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First published online 14 February 2006
doi: 10.1242/jcs.02779

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APC inhibits ERK pathway activation and cellular proliferation induced by RAS

¹ Division of Molecular and Cellular Biology, Department of Biotechnology, Yonsei University, Seoul 120-752, Korea
² Protein Network Research Center, Yonsei University, Seoul 120-752, Korea
³ Laboratory of Cell Signaling and Carcinogenesis, Van Andel Research Institute, Grand Rapids, MI 49503, USA
⁴ Department of Biology, Yonsei University, Seoul 120-752, Korea
⁵ Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Shinlim-dong, Kwanak-ku, Seoul 151-742, Korea

View larger version (37K): [in a new window]   Fig. 1. Effects of overexpressing APC on RAS-induced ERK pathway activation. (A) DLD-1 cells were transfected with 0.5 µg of the AP-1 (left panel) or ELK1 reporter (right panel) plasmids together with 0.5 µg of pCMV or pCMV-APC. Transfection with ELK1 trans-reporter was coupled with transfection with 0.5 µg of pFR-Luc or 25 ng of pFA2-ELK1 (Park et al., 2002). All cases except Mock (transfection with pCMV alone), were coupled with transfection with 0.1 µg of pMT3RAS-L61 (H-Ras-L61). Cells were harvested after 48 hours and the relative levels of expression of the reporters were measured by assaying luciferase. (B) DLD-1 cells were transfected with 0.5 µg of pCMV or pCMV-APC with or without 0.1 µg of pMT3RAS-L61. Cells were harvested after 72 hours. Western blot analyses were performed on whole-cell lysates to detect p-ERK, APC, ERK and -tubulin. The right bar graph shows quantitative analyses of the intensities of p-ERKs presented as the average of three independent identical experimental results from the left panel.

View larger version (42K): [in a new window]   Fig. 2. Effects of overexpression and deletion of APC on ERK activity. (A) Left, DLD-1 cells were transfected with 0.5 µg of pCMV or with 0.25 or 0.5 µg of pCMV-APC. Cells were harvested 72 hours after transfection, and p-ERK, APC, ERKs and -tubulin were detected in whole-cell lysates by western blotting. The right bar graph shows quantitative analyses of the intensities of p-ERKs shown as the average of three independent identical experimental results for transfection with 0.5 µg of pCMV-APC from the left panel. (B) Apcflox/flox primary mouse embryonic fibroblast (MEF) cells were infected with RCAS-Cre retrovirus (see Materials and Methods). ß-catenin, p-ERK, p-Raf1, p-Akt and -tubulin were detected in whole-cell lysates by western blotting. The right panels shows the Apc deletion within the Cre virus-infected MEF cells identified by RT-PCR analysis. Control is the result of PCR in the absence of template.

View larger version (20K): [in a new window]   Fig. 3. Effects of overexpression and reduction of ß-catenin on ERK activity. (A) DLD-1 cells were transfected with 1 µg of pcDNA3.0 or with different amounts (0.5 or 1.0 µg) of Flag-ß-catenin-pcDNA3.0. They were harvested 48 hours after transfection, and p-ERK, Flag-ß-catenin, and ERK were detected by western blotting. Anti-Flag antibody was used to detect Flag-ß-catenin. (B) DLD-1 cells were transfected with ß-catenin siRNA (or not transfected). They were harvested 72 hours later and p-ERK, ß-catenin, ERK, and -tubulin were detected by western blotting.

View larger version (38K): [in a new window]   Fig. 4. Effects of APC overexpression on ß-catenin-induced ERK and ELK1 reporter activation. (A) DLD-1 cells were transfected with 0.5 µg of pcDNA3.0 or Flag-ß-catenin-pcDNA3.0 with or without 0.5 µg of pCMV-APC. They were harvested 72 hours later and western blot analyses were performed to detect p-ERK, Flag-ß-catenin, APC and ERK. Anti-Flag antibody was used to detect Flag-ß-catenin. (B) DLD-1 cells were transfected with 0.5 µg of pCMV or pCMV-APC with or without Flag-ß-catenin-pcDNA3.0. (Left panel) Co-transfection of 0.5 µg of ELK1 with 0.5 µg of pFR-Luc and 25 ng of pFA2-ELK1; ELK1 trans-reporter activity was measured. (Right panel) pTOPFLASH, pFOPFLASH were co-transfected, and luciferase activities derived from the reporter genes were measured 48 hours later. Wnt/ß-catenin reporter activity was calculated by dividing the luciferase activity of pTOPFLASH by that of pFOPFLASH (Korinek et al., 1997).

