Regulation of Wnt Signaling by the Nuclear Pore Complex Miki Shitashige, Reiko Satow, Kazufumi Honda, Masaya Ono, Setsuo Hirohashi, Tesshi Yamada  Gastroenterology  Volume 134, Issue 7, Pages 1961-1971.e4 (June 2008) DOI: 10.1053/j.gastro.2008.03.010 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Interaction of NPC proteins with TCF-4. (A) Total cell lysates (Input) and immunoprecipitates of HCT-116 (left) and DLD1 (right) cells with anti-TCF-4 monoclonal antibody (TCF-4) or normal mouse IgG (IgG) were separated by SDS-PAGE and blotted with the indicated antibodies. (B) Total cell lysates (Input) and immunoprecipitates of HCT-116 (left) or DLD1 (right) cells with the indicated antibody or normal IgG were analyzed by immunoblotting with anti-TCF-4 monoclonal antibody. Gastroenterology 2008 134, 1961-1971.e4DOI: (10.1053/j.gastro.2008.03.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 In vitro sumoylation of TCF-4 by RanBP2. (A) Mixtures containing Aos1/Uba2, Ubc9, and the indicated recombinant proteins were incubated at 30°C for 30 minutes and analyzed by immunoblotting with anti-TCF-4, anti-SUMO1, and anti-GST antibodies. (B) GST-TCF-4 was incubated with the indicated proteins for 1, 5, or 30 minutes and analyzed by immunoblotting with the indicated antibodies. (C and D) Mixtures containing the indicated recombinant proteins were incubated for 30 minutes and analyzed by immunoblotting with the indicated antibodies. Gastroenterology 2008 134, 1961-1971.e4DOI: (10.1053/j.gastro.2008.03.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 In vivo sumoylation of TCF-4 by RanBP2. (A and B) HCT-116 (left) or DLD1 (right) cells were transfected with the indicated plasmids and control RNA or siRNA against RanBP2. Forty-eight hours after transfection, the whole cell lysates were immunoprecipitated with anti-TCF-4 antibody. The precipitated proteins (IP: TCF-4) and whole cell lysates (W:) were then analyzed by immunoblotting with anti-TCF-4, anti-HA (hemagglutinin), anti-RanBP2, anti-FLAG, and anti-β-actin (loading control) antibodies. Gastroenterology 2008 134, 1961-1971.e4DOI: (10.1053/j.gastro.2008.03.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Sumoylation of TCF-4 enhances its ability to bind β-catenin. GST-TCF-4 was incubated with the indicated proteins except for GST-β-catenin in 50-μL reaction mixtures at 30°C for 30 minutes. The reaction mixtures were further incubated for 30 minutes after addition of 0.1 (lanes 3 and 4) or 0.5 μg (lanes 5–7) of GST-β-catenin and immunoprecipitated (IP) with anti-TCF-4, anti-β-catenin, or anti-SUMO1 antibody. The precipitated proteins were analyzed by immunoblotting with anti-TCF-4, anti-β-catenin, and anti-SUMO1 antibodies. Gastroenterology 2008 134, 1961-1971.e4DOI: (10.1053/j.gastro.2008.03.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 NPC proteins regulate the nuclear import of β-catenin. (A and C) HCT-116 (A) or DLD1 (C) cells were transfected with 1 or 2 μg of pcDNA3.1-RanBP2, -RanGAP1, -Ubc9, or -SUMO1 (solid columns), or 2 μg of control (empty) pcDNA3.1 (open columns). Forty-eight hours after transfection, total cellular and nuclear proteins were analyzed by immunoblotting with the indicated antibodies. The amount of nuclear β-catenin relative to total cellular β-catenin is shown at the top of the Figure panel. (B and D) HCT-116 (B) or DLD1 (D) cells were transfected with 2 constructs (a and b) of control RNA (open columns) or 2 constructs (a and b) of siRNA against RanBP2, RanGAP1, Ubc9, or SUMO1. Forty-eight hours after transfection, total cellular and nuclear proteins were analyzed by immunoblotting with the indicated antibodies. The amount of nuclear β-catenin relative to total cellular β-catenin is shown at the top of the Figure panel. Please note that the 90-kilodalton RanGAP1 protein (the sumoylated form of RanGAP1) was resistant to the siRNA treatment. Gastroenterology 2008 134, 1961-1971.e4DOI: (10.1053/j.gastro.2008.03.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Regulation of TCF/LEF transcriptional activity by NPC proteins. (A) HEK293 cells were transfected in triplicate with canonical (TOP-FLASH, solid columns) or mutant (FOP-FLASH, open columns) TCF/LEF luciferase reporter; pFLAG-β-cateninΔN134 (+) or control (empty) pFLAG-CMV4 (−); and pcDNA3.1-RanBP2, -RanGAP1, -Ubc9, -SUMO1, or control pcDNA3.1. Luciferase activity was measured 48 hours after transfection. Bars, SD. (B) Colony formation by HEK293 cells transfected with pFLAG-β-cateninΔN134 (+) or control pFLAG-CMV4 and pcDNA3.1-RanBP2, -RanGAP1, -Ubc9, -SUMO1, or control pcDNA3.1. Transfectants were cultured in the presence of G418 for 8 days and stained. (C and D) HCT-116 (C) or DLD1 (D) cells were cotransfected in triplicate with TOP-FLASH (solid columns) or FOP-FLASH (open columns) and 1 or 2 μg of pcDNA3.1-RanBP2, -RanGAP1, -Ubc9, -SUMO1, or control pcDNA3.1. Luciferase activity was measured 48 hours after transfection. (E) Colony formation by HCT-116 (top) or DLD1 (bottom) cells transfected with pcDNA3.1-RanBP2, -RanGAP1, -Ubc9, -SUMO1, or control pcDNA3.1. Transfectants were cultured in the presence of G418 for 8 days and stained. Gastroenterology 2008 134, 1961-1971.e4DOI: (10.1053/j.gastro.2008.03.010) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Inhibition of TCF/LEF transcriptional activity of colorectal cancer cells by SENP2. (A and C) HCT-116 (A) or DLD1 (C) cells were transfected in triplicate with TOP-FLASH (solid columns) or FOP-FLASH (open columns); 1 μg of pcDNA3.1-RanBP2, -RanGAP1, -Ubc9, -SUMO1, or control pcDNA3.1; and 1 or 2 μg of pCMV-Tag2B-SENP1, -SENP2, -SENP3, or control (empty) pCMV-Tag2B. Luciferase activity was measured 48 hours after transfection. (B and D) Colony formation by HCT-116 (B) or DLD1 (D) cells transfected with pcDNA3.1-RanBP2, -RanGAP1, -Ubc9, -SUMO1, or control pcDNA3.1 and pCMV-Tag2B-SENP2 (+ SENP2) or control pCMV-Tag2B. Transfectants were cultured in the presence of G418 for 8 days and stained. Gastroenterology 2008 134, 1961-1971.e4DOI: (10.1053/j.gastro.2008.03.010) Copyright © 2008 AGA Institute Terms and Conditions