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. 2003 Oct 28;100(22):12729-34.
doi: 10.1073/pnas.2133261100. Epub 2003 Oct 16.

Eradication of pathogenic beta-catenin by Skp1/Cullin/F box ubiquitination machinery

Yunyun Su  1 , Shinji Ishikawa, Masayuki Kojima, Bo Liu
Affiliations

Eradication of pathogenic beta-catenin by Skp1/Cullin/F box ubiquitination machinery

Yunyun Su et al. Proc Natl Acad Sci U S A. .

Abstract

The use of Skp1/Cull 1/F box (SCF) ubiquitin-conjugation machinery as a potential knockout tool offers a means of eradicating disease-causing proteins. Here a chimeric F box protein (CFP) was engineered to achieve selective eradication of pathogenic beta-catenin in colorectal cancer. We show that CFP specifically searches for and subsequently links the abnormal beta-catenin to the cellular SCF ubiquitination complex. Introduction of the CFP to colorectal cancer cells induced targeted ubiquitination and proteolytic degradation of nuclear and cytoplasmic free beta-catenin while preserving its normal cellular adhesion counterpart. Elimination of pathogenic beta-catenin suppressed constitutive Wingless/Wnt signaling and inhibited in vitro and in vivo tumor cell growth. This study demonstrates a practical utility of a SCF-based knockout system as a tool in targeting an abnormal protein that affects growth and transformation.

