In the cytoplasm, Snail1 is quickly degraded; it has a half-life of only twenty-five minutes [33]. To protect from this degradation, Snail1 has nuclear localization signals (NLS): one monopartite from amino acids 151-152 and one bipartite overlapping the SNAG domain between amino acids 8 and 16 [38]. These signals are responsible for the nuclear transport of Snail1, which in turn is required for proper expression. β-catenin, Lef-1,

and IκB employ similar systems [38] (Figure 3, Table 1). Figure 3 Snail1 stability and localization. This figure shows the effects of GSK-3β and PAK1-mediated phosphorylation on Snail1 stability and subcellular localization. The outer circle represents the cell membrane, and the inner circle represents the #AZD6738 randurls[1|1|,|CHEM1|]# nucleus. Nuclear Snail1 is phosphorylated by GSK-3β at motif 2 and is BIBW2992 supplier consequently exported from the nucleus. If Snail1 remains in the cytoplasm, it is ultimately ubiquitinated and

degraded. By contrast, phosphorylation by PAK1 favors the nuclear localization of Snail1, which increases its stability. Table 1 Regulation of Snail1 expression Direct regulators Interaction location Upstream pathway(s) Reference(s) LOXL2/3 SNAG domain; K98 and K127 Notch/Lox [17] NF-κB Promoter: -194 to -78 bp TNFα, RANKL, PI3K/Akt [20,43,44] HIF-1α Promoter: -750 to -643 bp Hypoxic conditions [19] SMADs Promoter: -631 to -506 bp TGF-β1, Ras [45,46] IKKα Promoter: -631 to -506 bp (concurrent with SMADs) TGF-β1, Ras, PI3K/Akt [21,44,46] HMGA2 Promoter: 2 regions within -131 to -92 bp TGF-β1 [22] YY1 3’ Enhancer NF-κB [30] Egr-1 Promoter: 4 Anacetrapib sites between -450 and -50 bp HGF, MAPK [29] PARP-1 Promoter: SIRE

ILK [23] Gli1 There are 4 candidate GLI binding sites (consensus sequence for binding: 5′-GACCACCCA-3′) Shh, Wnt [26] STAT3 Promoter IL-6/JAK, HB-EGF/EGFR/MEK/ERK (mice) [24,25] MTA3 Promoter ER [27,28] PAK1 S246   [36] GSK-3β Motif 1 (S96, S100, S104) and Motif 2 (S107, S111, S115, S119) Wnt, PI3K/Akt, FGF [33,34] Snail1 Promoter: E box within SIRE Binds to own promoter [31] TNFα, NF-κB, FGF, Wnt, and microRNA signals also influence the regulation of GSK-3β-mediated phosphorylation. The TNFα/NF-κB pathway induces CSN2, which protects Snail1 from degradation by interfering with the binding of GSK-3β and β-Trcp. Thus, Snail1 is neither phosphorylated nor ubiquitylated [39]. FGF operates through the PI3K/Akt pathway to downregulate GSK-3β, and receptor tyrosine kinase induces EGF suppression of GSK-3β [34,40]. Wnt can also suppress GSK-3β and, thus, the phosphorylation of Snail1 [41]. Additionally, miR-148a causes the phosphorylation of AKT and GSK-3β, which results in less Snail1 localized in the nucleus. This, in turn, inhibited EMT in hepatocellular carcinoma [42]. Phosphorylation of upstream targets also influences the regulation of Snail1.