N-Cadherin Anti-Apoptotic Signaling Figure 11. Overexpression of Bim-EL S69G prevents N-cadherin induced cell survival. Expression vectors coding for wild type Bim-EL and Bim-EL S69G were lipofected in 24 hours starved GT1-7 cells together with pEGFP at a 2/1 ratio. Cell lysates of equivalent protein content were analyzed by western blotting at 8 hours and 26 hours post-transfection for Bim and GFP expression; actin was analyzed as loading control (A). Two hours after lipofection, cells were harvested from transfected cultures and seeded on N-cadherin. Preparations were fixed 6 hours and 24 hours later (corresponding to 8 hours and 26 hours post-transfection) and nuclei stained. The percentage of GFP positive cells with condensed nuclei was then determined for each condition in duplicates ($100 GFP+ cells counted each time). Represented data are the mean of two independent experiments. Asterisks indicate a significant difference (P,0.05) as compared with GFP expression alone. doi:10.1371/journal.pone.0033206.g011 down-regulation of Bim-EL, suggesting that integrin-mediated inhibition of anoikis may similarly rely, to some extent, on a Bim- EL down-regulation in neuronal cells as previously described in MCF-10A cells [14]. The MAP kinase pathway has been implicated in the regulation of Bim-EL expression in various other cell types like hematopoietic cells [52], fibroblasts [53], epithelial cells [14] and neurons [54]. Studies in PC12 neural cells have shown that NGF protects neurons against apoptosis by acute Mek1/2-dependent phosphor- ylation of Bim-EL and subsequent down-regulation of this specific isoform [54]. In epithelial [14] and cancer cells lines [51], inhibition of the MAP Kinase pathway was also reported to abolish Bim-EL phosphorylation and upregulate Bim protein levels in association with ano¨ıkis. Phosphorylation of Bim-EL by Erk1/2 on Ser-69 has been reported to promote its degradation via the proteasome pathway [33,55,56,57]. However, in our model, we were not able to detect an accumulation of Bim-EL upon treatment with proteasome inhibitors, neither we were able to detect Bim-EL ubiquitination (data not shown). Recently, an alternative mechanism was proposed by Ewings et al [58]. Erk1/2- dependent phosphorylation of Bim-EL promotes its rapid dissociation from Bcl-xL and Mcl-1 proteins and this dissociation may contribute to Bim-EL degradation [58]. Therefore, it remains to elucidate whether, in neuronal cells, Bim-EL phosphorylation drives its subsequent degradation and by which mechanisms. Nevertheless, in support to en essential role of Erk-dependent Bim- EL phosphorylation in triggering N-cadherin engagement-medi- ated neuronal cell survival, we observed an increased apoptosis on N-cadherin of cells overexpressing Bim-EL S69G. Interestingly, the overexpression of the wild type form of Bim-EL also impaired with a delay the survival of GT1-7 cells plated on N-cadherin. Altogether, these data indicate that the accumulation of Bim-EL prevents N-cadherin-induced cell survival, supporting the hypoth- esis that N-cadherin triggers neuronal cell survival through Bim- EL down-regulation. It has long been recognized that cadherin function in close cooperation with, and organize, the cytoskeleton (for review: [1]). In turn, cytoskeleton and in particular microtubules may serve as Figure 12. Hypothetical model for N-cadherin mediated cell survival signaling in neurons. doi:10.1371/journal.pone.0033206.g012 PLoS ONE | www.plosone.org 13 March 2012 | Volume 7 | Issue 3 | e33206