Limits...
A protein knockdown strategy to study the function of beta-catenin in tumorigenesis.

Cong F, Zhang J, Pao W, Zhou P, Varmus H - BMC Mol. Biol. (2003)

Bottom Line: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate.As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. congf@mskcc.org

ABSTRACT

Background: The Wnt signaling pathway plays critical roles in cell proliferation and cell fate determination at many stages of development. A critical downstream target of Wnt signaling is the cytosolic beta-catenin, which is stabilized upon Wnt activation and promotes transcription of a variety of target genes including c-myc and cyclin D. Aberrant Wnt signaling, which results from mutations of either beta-catenin or adenomatous polyposis coli (APC), renders beta-catenin resistant to degradation, and has been associated with multiple types of human cancers.

Results: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the beta-catenin binding domain of E-cadherin fused to betaTrCP ubiquitin-protein ligase, the stable beta-catenin mutant was recruited to the cellular SCF (Skp1, Cullin 1, and F-box-containing substrate receptor) ubiquitination machinery for ubiquitination and degradation. The DLD1 colon cancer cells express wild type beta-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of betaTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of beta-catenin. As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.

Conclusion: We have designed a novel approach to induce degradation of stabilized/mutated beta-catenin. Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells. The protein knockdown strategy can be utilized not only as a novel method to dissect the role of oncoproteins in tumorigenesis, but also as a unique tool to delineate the function of a subpopulation of proteins localized to a specific subcellular compartment.

Show MeSH

Related in: MedlinePlus

Engineering a βTrCP-E-cadherin chimera that targets oncogenic β-catenin for degradation. A. Schematic diagrams of F-TrCP-Ecad chimeras used in this study. The β-catenin binding domain (amino acids 794–883) of E-cadherin (Ecad) was fused to the C-terminus of FLAG-tagged βTrCP to form F-TrCP-Ecad. The N-terminus of βTrCP (amino acid 1–297) was deleted from F-TrCP-Ecad to make F-TrCP(ΔN)-Ecad. The transmembrane (TM) region and the β-catenin binding domain in E-cadherin are indicated. The F box of βTrCP is hatched. The FLAG epitope fused at the N-termini of βTrCP, βTrCP-Ecad, or βTrCP(ΔN)-Ecad is indicated by a solid rectangle. B. F-TrCP-Ecad binds to β-catenin S37A in vivo. HA-tagged β-catenin S37A and FLAG-tagged βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor MG132 for 4 hours before harvesting. Cell lysates were immunoprecipitated with the anti-FLAG antibody, precipitates were resolved by a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, and immunoblotted with the anti-HA antibody (top panel). Note that a nonspecific band that cross-reacted with the anti-HA antibody migrated slightly above β-catenin S37A in all lanes of this panel. The membrane was stripped and blotted with the anti-FLAG antibody (middle panel). The expression of β-catenin S37A in total cell lysates was examined by immunoblotting with the anti-HA antibody (bottom panel). C. F-TrCP-Ecad induces ubiquitination of β-catenin S37A in vivo. MYC-tagged β-catenin S37A, HA-tagged ubiquitin, and indicated βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor ALLN for 4 hours, and lysed in the denaturing buffer by boiling to disrupt non-covalent protein-protein interactions. Cell lysates were immunoprecipitated with the anti-HA antibody, and immunoprecipitates were resolved in SDS-PAGE and blotted with the anti-MYC antibody (top panel). The expression of β-catenin S37A in total cell lysates was determined by immunoblotting with the anti-MYC antibody (bottom panel). D. F-TrCP-Ecad reduces the steady state levels of β-catenin. HA-tagged β-catenin and CDK2 were co-expressed with indicated βTrCP derivatives in 293 cells. Total cell lysates were resolved by SDS-PAGE, and immunoblotted with the anti-HA antibody.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC222962&req=5

