JNK2 Interacts with b-Catenin Figure 5. Activated JNK2 interacts with b-catenin and GSK3b. (A) Active JNK2 binding to b-catenin and GSK3b was analyzed by immunoprecipitation. b-catenin (HA tagged) was co-transfected with empty vector or active JNK2 (Flag tagged) into HEK293T cells. Immunoprecipitation was performed with a Flag antibody. (B) Mammalian two-hybridization assays showed a strong binding of b-catenin and JNK2 protein. The experiments were triplicated independently. (C) Active JNK2 and b-catenin co-localized in the cell nucleus and cytoplasm. Active JNK2 (Flag tagged) and pEGFP-b-catenin were co-transfected into HEK293T cells. The cells were immunostained with a Flag antibody. Co-localization (yellow fluorescence) of active JNK2 (red fluorescence) and b-catenin (green fluorescence) was detected in the nucleus and cytoplasm. doi:10.1371/journal.pone.0006640.g005 (lane 4 versus 3), suggesting that GSK3b is required for JNK2- mediated b-catenin degradation. Post-translational modifications play critical role in the regulation of the b-catenin turnover by GSK3b [25]. To further confirm the role of GSK3b in b-catenin degradation induced by JNK2 activation, we generated series of constructs expressing b- catenin mutants [2] and co-transfected them with pMKK7-JNK2 into HKE293T cells. As shown in figure 3C, S33Y, S33F, or S37A mutation endowed b-catenin with less susceptibility to degradation induced by JNK2 activation, further demonstrating the require- ment of GS3b for JNK2-mediated degradation of b-catenin. Besides the indirect phosphorylation and degradation of b- catenin by JNK2 via GSK3b from our present study, a recent study has identified three more phosphorylation sites in b-catenin protein, two of them (Ser-191 and Ser-605) are critical for b- catenin stabilization and nuclear localization, and could be directly phosphorylated by JNK2, providing more evidences of the different roles of JNK2 in Wnt/b-catenin signaling [6]. However, JNK1 is not likely to have such a strong influence to phosphorylate Ser-191/605 of b-catenin [6]. Genetic Deficiency of JNK2 caused Upregulation of b- catenin, CDK4 and GSK3b Phosphorylation To validate the regulation b-catenin by JNK2 in vivo, JNK2-/- and +/+ mice were dissected. As shown in figure 4, b-catenin and its downstream target CDK4 were upregulated in intestinal epithelial cells from JNK2-/- mice, compared to the JNK2+/+ mice. Consistently, phosphorylated GSK3b was upregulated although total GSK3b was not changed. The in vivo data provided additional evidence that GSK3b is involved in JNK2-mediated b- catenin regulation. As to the effect of JNK2 on b-catenin downstream targets, our most recent in vitro study demonstrated that selenium-stimulated phosphorylation of JNK1/2 dramatically inhibited b-catenin and its downstream targets, such as c-myc, cyclin D1 and CDK4, in human colon cancer cells (Fang and Yang, et al, submitted), PLoS ONE | www.plosone.org 5 August 2009 | Volume 4 | Issue 8 | e6640