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First published online 24 February 2009
doi: 10.1242/jcs.040493

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H-Ras is degraded by Wnt/β-catenin signaling via β-TrCP-mediated polyubiquitylation

¹ National Research Laboratory of Molecular Complex Control and Department of Biotechnology, BK21 project for Medical Science, Yonsei University, Seoul 120-749, Korea
² Department of Biochemistry, BK21 project for Medical Science, Yonsei University, Seoul 120-749, Korea
³ Protein Network Research Center, BK21 project for Medical Science, Yonsei University, Seoul 120-749, Korea
⁴ Department of Pathology, Center for Chronic Metabolic Disease, BK21 project for Medical Science, Yonsei University, Seoul 120-749, Korea

View larger version (40K): [in this window] [in a new window]   Fig. 1. Both endogenous and overexpressed Ras are polyubiquitylated and degraded by the proteasome. (A) Upper panel: HEK293 cells were treated with cycloheximide (CHX) for the indicated times (0, 3, 6, 9 or 12 hours) with or without ALLN. Lower panel: HEK293 cells were treated with ALLN for 0, 4, 8 or 12 hours. Whole cell lysates (WCLs) were subjected to immunoblotting using indicated antibodies. (B) HEK293 cells were transfected with plasmids expressing H-Ras-Myc and HA-Ub. At 24 hours after transfection, cells were treated with ALLN for 12 hours before harvest. After lysis, immunoprecipitation was performed with anti-β-catenin or anti-Myc antibody. The ubiquitylation of either H-Ras or β-catenin was detected by immunoblotting with anti-HA antibody. WCLs were analyzed by immunoblotting for indicated proteins. (C) HEK293 cells were transfected with plasmid expressing HA-Ub. At 24 hours after transfection, cells were treated with ALLN for 12 hours. After lysis, immunoprecipitation was performed with anti-H-Ras or anti-β-catenin antibody. The polyubiquitylation of H-Ras or β-catenin was detected by anti-HA antibody. β-Catenin, Pan-Ras and ERK proteins were detected in WCLs.

View larger version (25K): [in this window] [in a new window]   Fig. 2. The β-TrCP degrades H-Ras. (A) HEK293 cells were transfected with H-Ras-Myc or Flag-β-TrCP expression plasmids or β-TrCP siRNA. Where indicated, cells were treated with ALLN for 12 hours before harvest. At 36 hours after transfection, cells were harvested and whole-cell lysates (WCLs) were subjected to immunoblotting. (B) HEK293 cells were transfected with H-Ras-Myc or Flag-β-TrCP expression plasmids or β-TrCP siRNA. At 24 hours after transfection, cells were treated with CHX for the indicated times (0, 3, 6, 9 and 12 hours) and harvested. WCLs were subjected to immunoblotting. Quantification of band densities is shown in the right panel. Error bars indicate standard deviation.

View larger version (43K): [in this window] [in a new window]   Fig. 3. β-TrCP interacts with H-Ras via its WD40 domain. (A) HEK293 cells were transfected with either vector or Flag-β-TrCP for 36 hours. Immunoprecipitation or pull-down analyses were performed with anti-H-Ras antibody or recombinant GST-H-Ras protein. β-TrCP and GST-H-Ras were detected by immunoblot analyses. (B) Schematic representation of the primary structure of the wild-type and mutant β-TrCP proteins is shown in the upper panel. Lower panels: HEK293 cells were transfected with vector or Flag-β-TrCP (Wt, F-box or WD40) plasmids together with H-Ras-Myc expression plasmid. Cell lysates were immunoprecipitated with anti-Flag or anti-Myc resin (left), or pulled down with GST-H-Ras recombinant protein (right) followed by immunodetection. β-TrCP, H-Ras, p-ERKs and -tubulin were detected in the whole-cell lysates (WCLs) by western blotting.

View larger version (41K): [in this window] [in a new window]   Fig. 4. β-TrCP polyubiquitylates H-Ras. (A-D) HEK293 cells were transfected with vector, H-Ras-Myc, Flag-β-TrCP (Wt or F-box) and HA-Ub expression plasmids and/or β-TrCP siRNA. At 24 hours after transfection, cells were treated with ALLN, and lysates were immunoprecipitated with anti-H-Ras antibody or anti-Myc resin followed by immunoblotting. β-TrCP, Pan-Ras, Myc-H-Ras and -tubulin were detected in whole-cell lysates (WCLs) by immunoblotting.

View larger version (54K): [in this window] [in a new window]   Fig. 5. Wnt/β-catenin signaling regulates polyubiquitylation and stability of H-Ras via modulation of β-TrCP binding affinity with H-Ras. (A,B) HEK293 cells were transfected with vector, H-Ras-Myc, Flag-β-TrCP, Axin, Apc, Flag-Ub and/or HA-Ub expression plasmids for 36 hours. Where indicated, transfected cells were treated with ALLN for 12 hours before harvest. Cell lysates were immunoprecipitated with anti-Myc resin or Pan-Ras antibody (for monitoring endogenous Ras). The immunoprecipitated lysates (IPLs) and whole-cell lysates (WCLs) were analyzed with indicated antibodies. (C-E) HEK293 cells were transfected with combination of vector, H-Ras-Myc, Flag-β-TrCP, HA-Ub and Apc expression plasmids and/or β-TrCP siRNA. At 24 hours after transfection, cells were treated with ALLN for 12 hours. Where required, cells were treated with Wnt3a (100 ng/ml), EGF (20 ng/ml) or different concentration of Wnt3a (0, 75 or 150 ng/ml) for 2 hours before harvest. Cell lysates were immunoprecipitated with anti-Myc resin or β-catenin antibody. IPLs or WCLs were analyzed by immunoblotting with indicated antibodies.

View larger version (40K): [in this window] [in a new window]   Fig. 6. The regulation of H-Ras stability via β-TrCP correlates with cellular transformation. (A,B) DLD-1 cells were transfected with vector and/or plasmids expressing H-Ras-Myc, Flag-β-TrCP and/or Axin. The transfected cells were selected with 800 µg/ml of G418 for 20 days and stained with 0.5% crystal violet in 20% ethanol, and the foci were photographed. The relative percentages of foci numbers were quantified. Error bars indicate standard deviation.

View larger version (98K): [in this window] [in a new window]   Fig. 7. In vivo regulation of Ras stability and ERK activity by Wnt/β-catenin signaling in mouse. The 12-week-old FVB mice were intravenously injected with PBS or Wnt3a for 12 hours, and then the colon tissues were harvested and frozen for biochemical analysis/fixed by paraformaldehyde as described in the Materials and Methods. (A,B) The whole-cell lysates (WCLs) were immunoprecipitated with anti-Pan-Ras antibody, and WCLs and immunoprecipitated lysates were immunoblotted with anti-β-catenin, anti-Pan-Ras, anti-β-actin, anti-p-ERK or anti-Ub antibody. (C-J) The immunofluorescence staining was performed as described in the Materials and Methods. Immunofluorescence staining patterns of β-catenin and Pan-Ras in the colon tissue from PBS- and Wnt3a-injected mouse were monitored by confocal microscope. The images in D-F and H-J are the higher magnification images of the boxed areas in C and G. Scale bars: 20 µm.

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