Figure 1: Engineering a βTrCP-E-cadherin chimera that targets oncogenic β-catenin for degradation. A. Schematic diagrams of F-TrCP-Ecad chimeras used in this study. The β-catenin binding domain (amino acids 794–883) of E-cadherin (Ecad) was fused to the C-terminus of FLAG-tagged βTrCP to form F-TrCP-Ecad. The N-terminus of βTrCP (amino acid 1–297) was deleted from F-TrCP-Ecad to make F-TrCP(ΔN)-Ecad. The transmembrane (TM) region and the β-catenin binding domain in E-cadherin are indicated. The F box of βTrCP is hatched. The FLAG epitope fused at the N-termini of βTrCP, βTrCP-Ecad, or βTrCP(ΔN)-Ecad is indicated by a solid rectangle. B. F-TrCP-Ecad binds to β-catenin S37A in vivo. HA-tagged β-catenin S37A and FLAG-tagged βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor MG132 for 4 hours before harvesting. Cell lysates were immunoprecipitated with the anti-FLAG antibody, precipitates were resolved by a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, and immunoblotted with the anti-HA antibody (top panel). Note that a nonspecific band that cross-reacted with the anti-HA antibody migrated slightly above β-catenin S37A in all lanes of this panel. The membrane was stripped and blotted with the anti-FLAG antibody (middle panel). The expression of β-catenin S37A in total cell lysates was examined by immunoblotting with the anti-HA antibody (bottom panel). C. F-TrCP-Ecad induces ubiquitination of β-catenin S37A in vivo. MYC-tagged β-catenin S37A, HA-tagged ubiquitin, and indicated βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor ALLN for 4 hours, and lysed in the denaturing buffer by boiling to disrupt non-covalent protein-protein interactions. Cell lysates were immunoprecipitated with the anti-HA antibody, and immunoprecipitates were resolved in SDS-PAGE and blotted with the anti-MYC antibody (top panel). The expression of β-catenin S37A in total cell lysates was determined by immunoblotting with the anti-MYC antibody (bottom panel). D. F-TrCP-Ecad reduces the steady state levels of β-catenin. HA-tagged β-catenin and CDK2 were co-expressed with indicated βTrCP derivatives in 293 cells. Total cell lysates were resolved by SDS-PAGE, and immunoblotted with the anti-HA antibody.

Mentions: The SCF ubiquitination machinery can be harnessed to degrade a specific target protein by fusing an F box protein with a peptide that is able to bind to the target protein [16]. Here we investigated whether an F-box protein can be redesigned to target their usual substrates that have become resistant to degradation (eg, by mutations in the N-terminus of β-catenin). Recognition of β-catenin by βTrCP normally requires phosphorylation of serine residues within the N-terminal DSGxxS motif of β-catein [13,15]. To target unphosphorylated and thus stabilized β-catenin to the core SCF for ubiquitination and degradation, we fused the β-catenin binding domain of E-cadherin (amino acids 794–883, designated Ecad) to the C-terminus of βTrCP (Fig. 1A). A glycine-serine-rich sequence was inserted between βTrCP and Ecad to relieve the potential steric hindrance between these two protein structures. Ser37 is one major GSK3 phosphorylation site of β-catenin that is recognized by βTrCP [13]. Substitution of Ser37 with Ala abrogates the association between β-catenin and βTrCP. It has been shown that β-catenin S37A is about 9-fold more stable than the wild-type β-catenin [17]. The binding between β-catenin S37A and βTrCP-Ecad was assayed in 293 cells using a co-immunoprecipitation assay. HA-tagged β-catenin S37A and FLAG-tagged βTrCP-Ecad were coexpressed in 293 cells. Total cell lysates were immunoprecipitated with the anti-FLAG antibody, and immunoprecipitates were subjected to SDS PAGE and immunoblotting with the anti-HA antibody. As shown in Fig. 1B, β-catenin S37A strongly interacted with F-TrCP-Ecad, but not F-TrCP, indicating that the intracellular domain of E-cadherin binds to the armadillo repeats of β-catenin in a phosphorylation-independent manner.


