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Chemical Genetic Analysis of FOXO Nuclear–Cytoplasmic Shuttling by Using Image‐Based Cell Screening
Author(s) -
Zanella Fabian,
Rosado Aranzazú,
García Beatriz,
Carnero Amancio,
Link Wolfgang
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200800255
Subject(s) - nuclear export signal , microbiology and biotechnology , pi3k/akt/mtor pathway , nuclear transport , signal transduction , biology , nuclear localization sequence , wortmannin , transcription factor , protein kinase b , chemistry , cytoplasm , biochemistry , cell nucleus , gene
FOXO proteins are direct targets of PI3K/Akt signaling and they integrate the signals of several other transduction pathways at the transcriptional level. FOXO transcription factors are involved in normal cell homeostasis and neoplasia, and they are regulated by multiple post‐transcriptional modifications. In cancer research, the regulation of the FOXO factors is receiving increasing attention as their activation has been linked to cell‐cycle arrest and apoptosis. Hence, FOXO proteins have been proposed to act as tumor suppressors. Here, we applied a chemical biology approach to study the mechanisms that influence the intracellular localization of the FOXO family member FOXO3a. We established a high‐throughput cellular‐imaging assay that monitors the nuclear–cytoplasmic translocation of a GFP–FOXO3a fusion protein in tumor cells. Nuclear accumulation of fluorescent signals upon treatment with the known PI3K inhibitors LY294002, wortmannin, PIK‐75, and PI‐103 was dose dependent and agreed well with the IC 50 values reported for PI3Kα inhibition in vitro. Additionally, we identified 17 compounds from a panel of 73 low‐molecular‐weight compounds capable of inducing the nuclear accumulation of GFP–FOXO. These compounds include chemicals known to interfere with components of the PI3K/Akt signaling pathway, as well as with nuclear export and Ca 2+ /calmodulin (CaM)‐dependent signaling events. Interestingly, the therapeutic agent vinblastine induced efficient nuclear translocation of the FOXO reporter protein. Our data illustrate the potential of chemical genetics when combined with robust and sensitive high‐content‐screening technology.