Wnt‐induced deubiquitination FoxM1 ensures nucleus β‐catenin transactivation

Article24 February 2016free access Transparent process Wnt-induced deubiquitination FoxM1 ensures nucleus β-catenin transactivation Yaohui Chen Yaohui Chen Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Yu Li Yu Li Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Cell Engineering Research Center and Department of Cell Biology, Fourth Military Medical University, Xi'an, China Search for more papers by this author Jianfei Xue Jianfei Xue Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Aihua Gong Aihua Gong Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Guanzhen Yu Guanzhen Yu Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Aidong Zhou Aidong Zhou Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Kangyu Lin Kangyu Lin Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Sicong Zhang Sicong Zhang Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Nu Zhang Nu Zhang Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Cara J Gottardi Cara J Gottardi Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Suyun Huang Corresponding Author Suyun Huang Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Program in Cancer Biology, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, USA Search for more papers by this author Yaohui Chen Yaohui Chen Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Yu Li Yu Li Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Cell Engineering Research Center and Department of Cell Biology, Fourth Military Medical University, Xi'an, China Search for more papers by this author Jianfei Xue Jianfei Xue Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Aihua Gong Aihua Gong Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Guanzhen Yu Guanzhen Yu Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Aidong Zhou Aidong Zhou Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Kangyu Lin Kangyu Lin Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Sicong Zhang Sicong Zhang Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Nu Zhang Nu Zhang Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Cara J Gottardi Cara J Gottardi Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Suyun Huang Corresponding Author Suyun Huang Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Program in Cancer Biology, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, USA Search for more papers by this author Author Information Yaohui Chen1,‡, Yu Li1,2,‡, Jianfei Xue1,‡, Aihua Gong1, Guanzhen Yu1, Aidong Zhou1, Kangyu Lin1, Sicong Zhang1, Nu Zhang1, Cara J Gottardi3 and Suyun Huang 1,4 1Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2Cell Engineering Research Center and Department of Cell Biology, Fourth Military Medical University, Xi'an, China 3Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 4Program in Cancer Biology, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 713 834 6232; Fax: +1 713 834 6257; E-mail: [email protected] The EMBO Journal (2016)35:668-684https://doi.org/10.15252/embj.201592810 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A key step of Wnt signaling activation is the recruitment of β-catenin to the Wnt target-gene promoter in the nucleus, but its mechanisms are largely unknown. Here, we identified FoxM1 as a novel target of Wnt signaling, which is essential for β-catenin/TCF4 transactivation. GSK3 phosphorylates FoxM1 on serine 474 which induces FoxM1 ubiquitination mediated by FBXW7. Wnt signaling activation inhibits FoxM1 phosphorylation by GSK3–Axin complex and leads to interaction between FoxM1 and deubiquitinating enzyme USP5, thereby deubiquitination and stabilization of FoxM1. FoxM1 accumulation in the nucleus promotes recruitment of β-catenin to Wnt target-gene promoter and activates the Wnt signaling pathway by protecting the β-catenin/TCF4 complex from ICAT inhibition. Subsequently, the USP5–FoxM1 axis abolishes the