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.

中文翻译：

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使用绝对蛋白质定量确定形成β-连环蛋白破坏复合物的限速因子

Wnt /β-catenin信号传导在本体发育和发育中都起着重要作用。在没有Wnt刺激的情况下，β-catenin被包括Axin，APC，GSK3B和FBXW11在内的多蛋白“破坏复合物”降解。尽管已确定了转导Wnt信号所需的关键分子，但仍缺乏对该途径的定量理解。在这里，我们使用LC-MS / MS通过绝对蛋白质定量计算了β-catenin破坏复合物的绝对数量。相似数量的破坏复合物构成蛋白和β-catenin相互作用，破坏复合物的数量约为1468个分子/细胞。我们证明了计算出的破坏复合物数量对于在稳态条件下控制β-catenin破坏速率是有效的。有趣的是，APC在破坏复合物组分中的最低表达水平约为2233个分子/细胞，该数目大约与计算出的破坏复合物数目相对应。siRNA转染降低APC表达可减少破坏复合物的数量，从而导致β-catenin积聚并刺激T细胞因子的转录活性。两者合计，我们的结果表明，APC表达的数量是构成β-catenin破坏复合物的速率限制因素。导致β-catenin积累并刺激T细胞因子的转录活性。两者合计，我们的结果表明，APC表达的数量是构成β-catenin破坏复合物的速率限制因素。导致β-catenin积累并刺激T细胞因子的转录活性。两者合计，我们的结果表明，APC表达的数量是构成β-catenin破坏复合物的速率限制因素。