Benetti Roberta, Copetti Tamara, Dell'Orso Stefania, Melloni Edon, Brancolini Claudio, Monte Martin, Schneider Claudio
Laboratorio Nazionale del Consorzio Interuniversitario per le Biotecnologie, Area Science Park, Padriciano 99, 34012 Trieste, Italy.
J Biol Chem. 2005 Jun 10;280(23):22070-80. doi: 10.1074/jbc.M501810200. Epub 2005 Apr 7.
Beta-catenin is a multifunctional protein serving both as a structural element in cell adhesion and as a signaling component in the Wnt pathway, regulating embryogenesis and tumorigenesis. The signaling fraction of beta-catenin is tightly controlled by the adenomatous polyposis coli-axin-glycogen synthase kinase 3beta complex, which targets it for proteasomal degradation. It has been recently shown that Ca(2+) release from internal stores results in nuclear export and calpain-mediated degradation of beta-catenin in the cytoplasm. Here we have highlighted the critical relevance of constitutive calpain pathway in the control of beta-catenin levels and functions, showing that small interference RNA knock down of endogenous calpain per se (i.e. in the absence of external stimuli) induces an increase in the free transcriptional competent pool of endogenous beta-catenin. We further characterized the role of the known calpain inhibitors, Gas2 and Calpastatin, demonstrating that they can also control levels, function, and localization of beta-catenin through endogenous calpain regulation. Finally we present Gas2 dominant negative (Gas2DN) as a new tool for regulating calpain activity, providing evidence that it counteracts the described effects of both Gas2 and Calpastatin on beta-catenin and that it works via calpain independently of the classical glycogen synthase kinase 3beta and proteasome pathway. Moreover, we provide in vitro biochemical evidence showing that Gas2DN can increase the activity of calpain and that in vivo it can induce degradation of stabilized/mutated beta-catenin. In fact, in a context where the classical proteasome pathway is impaired, as in colon cancer cells, Gas2DN biological effects accounted for a significant reduction in proliferation and anchorage-independent growth of colon cancer.
β-连环蛋白是一种多功能蛋白质，既作为细胞黏附中的结构成分，又作为Wnt信号通路中的信号成分，调节胚胎发育和肿瘤发生。β-连环蛋白的信号部分受到腺瘤性息肉病大肠杆菌-轴蛋白-糖原合酶激酶3β复合物的严格控制，该复合物将其靶向蛋白酶体降解。最近有研究表明，细胞内储存库释放的Ca(2+)会导致β-连环蛋白在细胞质中发生核输出和钙蛋白酶介导的降解。在这里，我们强调了组成型钙蛋白酶途径在控制β-连环蛋白水平和功能方面的关键相关性，表明内源性钙蛋白酶的小干扰RNA敲低本身（即在没有外部刺激的情况下）会导致内源性β-连环蛋白的游离转录活性池增加。我们进一步表征了已知的钙蛋白酶抑制剂Gas2和钙蛋白酶抑制蛋白的作用，证明它们也可以通过内源性钙蛋白酶调节来控制β-连环蛋白的水平、功能和定位。最后，我们提出Gas2显性阴性（Gas2DN）作为调节钙蛋白酶活性的新工具，提供证据表明它可以抵消Gas2和钙蛋白酶抑制蛋白对β-连环蛋白的上述影响，并且它通过钙蛋白酶起作用，独立于经典的糖原合酶激酶3β和蛋白酶体途径。此外，我们提供了体外生化证据，表明Gas2DN可以增加钙蛋白酶的活性，并且在体内它可以诱导稳定化/突变型β-连环蛋白的降解。事实上，在经典蛋白酶体途径受损的情况下，如在结肠癌细胞中，Gas2DN的生物学效应导致结肠癌细胞增殖和非锚定依赖性生长显著降低。