A protein knockdown strategy to study the function of beta-catenin in tumorigenesis.

Cong F, Zhang J, Pao W, Zhou P, Varmus H - BMC Mol. Biol. (2003)

Engineering a βTrCP-E-cadherin chimera that targets oncogenic β-catenin for degradation. A. Schematic diagrams of F-TrCP-Ecad chimeras used in this study. The β-catenin binding domain (amino acids 794–883) of E-cadherin (Ecad) was fused to the C-terminus of FLAG-tagged βTrCP to form F-TrCP-Ecad. The N-terminus of βTrCP (amino acid 1–297) was deleted from F-TrCP-Ecad to make F-TrCP(ΔN)-Ecad. The transmembrane (TM) region and the β-catenin binding domain in E-cadherin are indicated. The F box of βTrCP is hatched. The FLAG epitope fused at the N-termini of βTrCP, βTrCP-Ecad, or βTrCP(ΔN)-Ecad is indicated by a solid rectangle. B. F-TrCP-Ecad binds to β-catenin S37A in vivo. HA-tagged β-catenin S37A and FLAG-tagged βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor MG132 for 4 hours before harvesting. Cell lysates were immunoprecipitated with the anti-FLAG antibody, precipitates were resolved by a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, and immunoblotted with the anti-HA antibody (top panel). Note that a nonspecific band that cross-reacted with the anti-HA antibody migrated slightly above β-catenin S37A in all lanes of this panel. The membrane was stripped and blotted with the anti-FLAG antibody (middle panel). The expression of β-catenin S37A in total cell lysates was examined by immunoblotting with the anti-HA antibody (bottom panel). C. F-TrCP-Ecad induces ubiquitination of β-catenin S37A in vivo. MYC-tagged β-catenin S37A, HA-tagged ubiquitin, and indicated βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor ALLN for 4 hours, and lysed in the denaturing buffer by boiling to disrupt non-covalent protein-protein interactions. Cell lysates were immunoprecipitated with the anti-HA antibody, and immunoprecipitates were resolved in SDS-PAGE and blotted with the anti-MYC antibody (top panel). The expression of β-catenin S37A in total cell lysates was determined by immunoblotting with the anti-MYC antibody (bottom panel). D. F-TrCP-Ecad reduces the steady state levels of β-catenin. HA-tagged β-catenin and CDK2 were co-expressed with indicated βTrCP derivatives in 293 cells. Total cell lysates were resolved by SDS-PAGE, and immunoblotted with the anti-HA antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC222962&req=5