inhibitory effect of ICAT and is required for Wnt-mediated tumor cell proliferation. Therefore, Wnt-induced deubiquitination of FoxM1 represents a novel and critical mechanism for controlling canonical Wnt signaling and cell proliferation. Synopsis Wnt signaling stabilizes FoxM1, suppressing its phosphorylation by GSK3 and promoting its interaction with USP5. FoxM1 in turn interacts with beta-catenin and abolishes the inhibitory effect of ICAT, leading to Wnt target-gene expression and tumor cell proliferation. Wnts inhibit phosphorylation of FoxM1 by GSK3 to protect it from FBXW7-mediated ubiquitination-dependent degradation. Wnts induce the interaction between FoxM1 and the deubiquitinase USP5, leading to stabilization of FoxM1. Nuclear FoxM1/beta-catenin interaction controls Wnt target-gene expression by abolishing ICAT‘s inhibition. Introduction The canonical Wnt/β-catenin signaling pathway is transduced through Wnt receptors of the Frizzled family and stabilizes β-catenin, which enters the nucleus and forms a complex with TCF4/LEF-1 transcription factors (Behrens et al, 1996; Bhanot et al, 1996; Molenaar et al, 1996; Liu et al, 1999). Activation of TCF4/LEF-1 by binding to β-catenin induces the transcription of various target genes, including c-Myc and cyclin D1 (He et al, 1998; Shtutman et al, 1999; Tetsu & McCormick, 1999). The pathway is also persistently activated in many types of tumors and critically regulates tumor growth, invasion, and angiogenesis (Weeraratna et al, 2002; Clevers, 2006; Anastas & Moon, 2013). Unlike colorectal cancer, in which constitutive activation of β-catenin is due to inactivation of the tumor suppressor APC or mutations in β-catenin, many sporadic tumors have no such mutations (Morin et al, 1997; Paraf et al, 1997; Bafico et al, 2004). Therefore, it is important to elucidate whether aberration in other components of the Wnt signaling pathway causes the activation of β-catenin/TCF4-mediated transcription in such tumors, including glioma. We recently found that FoxM1 is a novel component of Wnt signaling (Zhang et al, 2011). FoxM1 is a member of the forkhead box (Fox) transcription factor family. Growing evidence indicates that FoxM1 plays important roles in mammal development and human tumorigenesis (Kalin et al, 2011; Raychaudhuri & Park, 2011). FoxM1 is a key cell cycle regulator of both the transition from G1 to S phase and progression to mitosis (Ye et al, 1997; Korver et al, 1998). FoxM1 is substantially elevated in most human tumors and contributes to oncogenesis in many tissue types, including liver, prostate, brain, breast, lung, colon, and pancreatic tumors (Kalin et al, 2011; Raychaudhuri & Park, 2011; Gong & Huang, 2012). Our recent studies also showed that expression of FoxM1 in high-grade glioma is significantly higher than that in low-grade glioma and that FoxM1 expression contributes to the growth of glioma, as well as pancreatic, colon, and breast cancer cells by promoting uncontrolled cell proliferation, invasion, and angiogenesis (Liu et al, 2006; Zhang et al, 2008; Li et al, 2013; Xue et al, 2014). Moreover, we showed that FoxM1 critically regulates stemness and tumorigenicity of glioma stem cells (Zhang et al, 2011). Although FoxM1 is commonly overexpressed in most human tumors, the molecular mechanisms for its overexpression remain unknown. Furthermore, it is well known that protein degradation can be regulated through both E3 ubiquitin ligases and deubiquitinases (DUBs). However, although a E3 ubiquitin ligase, APC/C-Cdh1, which regulates FoxM1 has been reported (Laoukili et al, 2008; Park et al, 2008), the role of DUB in FoxM1 protein stability has been virtually unexplored. The Wnt signaling pathway is one of the major signaling pathways in stem cells and cancer cells. Aberrant activation of the Wnt/β-catenin pathway is widespread in human cancers. A crucial but unclear step of Wnt/β-catenin pathway activation