Figure 7. 14-3-3s enhances Wnt/b-catenin signaling. (A) CCE cells were transfected with control or 14-3-3s vectors for 30 h followed by treatment with recombinant mouse Wnt3a (10 ng/mL) for 18 h. Recombinant mouse DKK-1 (1 ng/mL) was added 2 h before Wnt3a. Protein levels of b-catenin, GSK-3b and phosphorylated GSK-3b were detected by Western blot analysis. (B) 14-3-3s/GSK-3b complex was determined by IP with a GSK-3b antibody, and Flag and GSK-3b proteins were analyzed by Western blotting. doi:10.1371/journal.pone.0040193.g007 nous 14-3-3s and GSK-3b (Figure 7B). 14-3-3s binding of GSK- 3b was detected in 14-3-3s transfected cells which were enhanced by Wnt3a treatment (Figure 7B). DKK1 did not have a significant effect on interaction of 14-3-3s with GSK-3b in 14-3-3s transfected cells treated with Wnt3a (Figure 7B). These results suggest high levels of 14-3-3s augment the Wnt signaling to enhance GSK-3b inactivation and increase b-catenin stability. 14-3-3s Induces Mouse ES Cell Proliferation Discussion Our findings provide important information regarding the novel role of 14-3-3s in regulating mESC proliferation. Despite the expression of all seven isoforms of 14-3-3 proteins in mESCs, only 14-3-3s participates in mESC proliferation by binding, sequestration and inactivating GSK-3b. Our results demonstrate that 14-3-3s overexpression enhances GSK-3b phosphorylation and inactivation as well as increases interaction between 14-3-3s and GSK-3b. Furthermore, 14-3-3s over- expression triggers dissociation of b-catenin from the APC/ axin/GSK-3b complex, the so-called multiprotein destruction complex. Since the transcriptional bioavailability of b-catenin is tightly controlled by GSK-3b in the destruction complex, our data lead us to conclude that 14-3-3s is capable of sequestering GSK-3b and thereby releasing b-catenin from the multiprotein destruction complex which translocates into the nucleus and carries out the proliferative transcription. GSK-3b inactivation depends on phosphorylation within the multiprotein destruction complex. At resting state, GSK-3b is active in phosphorylating b-catenin to induce its degradation via ubiquitination/proteasome. When stimulated by Wnt, GSK-3b is phosphorylated and dissociated from the multiprotein destruction complex, thus releasing b-catenin. It is generally thought that phosphorylated GSK-3b is rapidly dephosphorylated and reasso- ciated with the APC/axin complex. In this study, we provide evidence that phosphorylated GSK-3b is controlled by 14-3-3s. High levels of 14-3-3s sequester and inactivate GSK-3b via which they enhance Wnt signaling to increase b-catenin. It is interesting that DKK blocks the effect of Wnt3a as expected but did not interfere with action of 14-3-3s on GSK-3b binding. These findings indicate that 14-3-3s provides a discrete pathway to control GSK-3b availability and activity. It is well recognized that RA induces ES cells to undergo differentiation and proliferation arrest. A number of mechanisms of RA actions have been proposed but the exact mechanisms are not clear. We show in this study that 14-3-3s/GSK-3b pathway is involved in RA-induced inhibition of mESC proliferation. RA selectively suppresses 14-3-3s. It increases b-catenin phosphory- lation and reduces b-catenin resulting in reduction of mESC proliferation. High levels of 14-3-3s confer resistance to RA by restoring GSK-3b phosphorylation and sequestration. Thus, 14-3- 3s is pivotal in regulating GSK-3b/b-catenin bioavailability as illustrated in Fig. 8. Our results reveal that knockdown of 14-3-3s with siRNA reduces mESC proliferation by only 30–40% compared to control (Figure 1C and 1D), suggesting that mESC proliferation does not depend entirely on 14-3-3s. A compensatory effect may be regulated by other signal pathways. This notion was supported by a recent report which indicates that 14-3-3s-deleted mESC give rise to viable mice with B-cell developmental defects [40]. It is interesting that of all seven 14-3-3 isoforms expressed in mESCs, only 14-3-3s is involved in regulating b-catenin-mediated mESC proliferation. In contrast, 14-3-3f was reported to bind GSK-3b and enhances Tau phosphorylation in brain [19], and 14-3-3f was reported to facilitate b-catenin export from the nucleus and thereby reduces b-catenin transcriptional activity [22]. Reasons for differential regulation of GSK-3b and b-catenin by different 14-3- 3 isoforms in different tissues and cells are unclear and require further investigation. According to the result in association of 14-3-3s with GSK-3b S9A mutant, our results indicate that 14-3-3s binds preferen- tially phosphorylated GSK-3b (Figure 5C). Using a pharmaco- logical inhibitor of PI-3K, we show that PI-3K/Akt is required PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e40193