GSK3b Is Involved in JNK2-Mediated b-Catenin Inhibition Dong Hu 1 , Xiuli Bi1, Wenfeng Fang 1 , Anjia Han 2 , Wancai Yang 1 * 1 Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, United States of America, 2 Department of Pathology, the First Affiliated Hospital and Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China Abstract Background: We have recently reported that mitogen-activated protein kinase (MAPK) JNK1 downregulates b-catenin signaling and plays a critical role in regulating intestinal homeostasis and in suppressing tumor formation. This study was designed to determine whether JNK2, another MAPK, has similar and/or different functions in the regulation of b-catenin signaling. Methodology and Principal Findings: We used an in vitro system with manipulation of JNK2 and b-catenin expression and found that activated JNK2 increased GSK3b activity and inhibited b-catenin expression and transcriptional activity. However, JNK2-mediated downregulation of b-catenin was blocked by the proteasome inhibitor MG132 and GSK3b inhibitor lithium chloride. Moreover, targeted mutations at GSK3b phosphorylation sites (Ser33 and Ser37) of b-catenin abrogated JNK2- mediated suppression of b-catenin. In vivo studies further revealed that JNK2 deficiency led to upregulation of b-catenin and increase of GSK3-b phosphorylation in JNK2-/- mouse intestinal epithelial cells. Additionally, physical interaction and co- localization among JNK2, b-catenin and GSK3b were observed by immunoprecipitation, mammalian two-hybridization assay and confocal microscopy, respectively. Conclusion and Significance: In general, our data suggested that JNK2, like JNK1, interacts with and suppresses b-catenin signaling in vitro and in vivo, in which GSK3b plays a key role, although previous studies have shown distinct functions of JNK1 and JNK2. Our study also provides a novel insight into the crosstalk between Wnt/b-catenin and MAPK JNKs signaling. Citation: Hu D, Bi X, Fang W, Han A, Yang W (2009) GSK3b Is Involved in JNK2-Mediated b-Catenin Inhibition. PLoS ONE 4(8): e6640. doi:10.1371/ journal.pone.0006640 Editor: Andreas Bergmann, University of Texas MD Anderson Cancer Center, United States of America. Received April 30, 2009; Accepted July 16, 2009; Published August 13, 2009 Copyright: 2009 Hu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grant (CA112081). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: wyang06@uic.edu Introduction The Wnt/b-catenin signaling plays a critical role in embryonic development and the regulation of homeostatic self-renewal in a number of adult tissues [1]. However, an aberrant activation of Wnt/b-catenin signaling, such as b-catenin accumulation and nuclear translocation, leads to transactivation of transcription of a broad range of genes and contributes to carcinogenesis, although this signaling pathway could be regulated by other signaling, molecules or pathways, including a destruction complex consisting of casein kinase Ia (CKIa), glycogen synthase kinase 3b (GSK3b), adenomatous polyposis coli (APC) and Axin. We recently reported that mitogen-activated protein kinase (MAPK) JNK1 interacts with and negatively regulates b-catenin signaling through GSK3b [2], and that the b-catenin alteration is likely to be associated with intestinal tumor formation induced by JNK1 inactivation [2]. Recently, an increasing body of evidence supported the crosstalk between MAPK and Wnt/b-catenin signaling [3–8]. It was reported that p38 MAPK regulates canonical Wnt/b-catenin signaling by inactivation of GSK3b [7,8]. However, JNK1, another important member of MAPK, was suggested to antagonize Wnt/b-catenin signaling by activating GSK3b [2]. The JNKs are encoded by three genes, namely JNK1, JNK2, and JNK3. JNK1 and JNK2 are ubiquitously expressed, whereas the expression of JNK3 is largely restricted to brain, heart, and testis [9]. Numerous studies have reported distinct functions of JNK1 and JNK2, for example, mice harboring JNK1 or JNK2 inactivation exhibited opposite susceptibility to tumor formation induced by 12-O-tetra- decanoylphorbol-13-acetate (TPA) [10,11]. In this study, we found that, like JNK1, JNK2 interacts with and inhibits b-catenin signaling, in which GSK3b functions upstream of proteasome pathway and plays a critical role in JNK2-mediate b-catenin degradation. Materials and Methods Cell Culture, Plasmids Construction and Transfection Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco’s modified Eagle’s medium, human lung cancer cells A549 were grown in minimal essential medium. pcDNA3-Flag- MKK7-JNK2 (active JNK2) and pcDNA3-Flag-MKK7-JNK1 (active JNK1) were provided by Dr.Davis [12], pcDNA3- hemagglutinin (HA)-b-catenin, HA-tagged b-catenin mutants (HA-S33Y b-catenin, HA-S33F b-catenin and HA-S37A b- catenin), pACT-JNK2 and pBIND- b-catenin were constructed as described previously [2,13–15]. TCF-4 reporter plasmid TOPFLASH and the control inactive reporter FOPFLASH were purchased from Upstate Biotechnology (Lake Placid, NY). Reporter plasmid pTK-Renilla was purchased from Promega PLoS ONE | www.plosone.org 1 August 2009 | Volume 4 | Issue 8 | e6640