is the assembly of a β-catenin/TCF transcription activation complex to the promoter of Wnt target gene. β-Catenin contains 12 armadillo repeats to form a superhelix that features a positively charged groove that can serve as a binding surface for its many partners, such as TCF4, FoxM1, and inhibitor of β-catenin and TCF4 (ICAT) (Huber et al, 1997; Tutter et al, 2001; Daniels & Weis, 2002; Graham et al, 2002). TCF4 binds to armadillo repeats 3–9 of β-catenin to form β-catenin/TCF transcription activation complex (Daniels & Weis, 2002). In contrast, ICAT is a newly identified physiological Wnt inhibitor that prevents the binding of TCF4 to β-catenin and thus the assembly of a β-catenin/TCF transcription activation complex. ICAT contains an N-terminal helical domain that binds to repeats 11 and 12 of β-catenin, and an extended C-terminal region that binds to repeats 5–10 in a manner similar to that of TCF4 (Daniels & Weis, 2002; Graham et al, 2002). Therefore, full-length ICAT dissociates complexes of β-catenin and TCF4, thus inhibiting the Wnt/β-catenin pathway (Daniels & Weis, 2002; Graham et al, 2002). In this study, we examined whether aberration of FoxM1 and/or ICAT is an important molecular mechanism for the activation of Wnt/β-catenin signaling in glioma cells. Our data revealed that FoxM1 is a novel downstream target of Wnt signaling and that USP5 is the first deubiquitylase that regulates FoxM1 deubiquitination and protein stabilization induced by Wnt activation. Wnt activation increased FoxM1 protein stabilization and thus nuclear accumulation. Nuclear FoxM1 then directly interacted with nuclear β-catenin, which released β-catenin from ICAT and enhanced recruitment of β-catenin to the promoter of Wnt target gene, hence increasing the expression of Wnt target gene. Furthermore, USP5 increased Wnt-mediated cell proliferation through FoxM1, which abrogated the inhibitory effect of ICAT. In human glioma tissues, high levels of FoxM1 and cyclin D1 are associated with high levels of USP5. Therefore, our study provides a novel mechanism for Wnt magnification of its own signaling activation. Results Wnt activation increases the stability of FoxM1 protein Recently, we reported that Wnt activation increased the expression level of FoxM1 (Zhang et al, 2011). To determine whether Wnt regulates FoxM1 protein stability, we first treated glioma cell lines LN229 and U87 with Wnt-3a at various doses (0–100 ng/ml) for 6 h. Wnt-3a treatments increased the levels of β-catenin (Fig 1A), indicating that Wnt signaling had been activated. The FoxM1 expression level was also increased by Wnt treatments in a dose-dependent manner in both cell lines (Fig 1A). Moreover, Wnt-3a treatment increased the half-life of FoxM1, measured by a pulse–chase assay (Fig 1B). However, Wnt-3a treatment for 6 h did not increase the mRNA level of FoxM1 in the U87 cells (Appendix Fig S1A), which excludes that FoxM1 transcription is regulated by Wnt-3a in the 6-h time frame. Furthermore, we determined the effect of inhibition of endogenous Wnt signaling on FoxM1 expression with use of a broadly efficacious Wnt antagonist, DKK1 (Glinka et al, 1998). DKK1 increased FoxM1 degradation compared with the control in both LN229 and U87 cells (Fig 1C). The above results indicated that activation of Wnt signaling increases the protein stability of FoxM1. Figure 1. Wnt signaling regulates the stability of FoxM1 and GSK3 kinase regulates the degradation of FoxM1 protein LN229 and U87 cells treated with Wnt-3a (0, 10, 20, 50, 100 ng/ml) for 6 h. The indicated proteins were analyzed by Western blotting. LN229 cells treated with or without Wnt-3a (50 ng/ml) were pulsed with [35S] methionine for 30 min and chased for the times indicated. Protein extracts were used for IP with anti-FoxM1 antibodies and subjected to SDS–PAGE and autoradiography. The intensities of the FoxM1 bands in autoradiography were then quantified by using