Akt signal transduction.…”

“…The loss of GSK3 protein explains the stable accumulation of nonphosphorylated β-catenin that is accessible to the adherens junction at the cell periphery (47). The Wnt/β-catenin pathway was very recently reported to protect cells from p53-mediated FoxO1-induced apoptosis, a mechanism involving the activation of the Akt survival pathway (27,29). Silencing of CD44 expression in SW620 cells, indicating decreased β-catenin, increased wild-type p53 and FoxO1 expression.…”

“…Silencing of CD44 expression in SW620 cells, indicating decreased β-catenin, increased wild-type p53 and FoxO1 expression. Activation of FoxO1 induces apoptosis by up-regulating a number of cell death genes, including those encoding the ligand for the death receptor (Fas) and the Bcl-2-interacting mediator (Bim) of cell death, and the tumor necrosis factor-related apoptosis-inducing ligand (27,33). Sub-cellular localization of wild-type FoxO1 is controlled primarily by phosphorylation, leading to nuclear export and subsequent repression of transcriptional activity (25,26).…”

“…Nuclear localization of forkhead box transcription factor O (FoxO) proteins is required for transcriptional regulatory functions, which include the control of genes involved in apoptosis, such as Bim (18,19) and FasL (20), and genes involved in cell cycle regulation, such as p27 (21,22) and cyclin D1 and D2 (23,24). As major direct substrates of FoxO factors are negatively regulated by Akt phosphorylation in the presence of growth factor signaling (25,26), which results in binding to 14-3-3 proteins followed by nuclear export (27). Akt is recruited to the plasma membrane through the binding of its pleckstrin homology domain to the phosphatidylinositol 3,4,5-triphosphate (PIP3), which is a product of PI3K that is anchored to the plasma membrane.…”

Loss of expression and nuclear/cytoplasmic localization of the FOXP1 forkhead transcription factor are common events in early endometrial cancer: relationship with estrogen receptors and HIF-1α expression

“…Nuclear/cytoplasmic shuttling of members of the forkhead protein family (ie FOXO1) has been reported to occur as a result of protein phosphorylation. 28 Phosphorylation of FKHRL1 via a phosphatidylinositol 3-kinase/Akt kinase results in cytoplasmic shuttling of the protein, 29 which suggests that similar mechanisms may be also involved in FOXP1 cytoplasmic localization. Given that hypoxia both enhances Act activity 30 and induces HIF-1a and -2a overexpression, the significant association between the cytoplasmic localization of FOXP1 and HIF-1a overexpression may be the result of a single cause, namely hypoxia.…”