View larger version (34K): [in a new window]   Fig. 5. Effects of ß-catenin overexpression on activation of the RAF1MEKERK cascade, and effects of PD98059 and constitutively active MEK on ß-catenin-induced ERK activation. (A) DLD-1 cells were transfected with 1.0 µg of pcDNA3.0 or Flag-ß-catenin-pcDNA3.0. After 48 hours, they were harvested and p-ERK, p-MEK, p-Raf (Ser-338), Flag-ß-catenin and -tubulin were detected by western blotting. (B) DLD-1 cells were transfected with 1.0 µg of pcDNA3.0, Flag-ß-catenin-pcDNA3.0 or Flag-S33Y-ß-catenin-pcDNA3.0. Where required, cells were exposed to 20 µM PD98059 for 24 hours before harvesting (upper panel), or 0.2 µg of MEK-CA-pcDNA3.0 was co-transfected with the Flag-ß-catenin-pcDNA3.0 (lower panel). After 48 hours p-ERK, Flag-ß-catenin and ERK were detected by western blotting.

View larger version (24K): [in a new window]   Fig. 6. Effects of a dominant negative TCF4 on RAS-induced activation of ERK and ELK1 reporters. (A) DLD-1 cells were grown and transfected with 0.1 µg of empty pMT3 vector or 0.1 µg of pMT3 RAS-L61. Where required, they were co-transfected with 0.5 µg of the MYC-tagged dnTCF4 expression vector, N-TCF4E (Tetsu and McCormick, 1999). 48 hours after transfection, cell extracts were prepared, and p-ERK, RAS-L61, N-TCF4E (dn-TCF4-MYC), ERK and -tubulin proteins were detected by western blotting. Anti-MYC antibody was used to detect dn-TCF4-MYC. (B) DLD-1 cells were grown and transfected with 0.1 µg of empty pMT3 vector or 0.1 µg of pMT3 RAS-L61. Where required, they were co-transfected with 0.5 µg of MYC-tagged dn-TCF4 expression vector, N-TCF4E. 0.5 µg of ELK1 trans-reporter was co-transfected, as described in Fig. 1B. Extracts were prepared 48 hours after transfection, and luciferase activities measured. Each data point represents the average of three independent analyses.

View larger version (106K): [in a new window]   Fig. 7. Effects of APC transfection on RAS- and ß-catenin-induced foci of transformation in DLD-1 cells. 1x104 DLD-1 cells were transfected with 0.5 µg each of different combinations of pCMV, pCMV-APC, pcDNA3.0-Flag-ß-catenin, or pMT3 RAS-L61, and selected with G418. After 12 days, the cells were stained with 0.5% crystal Violet in 20% ethanol and the foci photographed.

View larger version (76K): [in a new window]   Fig. 8. Effects of APC on RAS-induced morphological transformation in NIH 3T3 cells. (A) NIH 3T3 cells containing doxycyclin (Dox)-inducible H-Ras (G12R) (Lim et al., 2004) were grown in DMEM containing 10% FBS, and were treated (or not) with 2 µg/ml Dox of for 24 hours. They were examined by phase-contrast microscopy, photographed (x200), and p-ERK, ERK, -tubulin and RAS were detected by western blotting. (B) RAS-inducible NIH 3T3 cells were grown as in A, and transfected with 0.5 µg of either pEGFP-C1 or pEGFP-C1-hAPC. Cells were visualized by fluorescence microscopy. Arrows indicate cells expressing APC-EGFP.

View larger version (35K): [in a new window]   Fig. 9. Effects of APC on RAS- or ß-catenin-induced proliferation in NIH 3T3 cells. (A) RAS-inducible NIH 3T3 cells were grown and transfected with 0.5 µg of either pCMV or pCMV-APC, and RAS was induced by doxcyclin treatment as described in Fig. 8A. The cells were fixed, permeabilized and incubated with BrdU antibody and subsequently with TRITC-conjugated goat anti-rabbit IgG. The relative intensities of TRITC-conjugated BrdU were measured using a FACS Vantage system (Becton-Dickinson Immunocytometry systems). The lower panel shows a quantitative analysis of the data in the upper panels. Each data point represents the average of three independent analyses. (B) RAS-inducible NIH 3T3 cells were grown and transfected with 0.5 µg of either pCMV or pCMV-APC together with or without 0.5 µg of pcDNA3.0 or pcDNA3.0-Flag-ß-catenin. Immunofluorescence staining and FACS analyses were performed and analyzed as described in A. The lower panel shows a quantitative analysis of the data in the upper panels. Each data point represents the average of three independent analyses.

View larger version (39K): [in a new window]   Fig. 10. The effects of APC on GTP-loading and the protein level of Ras. (A) DLD-1 cells were transfected with 0.5 µg of either pCMV or pCMV-APC with or without 0.1 µg of pMT3-RAS-L61. Cells were harvested 72 hours after transfection. RAS pull-down analysis was performed to detect GTP-loaded active RAS (De-Rooij and Bos, 1997) as described in Materials and Methods. Western blot analyses were performed on whole-cell lysates to detect p-ERK, APC, Pan-RAS and -tubulin. The GTP-loaded Pan-RAS proteins (RAS-GTP) were also detected by western blot analysis using anti-Pan-RAS antibody. (B) DLD-1, SW480 or NIH 3T3 cells were transfected with 0.5 µg of pCMV or pCMV-APC. Cells were harvested 72 hours after transfection, and p-ERK, APC, Pan-RAS and -tubulin were detected in whole-cell lysates by western blotting. RAS pull-down analysis and detection of RAS-GTP were performed as described in A.