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Figures

Fig. 1.
Fig. 1.
Generation of CFP-targeting constructs. (a Left) Schematic representation of β-catenin-binding motifs expressed in 293T cells and a summary of their ability to bind free β-catenin and the E-cadherin-associated β-catenin. (a Right) Coimmunoprecipitation of non-E-cadherin and E-cadherin-associated β-catenin with pShuttleHA (control vector), APCbc2, APCbc4, and APCbc6. Lysates from transfected 293T cells were first immunoprecipitated with anti-HA antibody, and the amount of β-catenin recognized by APCbc2, APCbc4, and APCbc6 was revealed by Western blot analysis with mouse anti-β-catenin antibody (second blot from the top). The anti-HA antibody-cleared protein lysates were subsequently precipitated with anti-E-cadherin antibody, and the amount of β-catenin still associated with E-cadherin was detected by Western blot analysis with mouse anti-β-catenin antibody (third blot from the top). The expression levels of APCbc2, APCbc4, and APCbc6 in the transfected 293T cell lysates are shown on the bottom. Protein lysates with equal amounts of β-catenin from transfected cells were used for the IP (displayed in the top row). (b Left) schematic of F box motifs expressed in 293T cells and summaries of their binding abilities to Skp1 protein. (b Right) Coimmunoprecipitation of Skp1 protein with F1, F2, and F3. Protein lysates from transfected 293T cells were immunoprecipitated with anti-HA or anti-Flag monoclonal antibody, and the amount of Skp1 protein associated with F1, F2, F3, and β-TrCP was revealed by Western blot analysis with rabbit anti-Skp1 antibody (shown in the top row). The expression levels of F1EGFP, F2EGFP, F3EGFP, and β-TrCP in the transfected cells are shown in the second row. The presence of similar levels of α-actin protein in each lane indicates that an equal amount of protein lysates was used for the IP and Western blot analyses (bottom blot). (c Left) Schematic of F2APCbc4 and F3APCbc4 constructs used for transient expression in 293T cells. (c Right) Coimmunoprecipitation of Skp1 and β-catenin proteins with APCbc4, F2APCbc4, F3APCbc4, or Flag-tagged β-TrCP. Protein lysates from transfected 293T cells were immunoprecipitated with anti-HA or anti-Flag antibody, and the amount of Skp1 and β-catenin proteins associated with APCbc4, F2APCbc4, F3APCbc4, or β-TrCP was revealed by Western blot analysis with respective antibodies as designated (top and second blots). The expression levels of APCbc4, F2APCbc4, F3APCbc4, or β-TrCP in the transfected 293T cells are shown in the third blot. The presence of similar levels of α-actin protein in each lane indicates that an equal amount of protein lysates was used for the IP and Western blot analyses (bottom row).
Fig. 2.
Fig. 2.
F3APCbc4 targets both mutant and wild-type β-catenin and suppresses β-catenin/TCF4-mediated transcription activities. (a) Coimmunoprecipitation of Skp1, wild-type, or mutant β-catenin, with vector control, Flag-tagged β-TrCP, APCbc4, or F3APCbc4 in CRC cell lines. Protein lysates from transiently transfected HAβ18 (contained a mutant β-catenin allele) and DLD1 (contained wild-type β-catenin but mutant APC) were immunoprecipitated with anti-Flag or anti-HA antibody. Amount of Skp1 or β-catenin protein associated with β-TrCP, APCbc4, or F3APCbc4 was revealed by Western blot analysis with anti-Skp1 or anti-β-catenin antibody (top and second blots). The expression levels of Flag-tagged β-TrCP, APCbc4, or F3APCbc4 in the transfected cells are shown in the third row. The presence of similar levels of α-actin protein in each lane indicates that an equal amount of protein lysates was used for the IP and Western blot analyses (bottom blot). (b) Luciferase activities were suppressed in both HCT116 and DLD1 transfected with F3APCbc4 as compared with the vector control, β-TrCP, wild-type APC, and APCbc4. Luciferase activities from two independent transfections are shown.
Fig. 3.
Fig. 3.
F3APCbc4-mediated linkage of abnormal β-catenin to ubiquitin-conjugation machinery. IP and Western blot analyses of DLD1-APCbc4 and DLD1-F3APCbc4 show F3APCbc4 (clone F5)-mediated ubiquitination and degradation of pathogenic β-catenin in CRC cells. Reduction of β-catenin in CRC cells mediated by F3APCbc4, but not by APCbc4, is shown in the second row (compare lanes 3, 4, 5, and 6). For detection of F3APCbc4-mediated ubiquitination of β-catenin in CRC cells, DLD1-APCbc4 or DLD1-F3APCbc4 was cultured in the presence of 12.5 μM ALLN for 12 h under the induced or uninduced condition. The second row shows that ubiquitinized β-catenin is present in DLD1-F3APCbc4 but not in DLD1-APCbc4. The presence of ubiquitin-conjugated β-catenin is further demonstrated when the same protein lysates were immunoprecipitated with anti-β-catenin monoclonal antibody and the amount of ubiquitinizedβ-catenin was probed with anti-ubiquitin antibody (third blot as designated). The top row shows levels of APCbc4 or F3APCbc4 under induced or noninduced conditions. Levels of α-actin in the bottom row indicate that equal amounts of protein lysate were used for the IP and Western blot analyses.
Fig. 4.
Fig. 4.
Targeted destruction of pathogenic β-catenin suppresses Wingless/Wnt signaling. (a) IP and Western blot analyses show the reduction of nuclear β-catenin and suppression of c-MYC mediated by F3APCbc4 in CRC cells. The expression level of APCbc4 or F3APCbc4 after the removal of doxycycline for the indicated times is shown in the top blot. Levels of nuclear β-catenin reduction and subsequent suppression of c-MYC associated with induced expression of APCbc4 or F3APCbc4 are shown in the second and third blots. Nuclear fraction from HFF cells was used as control (lane 11). The presence of similar levels of nuclear lamin B1 protein in the nuclear fraction indicates that equal amounts of nuclear lysates were used for the Western blot analysis (fourth blot). Western blot analysis of the cytoplasmic fraction shows that the level of membrane E-cadherin-associated β-catenin was not affected by F3APCbc4 (second blot from the bottom). Absence of nuclear lamin B1 protein in the cytoplasmic fraction indicates that no crosscontamination occurred between the nuclear and cytoplasmic fractions (third blot from the bottom). For detection of F3APCbc4-mediated ubiquitination of β-catenin in CRC cells, DLD1-F3APCbc4 and DLD1-APCbc4 were cultured in the presence of 12.5 μM ALLN for 6 h under induced or uninduced conditions. The bottom blot shows the presence of ubiquitinized β-catenin in CRC cells associated with the induced expression of F3APCbc4 (lanes 7–10) but not with that of APCbc4 (lanes 2–5). HFF cells were included as a positive control (lane 11). (b) Immunofluorescence staining shows the disappearance of nuclear and cytoplasmic β-catenin but not the E-cadherin-associated β-catenin in DLD1-APCbc4. No changes in nuclear or cytoplasmic β-catenin are evident in DLD1-APCbc4.
Fig. 5.
Fig. 5.
Targeted destruction of pathogenic β-catenin inhibits the growth of DLD1 cells in vitro and in vivo.(a) Growth kinetics of CRC cells with or without induced expression of APCbc4 (growth chart on the left) or F3APCbc4 (growth chart on the right). Numbers were derived from an average of the results of three different culture dishes. (b) Colony formation in collagen gel. Proliferation was inhibited in DLD1 by F3APCbc4 but not by APCbc4. This proliferation was visualized by crystal violet staining of in vitro cultured DLD1 cells with or without induced expression of APCbc4 or F3APCbc4 for 9 days until the individual colonies were visible. Numbers of colonies are illustrated.
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