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Molecular Association Between B-Catenin Degradation Complex and Rac Guanine Exchange Factor DOCK4 Is Essential for Wnt/B-Catenin Signaling

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Molecular Association Between B-Catenin Degradation Complex and Rac Guanine Exchange Factor DOCK4 Is Essential for Wnt/B-Catenin Signaling Oncogene (2008) 27, 5845–5855 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Molecular association between b-catenin degradation complex and Rac guanine exchange factor DOCK4 is essential for Wnt/b-catenin signaling G Upadhyay1,3, W Goessling1,2, TE North2, R Xavier1, LI Zon2 and V Yajnik1 1Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA and 2Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA The canonical Wnt/b-catenin pathway is a highly (Clevers, 2006). Depending on the receptor repertoire of conserved signaling cascade that is involved in develop- the cell, Wnts induce either b-catenin-dependent (cano- ment and stem cell renewal. The deregulation ofthis nical) or b-catenin-independent (noncanonical) signal- pathway is often associated with increased cell growth and ing cascades (Mikels and Nusse, 2006). In canonical neoplasia. The small GTPase Rac has been shown to Wnt/b-catenin pathway, Wnt ligand Wnt3A interacts influence canonical Wnt signaling by regulating b-catenin with cell-surface receptors leading to stabilization of stability through an unknown mechanism. We report that b-catenin in the cytosol, followed by nuclear transloca- DOCK4, a guanine nucleotide exchange factor (GEF) for tion where it acts as a transcriptional coactivator. In the Rac and a member ofthe CDM familyofunconventional absence of canonical Wnt signals, b-catenin is bound to GEFs, mediates Wnt-induced Rac activation in the a multi-protein degradation complex consisted of the canonical Wnt/b-catenin pathway. DOCK4 expression tumor suppressor protein, adenomatosis polyposis coli regulates cellular b-catenin levels in response to the Wnt (APC), the signaling scaffold protein, Axin and the signal, in vitro. Biochemical studies demonstrate that serine-threonine kinase, glycogen synthase kinase 3b DOCK4 interacts with the b-catenin degradation com- (GSK3b) (Moon et al., 2002; Kimelman and Xu, 2006). plex, consisting ofthe proteins adenomatosis polyposis When bound to this complex, b-catenin is constitutively coli, Axin and glycogen synthase kinase 3b (GSK3b). This phosphorylated by GSK3b, which serves as an earmark molecular interaction enhances b-catenin stability and for its ubiquitin-mediated degradation (Yost et al., 1996; Axin degradation. Furthermore, we observe that DOCK4 Aberle et al., 1997; Willert et al., 1999; Ha et al., 2004). is phosphorylated by GSK3b, which enhances Wnt- Wnt stimulation results in the inhibition of GSK3b- induced Rac activation. Using a T-cell factor reporter mediated b-catenin phosphorylation and activation of zebrafish we confirm that DOCK4 is required for Wnt/b- GSK3b-mediated phosphorylation of Axin and the Wnt catenin activity, in vivo. These results elucidate a novel co-receptor LRP5/6 (Zeng et al., 2005). Axin phospho- intracellular signaling mechanism in which a Rac GEF, rylation triggers its membrane translocation and sub- DOCK4 acts as a scaffold protein in the Wnt/b-catenin sequent degradation (Zeng et al., 2005). These molecular pathway. events ultimately result in the disassembly of the Oncogene (2008) 27, 5845–5855; doi:10.1038/onc.2008.202; b-catenin degradation complex and an increase in published online 21 July 2008 b-catenin levels in the cytosol. The formation and disassembly of the b-catenin degradation complex is a Keywords: Wnt/b-catenin; Rac; GSK3b; Axin; APC; tightly controlled process and several regulators of this DOCK4 process have been described such as disheveled, protein phosphatase 1, presenilin and casein kinase1 (Amit et al., 2002; Kang and Massague, 2004; Luo et al., 2007; Macdonald et al., 2007). Introduction The small GTPase Rac, a regulator of actin cyto- skeleton, has been recently shown to be important for Wnts are extracellular glycoproteins that bind and nuclear translocation of b-catenin and T-cell factor activate cell-surface receptors initiating a signaling (TCF) activation (Esufali and Bapat, 2004; Wu et al., cascade that is important in development and disease 2008). Furthermore, the neurotropic JC virus T antigen has been shown to recruit Rac to stabilize b-catenin (Bhattacharyya et al., 2007). Prior to these reports, Rac Correspondence: Dr V Yajnik, Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, was believed to be a critical regulator of b-catenin- 55 Fruit Street, GRJ-825, Boston, MA 02114, USA. independent, noncanonical signaling (Habas et al., 2003; E-mail: [email protected] Veeman et al., 2003). 