Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, USA.
J Biol Chem. 2010 Apr 30;285(18):13507-16. doi: 10.1074/jbc.M109.049411. Epub 2010 Feb 24.
Beta-catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt target genes. Upon UV-induced DNA damage, beta-catenin is recruited for polyubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin beta-like 1 (TBL1), and adenomatous polyposis coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated beta-catenin substrate, we have investigated Siah-1-mediated ubiquitination of beta-catenin in vitro and in cells. Overexpression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of beta-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to polyubiquitinate beta-catenin. In addition, TBL1 was shown to play a role in protecting beta-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of beta-catenin, suggesting that polyubiquitination of beta-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.
β-连环蛋白是 Wnt 信号通路的关键组成部分，作为 Wnt 靶基因的转录共激活因子发挥作用。在紫外线诱导的 DNA 损伤后，β-连环蛋白被募集进行多泛素化，并随后通过独特的、由 p53 诱导的 SCF 样复合物（SCF(TBL1)）进行蛋白酶体降解，该复合物由 Siah-1、Siah-1 相互作用蛋白（SIP）、Skp1、转导素β样 1（TBL1）和腺瘤性结肠息肉病基因（APC）组成。鉴于涉及的各种因素的复杂性和非磷酸化 β-连环蛋白底物泛素化的新颖性，我们已经在体外和细胞中研究了 Siah-1 介导的 β-连环蛋白泛素化。为每个 SCF(TBL1) 蛋白开发了过表达和纯化方案，使我们能够使用体外泛素化测定法对 β-连环蛋白的泛素化进行系统分析。这项研究表明，Siah-1 本身能够多泛素化 β-连环蛋白。此外，还表明 TBL1 在体外保护 β-连环蛋白免受 Siah-1 泛素化以及细胞中 Siah-1 靶向的蛋白酶体降解方面发挥作用。发现 Siah-1 和 TBL1 与 β-连环蛋白的相同角蛋白重复结构域结合，这表明在 Wnt 信号转导过程中，β-连环蛋白的多泛素化受到 Siah-1 和 TBL1 之间竞争的调节。
