(B) WB of lysates from 293T cells co-transfected with single D-box mutants of Flag-conductin (Flag-D1, -D2, -D3, -D4), as well as compound mutants (Flag-D134, Flag-D1234) together with GFP or GFP-CDC20 (arrowheads). (Fig 4A). We generated single and compound mutants (Flag D1CD4) by substituting arginine and lysine residues with alanine, and assessed degradation by CDC20. Whereas single mutants Flag-D2, -D3, -D4 were degraded by GFP-CDC20, Flag-D1 TRAM-34 and compound mutants Flag-D134 and Flag-D1234 were resistant (Fig 4B). The conserved D-box1 might therefore be a functional CDC20 degradation motif. Indeed, immunoprecipiation experiments indicated that D-box mutant conductin binds weakly to CDC20 (Fig 4C). Collectively, the results suggest that conductin is usually a bona fide substrate TRAM-34 for CDC20-mediated degradation during mitotic exit. Open in a separate window Physique 4 CDC20 mediates degradation of conductin via a conserved degradation domain name. (A) Schematic representation of mouse conductin protein and conversation domains for Wnt-signalling components, as well as putative D-boxes. Below, alignment of putative D-boxes (in strong) and surrounding amino acids is usually shown for human, mouse, zebrafish and sequences. Asterisks show conservation. (B) WB of lysates from 293T cells co-transfected with single D-box mutants of Flag-conductin (Flag-D1, -D2, -D3, -D4), as well as compound mutants (Flag-D134, Flag-D1234) together with GFP or GFP-CDC20 (arrowheads). (C) WB for GFP and Flag after IP with a GFP antibody from lysates of 293T cells co-transfected with indicated plasmids. Expression of Flag-tagged constructs in lysates is usually shown in lower panel (INPUT). CDC20, cell division cycle 20; GFP, green fluorescent protein; IP, immunoprecipiation; WB, western blot. CDC20 regulates Wnt/-catenin signalling via conductin To analyse whether activation of APC/C by CDC20 influences Wnt/-catenin signalling, we assessed the activity of TOP/FOPFlash reporters in mitotic SW480 cells after coexpression of GFP-CDC20. CDC20 increased TOP/FOP activity as compared with control GFP transfection (Fig 5A). Reciprocally, knockdown of CDC20 reduced reporter activity in G1 cells and concurrent knockdown of conductin blocked this effect, suggesting that during the cell cycle CDC20 regulates Wnt/-catenin signalling through conductin (Fig 5B). Knockdown of CDC20 in asynchronous HCT116 cells also decreased reporter activity (supplementary Fig S2F online). We presume that in HCT116 cells conductin functions mainly by cytoplasmic retention of mutated -catenin [24]. Importantly, knockdown of CDC20, which led to increased conductin levels and -catenin phosphorylation, reduced expression of all -catenin target genes tested, whereas concurrent knockdown of conductin, which increased activated -catenin, alleviated the reduction in target gene expression (Figs 5C,D). Overexpression of Flag-conductin in SW480 cells reduced TOP/FOP reporters, and coexpression of GFP-CDC20 counteracted this effect (Fig 5E). Importantly, GFP-CDC20 could not counteract the reduction of TOP/FOP in response to coexpressed CDC20-resistant mutant Flag-D1 (Fig 5E). We next assessed the ability of wild-type, as well as CDC20-resistant, conductin to inhibit proliferation of colon cancer cells. Expression of Flag-D1 mutant, but not of wild-type Flag-conductin or Flag-D2, significantly inhibited colony formation of SW480 cells but did not impact that of human osteosarcoma (U2OS) cells, which do not rely on aberrant Wnt signalling for cell growth (Fig 5F,G). Transfection efficiencies were similar for all those plasmids (about 33% for SW480 and 40% for U2OS cells). Our data suggest that CDC20 regulates Wnt/-catenin signalling and growth of colon cancer cells by controlling protein levels of conductin during the cell cycle. Open in a separate window Physique 5 CDC20 regulates Wnt signalling through conductin. TOP/FOP ratios of luciferase activities in SW480 cells transfected with reporters and GFP-CDC20, or GFP, collected 9 h after release from aphidicolin synchronization (G2/M) (A), or with indicated siRNAs collected 9 h after release from nocodazole arrest (G1/S) (B). (C) Western blotting for endogenous proteins in lysates of TRAM-34 SW480 cells transfected with indicated combinations of siRNAs against GFP, CDC20 and conductin. (D) RTCPCR for indicated target genes in cells from C. (E) TOP/FOP ratios of luciferase activities in SW480 cells transfected with reporters IFNB1 and indicated combinations of expression plasmids. Asterisks show statistically significant differences from control (GFP; [27]. Main antibodies rabbit anti-axin1, anti-phospho–catenin (Ser33/37/Thr41), mouse anti-HA (Cell Signalling), mouse anti-active–catenin (anti-ABC; Millipore), mouse anti-Flag, mouse anti–actin (Sigma), TRAM-34 mouse anti-GFP (Roche), mouse anti-APC (Ali12-28; Abcam), goat anti-p55 CDC20 (C-19), rabbit anti–catenin (H102; Santa Cruz) and mouse anti-Cyclin B1 (Upstate) were used according to the.