Nuclear Localization Signals of the E-Cadherin Transcriptional Repressor Snail

Abstract

The E-cadherin transcriptional repressor, Snail, plays a critical role in driving the epithelial-mesenchymal transition programs that mark gastrulation as well as invasion of cancer cells. Recent data suggest that Snail is phosphorylated by GSK3-β, resulting in β-TRCP-mediated ubiquitination and proteasomal degradation. Accordingly, Wnt signaling inhibits Snail phosphorylation, and consequently increases Snail protein levels. In the present study, we examine the function of nuclear localization motifs embedded within the Snail sequence. A typical bipartite nuclear localization signal (NLS) motif is located at the N-terminal of Snail, where it overlaps with the SNAG domain (residues 8–16), while a basic cluster NLS motif is found proximal to zinc finger domains (residues 151–152). Mutational inactivation of these NLS signals resulted in decreased levels of nuclear and total Snail protein as well as attenuated Snail repressor activity on an E-cadherin promoter construct, suggesting that NLS motifs are essential for proper function. In the presence of GSK3 inhibitor LiCl, the cytoplasmic levels of the NLS mutants increased, suggesting that cytosolic Snail undergoes rapid phosphorylation and degradation. Given the highly conserved nature of the Snail NLS motifs (from Xenopus to human), these results indicate that nuclear localization signals regulate Snail expression and subcellular localization via GSK3-β-dependent phosphorylation.