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The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell
M Hart1, J P Concordet, I Lassot
1Onyx Pharmaceuticals 3031 Research Drive Richmond California 94806 USA.
Abstract:
Defects in beta-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of beta-catenin can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3beta (GSK3beta). Given that phosphorylation can regulate targeted degradation of beta-catenin by the proteasome, beta-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the beta-catenin homolog Armadillo. We reasoned that the human homologs of Slimb - beta-TrCP and its isoform beta-TrCP2 (KIAA0696) - might interact with beta-catenin. We found that the binding of beta-TrCP to beta-catenin was direct and dependent upon the WD40 repeat sequences in beta-TrCP and on phosphorylation of the GSK3beta sites in beta-catenin. Endogenous beta-catenin and beta-TrCP could be coimmunoprecipitated from mammalian cells. Overexpression of wild-type beta-TrCP in mammalian cells promoted the downregulation of beta-catenin, whereas overexpression of a dominant-negative deletion mutant upregulated beta-catenin protein levels and activated signaling dependent on the transcription factor Tcf. In contrast, beta-TrCP2 did not associate with beta-catenin. We conclude that beta-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated beta-catenin.
Insights
Beta-catenin regulation is crucial for preventing cell transformation. This study identifies beta-transducin repeat containing E3 ubiquitin protein ligase (beta-TrCP) as a key E3 ubiquitin ligase targeting phosphorylated beta-catenin for degradation.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Beta-catenin dysregulation, often due to mutations affecting glycogen synthase kinase 3beta (GSK3beta) phosphorylation sites, contributes to cancer development.
- Phosphorylation regulates beta-catenin degradation via the proteasome, suggesting interaction with E3 ubiquitin ligase complexes.
- The Drosophila F-box protein Slimb targets beta-catenin homologs for degradation, indicating a conserved mechanism.
Purpose of the Study:
- To investigate the interaction between human beta-catenin and its potential E3 ubiquitin ligase partners, specifically the F-box protein homologs beta-TrCP and beta-TrCP2.
- To determine if beta-TrCP mediates the degradation of phosphorylated beta-catenin.
Main Methods:
- Co-immunoprecipitation assays to detect protein-protein interactions between beta-catenin and beta-TrCP/beta-TrCP2.
- Analysis of beta-catenin binding to beta-TrCP, focusing on WD40 repeat sequences and GSK3beta phosphorylation sites.
- Overexpression studies in mammalian cells to assess the impact of wild-type and mutant beta-TrCP on beta-catenin protein levels and Tcf signaling.
Main Results:
- Direct binding was observed between beta-catenin and beta-TrCP, dependent on beta-TrCP's WD40 repeats and beta-catenin phosphorylation at GSK3beta sites.
- Endogenous beta-catenin and beta-TrCP were co-immunoprecipitated from mammalian cells.
- Overexpression of wild-type beta-TrCP reduced beta-catenin levels, while a dominant-negative mutant increased them and activated Tcf signaling; beta-TrCP2 did not interact with beta-catenin.
Conclusions:
- Beta-transducin repeat containing E3 ubiquitin protein ligase (beta-TrCP) directly binds to phosphorylated beta-catenin.
- Beta-TrCP functions as an E3 ubiquitin ligase, targeting phosphorylated beta-catenin for proteasomal degradation.
- This mechanism is critical for regulating beta-catenin levels and preventing neoplastic transformation.