NIH Image software, and the densities of the FoxM1 bands at time 0 were set as 100%. Values are mean ± SD from two independent experiments. Levels of FoxM1 were analyzed by Western blotting in LN229 and U87 cells treated with Wnt inhibitor DKK1 (100 ng/ml) for the indicated times. Note that the basal levels of FoxM1 and β-catenin in (A) and (C) are similar but look different due to variations in the exposure time of the Western blots. FoxM1 expression levels were determined in LN229 cells treated with or without GSK3 inhibitor LiCl (10 mM) and CHX (100 μg/ml) for the indicated times. GSK3 wild-type (WT) and knockout (KO) cells were treated with CHX (100 μg/ml) for the indicated times, and FoxM1 and GSK3α/β expression levels were determined by Western blotting. GSK3βCA or GSK3βKD plasmids were transfected into 293T cells for 48 h. The cells were then treated with CHX (100 μg/ml) for the indicated times. Endogenous FoxM1 expression level was determined by Western blotting. Data information: All Western blots in (A, C–F) are representative of three independent experiments. Download figure Download PowerPoint GSK3 kinase regulates the degradation of FoxM1 protein Since inhibition of GSK3-mediated β-catenin degradation is a key process in canonical Wnt signaling (Clevers, 2006; Kim et al, 2013), we explored whether GSK3 also mediates FoxM1 degradation. We first treated LN229 cells with a GSK3 inhibitor, LiCl, along with a protein synthesis inhibitor, cycloheximide. LiCl stabilized FoxM1 protein in the presence of CHX (Fig 1D). To confirm that GSK3 regulates FoxM1 stability, the protein degradation of endogenous FoxM1 was measured in GSK3 wild-type (GSK3α+/+;GSK3β+/+) and knockout (GSK3α−/−;GSK3β−/−) mouse embryonic stem cells. The levels of FoxM1 in GSK3α−/−;GSK3β−/− cells were increased compared with those in GSK3α+/+;GSK3β+/+cells; also, the FoxM1 underwent degradation with time in GSK3α+/+;GSK3β+/+cells but was quite stable in GSK3α−/−;GSK3β−/− cells (Fig 1E). Furthermore, to determine whether GSK3 kinase activity impacts FoxM1 protein stability, FoxM1 protein degradation was analyzed in 293T cells transfected with GSK3β-CA (constitutively active) or GSK3β-KD (kinase-inactive) plasmid. FoxM1 protein degradation was inhibited by GSK3β-KD but not by GSK3β-CA transfection in the cells (Fig 1F). The above results indicated that GSK3 kinase activation increases the degradation of FoxM1 protein. GSK3 phosphorylates FoxM1 protein at the S474 site, which promotes ubiquitination of FoxM1 To understand the mechanisms responsible for the regulation of FoxM1 protein expression by the Wnt pathway through inhibition of GSK3, we determined whether GSK3 interacts with and phosphorylates FoxM1 protein. We constructed deletion mutants of Flag-tagged FoxM1 (Fig 2A). Subsequently, with use of the Flag-tagged FoxM1 full-length or deletion mutant proteins (Fig 2B) in 293T cells by co-IP assay, we found that full-length and C-terminal of FoxM1 interact with endogenous GSK3 (Fig 2B). Figure 2. Phosphorylation of FoxM1 by GSK3β at S474 promotes the ubiquitination of FoxM1 Schematic of Flag-FoxM1 deletion mutants in a mammalian expression system. FL: full length; NT: NH2-terminal domain; DBD: DNA-binding domain; CT: COOH-terminal domain. Lysates from 293T cells expressing Flag-FoxM1 or its mutants were subjected to IP using mouse anti-Flag antibody, followed by IB with anti-GSK3α/β antibody (upper panel) and rabbit anti-Flag antibody (middle panel). Sequence analysis of human FoxM1 identified three putative GSK3β target sites at S228, T309, and S474. HA-ubiquitin and Flag-tagged FoxM1 (WT) or mutants FoxM1S228A, FoxM1T309A, or Flag-FoxM1S474A plasmids were co-transfected into 293T cells. After 36 h, cells were treated with 25 nM MG132 for 6 h. Cell lysates were subjected to IP with anti-Flag antibody, followed by IB with anti-Flag and anti-HA antibody. HA-ubiquitin was co-transfected into 293T