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Published on: June 17, 2014
Determination of Rate-Limiting Factor for Formation of Beta-Catenin Destruction Complexes Using Absolute Protein
Masashi Kitazawa1, Tomohisa Hatta1, Koji Ogawa1
1Molecular Profiling Reserch Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST) , 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
Abstract:
Wnt/β-catenin signaling plays important roles in both ontogenesis and development. In the absence of a Wnt stimulus, β-catenin is degraded by a multiprotein "destruction complex" that includes Axin, APC, GSK3B, and FBXW11. Although the key molecules required for transducing Wnt signals have been identified, a quantitative understanding of this pathway has been lacking. Here, we calculated the absolute number of β-catenin destruction complexes by absolute protein quantification using LC-MS/MS. Similar amounts of destruction complex-constituting proteins and β-catenin interacted, and the number of destruction complexes was calculated to be about 1468 molecules/cell. We demonstrated that the calculated number of destruction complexes was valid for control of the β-catenin destruction rate under steady-state conditions. Interestingly, APC had the minimum expression level among the destruction complex components at about 2233 molecules/cell, and this number approximately corresponded to the calculated number of destruction complexes. Decreased APC expression by siRNA transfection decreased the number of destruction complexes, resulting in β-catenin accumulation and stimulation of the transcriptional activity of T-cell factor. Taken together, our results suggest that the amount of APC expression is the rate-limiting factor for the constitution of β-catenin destruction complexes.
Insights
The Wnt/β-catenin pathway
Area of Science:
- Cellular biology
- Molecular signaling pathways
- Developmental biology
Background:
- Wnt/β-catenin signaling is crucial for development and ontogenesis.
- β-catenin degradation is mediated by a destruction complex in the absence of Wnt.
- Quantitative insights into this pathway have been limited.
Purpose of the Study:
- To quantitatively determine the number of β-catenin destruction complexes.
- To identify the rate-limiting component of the destruction complex.
Main Methods:
- Absolute protein quantification using Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS).
- Analysis of protein-protein interactions within the destruction complex.
- siRNA-mediated knockdown of Adenomatous Polyposis Coli (APC) expression.
Main Results:
- Approximately 1468 β-catenin destruction complexes exist per cell.
- Adenomatous Polyposis Coli (APC) expression level (approx. 2233 molecules/cell) was the minimum among complex components.
- Reduced APC levels decreased destruction complex numbers, leading to β-catenin accumulation and T-cell factor activation.
Conclusions:
- APC expression level is the rate-limiting factor for β-catenin destruction complex formation.
- This finding provides a quantitative understanding of Wnt/β-catenin signaling regulation.
- APC quantity directly impacts β-catenin stability and downstream signaling.