3Current address: Dr G Upadhyay, Cancer Genetics, Georgetown In cell signaling, Rac is activated by regulatory University, Washington, DC 20007, USA. E-mail: [email protected] proteins called guanine exchange factors or GEFs Received 25 March 2008; revised 12 May 2008; accepted 19 May 2008; (Burridge and Wennerberg, 2004). We previously published online 21 July 2008 identified and cloned DOCK4, a member of DOCK b-catenin degradation complex and DOCK4 G Upadhyay et al 5846 family of GEFs, which regulates b-catenin at adherence junctions by Rap activation (Yajnik et al., 2003). DOCK4 is a multi-domain protein, which is a part of the DOCK superfamily of 11 unconventional GEFs, characterized by the presence of a DHR1 and DHR2 (DOCK homology regions 1 and 2) domains (Brugnera et al., 2002; Cote and Vuori, 2002). The DHR1 domain binds phospholipid PIP3 and the DHR2 domain activates Rac and together regulate protein function in cell migration (Cote et al., 2005; Lu et al., 2005). The precise mechanism of Rap1 regulation by DOCK4 remains presently unclear but we demonstrated that like other DOCK family members, DOCK4 activates Rac1 through its DHR2 domain (Lu et al., 2005; Yan et al., 2006). In light of the fact that Rac regulates b-catenin, we hypothesized that DOCK4 regulates the cytosolic pool of b-catenin by Rac activation in canonical Wnt signaling. Here we report that DOCK4 is required for Wnt-induced Rac activation, TCF transcription and cell migration. Furthermore, we illustrate the molecular mechanism of DOCK4 action in canonical Wnt signaling. Results DOCK4 enhances Wnt/b-catenin signaling To determine whether DOCK4 influences Wnt/b- catenin signaling, we used HEK293 cells, which have Figure 1 DOCK4 is required for a transcriptionally active pool of an intact Wnt pathway (Liu et al., 2007). HEK293 cells b-catenin. (A) DOCK4 enhances b-catenin-induced T-cell factor were transfected with a constant amount of Flag b- (TCF) transcription. b-Catenin (200 ng) was co-transfected with increasing amounts of DOCK4 for relative TCF reporter assay (a), catenin with increasing concentration of Flag-DOCK4. western blot analysis performed in parallel demonstrates DOCK4- TopFlash reporter assays revealed that b-catenin- dependent increase in the total levels of b-catenin but not actin (b). induced TCF activation was enhanced 2- to 5-fold by (B) Silencing of DOCK4 abrogates Wnt3A-induced TCF activa- DOCK4 in a dose-dependent manner (Figure 1A, a). In tion demonstrated by relative TCF reporter assay (a) cytosolic and membrane fractions were isolated in parallel from these samples. parallel, western blot analysis of lysates showed that Western blot analysis of these fractions showed that Wnt3A but steady-state levels of b-catenin were also increased (2- to not Wnt5A treatment leads to cytosolic accumulation of free 5-fold) upon DOCK4 expression (Figure 1A, b). Thus, b-catenin, which is inhibited in the presence of DOCK4 SiRNA (b). ectopic coexpression of DOCK4 with b-catenin DOCK4 is abbreviated as D4 in the figure. enhances b-catenin stability and TCF activation, sug- gesting that DOCK4 may be an intracellular regulator These SiRNAs showed a greater than 50% reduction in of canonical Wnt signaling. DOCK4 expression and threefold reduction in TCF activation, further confirming that DOCK4 expression DOCK4 is required for cytosolic accumulation is required for TCF activation. Cells transfected with the of b-catenin SH3-domain-deleted DOCK4 are resistant to down- To test whether DOCK4 is required in Wnt/b-catenin regulation by the DOCK4 SiRNA as it targets the signaling, we used NIH3T3 cells, which also have an nucleotides within the SH3-domain-coding segment of intact Wnt pathway (Hocevar et al., 2003). NIH3T3 the DOCK4 mRNA (Yajnik et al., 2003). Thus, co- cells were transfected with DOCK4 SiRNA and treated transfection of SH3-domain-deleted DOCK4 along with with either Wnt3A (canonical ligand) or Wnt5A DOCK4 SiRNA rescued the Wnt3A-mediated TCF (noncanonical ligand). Transient transfection with activation (Supplementary Figure1B). DOCK4 SiRNA reduced DOCK4 expression by over To confirm whether DOCK4 expression is required 90% (Figure 1B, a , inset) without affecting cell viability for TCF activation we tested a panel of cell lines (Supplementary Figure 1A). A significant inhibition including mouse intestinal epithelial cell IEC6 and colon (fivefold) in the Wnt3A-induced TCF activation was cancer cell lines with constitutively activated Wnt observed when cells were treated with DOCK4 SiRNA signaling that included Hct116, SW480, DLD-1 and using TopFlash reporter assays (Figure 1B, a). In HT29. TopFlash reporter studies revealed that silencing addition, two commercially available SiRNA for DOCK4 expression reduced Wnt3A-induced TCF DOCK4 (Dharmacon Technologies, Lafayette, CO, activation fivefold in the IEC6 cell line (Po0.001). USA) were tested for Wnt3A-induced TCF activation. Among colon cancer cell lines, we found inhibition of Oncogene b-catenin degradation complex and DOCK4 G Upadhyay et al 5847 TCF activity in DLD-1 and HT29 cells but not in Hct116 (DSH3 DOCK4), and to C-terminal-deleted DOCK4 and SW480 cells (Supplementary Table 1). DLD-1 and (DC-term DOCK4); whereas, it did not bind to DHR2- HT-29 cells contain an active cellular machinery for domain-deleted DOCK4 (DDHR2 DOCK4; Figure 2B, ubiquitin-mediated degradation of b-catenin and this b). To determine whether the DHR2 domain is not only mechanism is nonfunctional in HCT116 and SW480 cell necessary but also sufficient to bind APC, a glutathione lines (Ilyas et al., 1997; Yang et al., 2006).
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