cells. After 36 h, cells were treated with 25 nM MG132 for 6 h. Cell lysates were subjected to IP with IgG or anti-FoxM1 phospho-S474 antibody, followed by IB with anti-FoxM1 and anti-HA antibody. HA-tagged K48-only ubiquitin or K63-only ubiquitin construct was transfected into 293T cells. After 36 h, cells were treated with 25 nM MG132 for 6 h. Cell lysates were subjected to IP with IgG or anti-FoxM1 antibody, followed by IB with anti-FoxM1 and anti-HA antibody. GSK3β CA or vector and Flag-tagged FoxM1 (WT) or mutant S474A were co-transfected into 293T cells. Lysates of the cells were subjected to IP with anti-Flag antibody and followed by IB with anti-FoxM1 phospho-S474 antibody. Data information: All data are representative of three independent experiments. Download figure Download PowerPoint We then sought to determine whether FoxM1 protein contains consensus sequence for GSK3 substrates by using the GSK3 substrates consensus sequence S/T–X–X–X-S/T, in which the first S (serine) or T (threonine) is the target residue (Cohen & Frame, 2001; Doble & Woodgett, 2003). Analysis of the FoxM1 coding sequence identifies three motifs that resemble the GSK target sequence: S228-XXX-S232, T309-XXX-S313, and S474-XXX-S478 (Fig 2C). To determine the importance of these putative sites in protein stability, we generated T309A, S474A, and S228A mutants by mutating the serine or threonine at the sites of Flag-FoxM1 plasmid to alanine. Meanwhile, we determined whether the ubiquitin proteasome pathway is involved in Wnt signaling-mediated FoxM1 stability by detecting the FoxM1 level in LN229 cells treated with DKK1 and MG132 (a proteasome inhibitor). MG132 inhibited FoxM1 degradation induced by DKK1 (Appendix Fig S2). Thus, we analyzed ubiquitination of FoxM1 wild-type (WT) and S228A, S309A, or S474A mutants in 293T cells and found that S474A had the lowest ubiquitination level among the FoxM1 mutants and WT (Fig 2D), suggesting that phosphorylation at the S474 site is important for the ubiquitination of FoxM1. To confirm this point, we generated an antibody that specifically recognizes FoxM1 phosphorylated at S474 residues, named p-S474 antibody. Then, we used this antibody to pull down the ubiquitinated FoxM1 and found that most of the S474 phosphorylated FoxM1 protein was ubiquitinated compared with wild-type FoxM1 (Fig 2E). Next, we determine the type of ubiquitin chains for FoxM1 ubiquitination. Previous studies showed that ubiquitination through lysine-48 (K48)-linked poly-ubiquitin chains generally targets proteins for degradation, whereas ubiquitination through K63-linked poly-ubiquitin chains play a critical role in signaling activation (Nathan et al, 2013). Thus, we transfected 293T cells with HA-tagged K48-only or K63-only ubiquitin constructs and then analyzed the ubiquitination of FoxM1. We found that only poly-ubiquitin-K48 in FoxM1 was detected by anti-HA antibody, suggesting that FoxM1-ubiquitin chains are K48-linked poly-ubiquitin chains (Fig 2F). Furthermore, consistent with the above results, mutation in the S474 site prevented GSK3β-CA-mediated phosphorylation of FoxM1, which was recognized by the p-S474 antibody in 293T cells (Fig 2G). These results suggest that phosphorylation of FoxM1 by GSK3 at the S474 site promotes ubiquitination of FoxM1. Moreover, these results suggest that serine 474 followed by a proline is a classical targeted site for GSK3 which is established as a proline-directed serine/threonine kinase (Hooper et al, 2008). Wnt activation inhibits the phosphorylation of FoxM1 mediated by Axin–GSK3 complex It is known that Wnt activation disrupts the interaction of β-catenin with Axin which scaffolds between β-catenin and GSK3, thereby inhibiting phosphorylation of β-catenin by GSK3 and causing β-catenin stabilization (Clevers & Nusse, 2012; Kim et al, 2013). We thus determined whether Axin is a scaffold protein between FoxM1 and GSK3. First, we found that