Figure 1: Engineering a βTrCP-E-cadherin chimera that targets oncogenic β-catenin for degradation. A. Schematic diagrams of F-TrCP-Ecad chimeras used in this study. The β-catenin binding domain (amino acids 794–883) of E-cadherin (Ecad) was fused to the C-terminus of FLAG-tagged βTrCP to form F-TrCP-Ecad. The N-terminus of βTrCP (amino acid 1–297) was deleted from F-TrCP-Ecad to make F-TrCP(ΔN)-Ecad. The transmembrane (TM) region and the β-catenin binding domain in E-cadherin are indicated. The F box of βTrCP is hatched. The FLAG epitope fused at the N-termini of βTrCP, βTrCP-Ecad, or βTrCP(ΔN)-Ecad is indicated by a solid rectangle. B. F-TrCP-Ecad binds to β-catenin S37A in vivo. HA-tagged β-catenin S37A and FLAG-tagged βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor MG132 for 4 hours before harvesting. Cell lysates were immunoprecipitated with the anti-FLAG antibody, precipitates were resolved by a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, and immunoblotted with the anti-HA antibody (top panel). Note that a nonspecific band that cross-reacted with the anti-HA antibody migrated slightly above β-catenin S37A in all lanes of this panel. The membrane was stripped and blotted with the anti-FLAG antibody (middle panel). The expression of β-catenin S37A in total cell lysates was examined by immunoblotting with the anti-HA antibody (bottom panel). C. F-TrCP-Ecad induces ubiquitination of β-catenin S37A in vivo. MYC-tagged β-catenin S37A, HA-tagged ubiquitin, and indicated βTrCP derivatives were co-expressed in 293 cells. Cells were treated with the proteasome inhibitor ALLN for 4 hours, and lysed in the denaturing buffer by boiling to disrupt non-covalent protein-protein interactions. Cell lysates were immunoprecipitated with the anti-HA antibody, and immunoprecipitates were resolved in SDS-PAGE and blotted with the anti-MYC antibody (top panel). The expression of β-catenin S37A in total cell lysates was determined by immunoblotting with the anti-MYC antibody (bottom panel). D. F-TrCP-Ecad reduces the steady state levels of β-catenin. HA-tagged β-catenin and CDK2 were co-expressed with indicated βTrCP derivatives in 293 cells. Total cell lysates were resolved by SDS-PAGE, and immunoblotted with the anti-HA antibody.
Mentions: The SCF ubiquitination machinery can be harnessed to degrade a specific target protein by fusing an F box protein with a peptide that is able to bind to the target protein [16]. Here we investigated whether an F-box protein can be redesigned to target their usual substrates that have become resistant to degradation (eg, by mutations in the N-terminus of β-catenin). Recognition of β-catenin by βTrCP normally requires phosphorylation of serine residues within the N-terminal DSGxxS motif of β-catein [13,15]. To target unphosphorylated and thus stabilized β-catenin to the core SCF for ubiquitination and degradation, we fused the β-catenin binding domain of E-cadherin (amino acids 794–883, designated Ecad) to the C-terminus of βTrCP (Fig. 1A). A glycine-serine-rich sequence was inserted between βTrCP and Ecad to relieve the potential steric hindrance between these two protein structures. Ser37 is one major GSK3 phosphorylation site of β-catenin that is recognized by βTrCP [13]. Substitution of Ser37 with Ala abrogates the association between β-catenin and βTrCP. It has been shown that β-catenin S37A is about 9-fold more stable than the wild-type β-catenin [17]. The binding between β-catenin S37A and βTrCP-Ecad was assayed in 293 cells using a co-immunoprecipitation assay. HA-tagged β-catenin S37A and FLAG-tagged βTrCP-Ecad were coexpressed in 293 cells. Total cell lysates were immunoprecipitated with the anti-FLAG antibody, and immunoprecipitates were subjected to SDS PAGE and immunoblotting with the anti-HA antibody. As shown in Fig. 1B, β-catenin S37A strongly interacted with F-TrCP-Ecad, but not F-TrCP, indicating that the intracellular domain of E-cadherin binds to the armadillo repeats of β-catenin in a phosphorylation-independent manner.

Bottom Line: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate.As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. congf@mskcc.org

ABSTRACT

Background: The Wnt signaling pathway plays critical roles in cell proliferation and cell fate determination at many stages of development. A critical downstream target of Wnt signaling is the cytosolic beta-catenin, which is stabilized upon Wnt activation and promotes transcription of a variety of target genes including c-myc and cyclin D. Aberrant Wnt signaling, which results from mutations of either beta-catenin or adenomatous polyposis coli (APC), renders beta-catenin resistant to degradation, and has been associated with multiple types of human cancers.

Results: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the beta-catenin binding domain of E-cadherin fused to betaTrCP ubiquitin-protein ligase, the stable beta-catenin mutant was recruited to the cellular SCF (Skp1, Cullin 1, and F-box-containing substrate receptor) ubiquitination machinery for ubiquitination and degradation. The DLD1 colon cancer cells express wild type beta-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of betaTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of beta-catenin. As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.

Conclusion: We have designed a novel approach to induce degradation of stabilized/mutated beta-catenin. Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells. The protein knockdown strategy can be utilized not only as a novel method to dissect the role of oncoproteins in tumorigenesis, but also as a unique tool to delineate the function of a subpopulation of proteins localized to a specific subcellular compartment.

Show MeSH
Related in: MedlinePlus