FoxM1 interacted with Axin and formed a complex with Axin and GSK3 (Fig 3A). Knockdown of Axin inhibited the interaction of FoxM1 with GSK3 (Fig 3B). Knockdown of Axin also inhibited the phosphorylation of FoxM1 at S474 (Fig 3C). These results suggest that Axin plays a scaffold role in the interaction of FoxM1 with GSK3. Moreover, we found that Wnt-3a treatment induced the dissociation of Axin with FoxM1 (Fig 3D), suggesting that Wnt-3a regulates FoxM1 phosphorylation by GSK3 through Axin–GSK3 complex. Furthermore, we found that knockdown of Axin led to significantly reduction of FoxM1 poly-ubiquitination (Fig 3E). Knockdown of Axin also further enforced the Wnt-3a-induced reduction of FoxM1 poly-ubiquitination (Fig 3E). These results indicated that Wnt activation inhibits the phosphorylation of FoxM1 mediated by Axin–GSK3 complex which is required for the FoxM1 ubiquitination. Figure 3. Wnt activation inhibits the phosphorylation of FoxM1 mediated by Axin–GSK3 complex and the ubiquitination of FoxM1 mediated by FBXW7 Cell lysates of LN229 cells were subjected to IP using anti-Axin antibody or control IgG, followed by IB with the indicated antibodies. Axin siRNA or control siRNA was transfected into LN229 cells and, after 36 h, co-IP was performed with lysates of the cells using anti-FoxM1 antibody followed by IB with the indicated antibodies. Axin siRNA or control siRNA was transfected into LN229 cells for 36 h. Lysates of the cells were subjected to IB with anti-FoxM1 phospho-S474 antibody. Cell lysates of LN229 cells with or without Wnt-3a treatment for 4 h were subjected to IP using anti-Axin antibody or control IgG, followed by IB with anti-FoxM1 and anti-Axin antibodies. Axin siRNA or control siRNA with HA-ubiquitin were transfected into 293T cells and, after 36 h, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for 6 h. Then, IP was performed with lysates of the cells using anti-FoxM1 antibody followed by IB with the indicated antibodies. Wild-type Flag-FoxM1 or S474A mutant was transfected into 293T cells after 36 h, and cells were treated with MG132 for 6 h. Then, IP was performed with lysates of the cells using anti-Flag antibody followed by IB with the indicated antibodies. HA-ubiquitin and/or FBXW7, GSK3β CA, Flag-FoxM1 were co-transfected into 293T cells and, after 36 h, cells were treated with MG132 for 6 h. Then, IP was performed with lysates of the cells using anti-Flag antibody followed by IB with the indicated antibodies. FBXW7 siRNA or control siRNA and HA-ubiquitin were transfected into 293T cells and, after 36 h, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for 6 h. Then, IP was performed with lysates of the cells using anti-FoxM1 antibody followed by IB with the indicated antibodies. Data information: All data are representative of three independent experiments. Download figure Download PowerPoint FBXW7 mediates FoxM1 ubiquitination, which is inhibited by Wnt/GSK3 signaling It has been reported that Wnt activation leads to stabilization of proteins by β-catenin-independent Wnt/STOP (Wnt-dependent stabilization of proteins) signaling (Acebron et al, 2014; Koch et al, 2015). The roles of GSK3 as well as Axin/APC complex in Wnt/STOP signaling have been explored previously (Taelman et al, 2010; Stolz et al, 2015). Once phosphorylated by GSK3, proteins can be ubiquitinated by E3 ubiquitin ligases, including FBW7 (F box/WD repeat-containing protein 7) and NEDD4L (neural precursor cell expressed, developmentally down-regulated 4-like), which lead to degradation of substrate proteins (Kim et al, 2009; Taelman et al, 2010; Acebron et al, 2014; Stolz et al, 2015). When FoxM1 protein was examined to identify putative degrons for the above E3 ubiquitin ligases, the motif LWEWPS(474)PAPS of FoxM1 was found to be closed to a consensus degron for FBXW7: ΩxΩΩΩ(S/T)Pxx(S/T/E) [Ω = hydrophobic] (Welcker & Clurman, 2008). Thus, we determined whether FBXW7 is an E3 ligase for FoxM1. We found that FBXW7 interacted with wild type of FoxM1 but not with S474A mutant of FoxM1 in the presence of MG132 (Fig 3F). Moreover, overexpression of FBXW7 induced FoxM1 ubiquitination as compared with control (Fig 3G, lane 4 versus lane 2). Next, since S474 is phosphorylated by GSK3, we determined whether FBXW7-mediated FoxM1 ubiquitination is regulated by GSK3 activation, by using GSK3β CA construct. We found that FBXW7-induced FoxM1 ubiquitination was increased by GSK3β CA transfection (Fig 3G), suggesting that GSK3 activity enhances FBXW7-mediated FoxM1 ubiquitination. Furthermore, we found that knockdown of FBXW7 by siRNA inhibited FoxM1 ubiquitination as compared with control siRNA (Fig 3H). More importantly, knockdown of FBXW7 reversed the inhibitory effect of Wnt treatment on FoxM1 ubiquitination (Fig 3H). Collectively, these results suggest that FBXW7-mediated FoxM1 ubiquitination is regulated by Wnt signaling and that Wnt induced FoxM1 stabilization via a Wnt/STOP mechanism. USP5 regulates stabilization of FoxM1 by deubiquitination in response to Wnt stimulation Ubiquitin–ubiquitin and ubiquitin–protein bonds can be cleaved by the action of DUB enzymes including ubiquitin-specific proteases (USPs). Since the S478A mutation suppresses ubiquitination of FoxM1, we hypothesized that Wnt activation may function in cooperation with an unknown DUB. To identify a DUB that targets the ubiquitin of FoxM1, we screened a panel of USPs in which a total of 32 USP cDNA plasmids were transfected into 293T cells. Strikingly increased endogenous FoxM1 expression under Wnt-3a treatment was observed in the cells transfected with USP5 (Fig 4A). Moreover, when USP5 was overexpressed in 293T cells, FoxM1 degradation was inhibited (Fig 4B), suggesting a role of USP5 in FoxM1 protein stability. Figure 4. USP5 deubiquitylates and stabilizes FoxM1 in response to Wnt stimulation 293T cells were transfected with USPs cDNA or control plasmid. After 36 h of transfection, cells were treated with 50 ng/ml Wnt-3a for 4 h. FoxM1 protein levels in the cells were then determined by Western blotting. 293T cells were transfected with 1 μg control plasmid or USP5 cDNA in 6-well plates. After 36 h of transfection, cells were treated with 100 μg/ml CHX for 0, 3, or 6 h. FoxM1 protein levels in the cells were then determined by Western blotting. GSK3β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 into 293T cells. After 36 h of transfection, cells were treated with 50 ng/ml Wnt-3a or control PBS for 4 h. Cell lysates were subjected to IP with anti-Myc antibody and followed by IB with anti-Flag or anti-Myc antibody. GSK3β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 into 293T cells. After 36 h of transfection, cells were treated with 50 ng/ml Wnt-3a or control PBS for 6 h. Cell lysates were subjected to IP with anti-FoxM1 antibody and followed by IB with anti-FoxM1 or USP5 antibody. USP5 and Flag-FoxM1 (WT) or mutants Flag-FoxM1S474A or Flag-FoxM1S474E plasmids were co-transfected into 293T cells for 36 h. Cell lysates were subjected to IP with anti-Flag antibody and followed by IB with anti-Flag or anti-USP5 antibody. GSK3β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 (WT) or mutant Flag-FoxM1S474A into 293T cells for 36 h. Cell lysates were subjected to IP with anti-Flag antibody and followed by IB with anti-Flag or anti-USP5 antibody. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without GSK3β CA into 293T cells. After 36 h, cells were treated with 25 nM MG132 for 6 h. Cell lysates were subjected to IP with anti-Flag followed by IB with anti-HA or anti-FoxM1 antibody. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without siUSP5 into U87 cells. After 36 h, cells were treated with 50 ng/ml Wnt-3a and 25 nM MG132 for 6 h. Cell lysates were subjected to IP with anti-Flag antibody followed by IB with anti-HA or anti-FoxM1 antibody. Data informat