The serine/threonine protein kinase casein kinase 1α (CK1α) functions as a negative regulator of Wnt signaling, phosphorylating β-catenin at serine 45 (P-S45) to initiate its eventual ubiquitin-mediated degradation. We previously showed that the repurposed, FDA-approved anthelminthic drug pyrvinium potently inhibits Wnt signaling in vitro and in vivo. Moreover, we proposed that pyrvinium's Wnt inhibitory activity was the result of its function as an activator of CK1α. An understanding of the mechanism by which pyrvinium activates CK1α is important because pyrvinium was given an orphan drug designation by the FDA to treat familial adenomatous polyposis, a precancerous condition driven by constitutive Wnt signaling. In the current study, we show that pyrvinium stimulates the phosphorylation of S45 β-catenin, a known CK1α substrate, in a cell-based assay, and does so in a dose- and time-dependent manner. Alternative splicing of CK1α results in four forms of the protein with distinct biological properties. We evaluated these splice products and identified the CK1α splice variant, CK1αS, as the form that exhibits the most robust response to pyrvinium in cells. Kinetic studies indicate that pyrvinium also stimulates the kinase activity of purified, recombinant CK1αS in vitro, increasing its catalytic efficiency (kcat/Km) toward substrates. These studies provide strong and clear mechanistic evidence that pyrvinium enhances CK1α kinase activity.

中文翻译：

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CK1α 激活剂 Pyrvinium 可提高 CK1α 的催化效率 (kcat/Km)。

丝氨酸/苏氨酸蛋白激酶酪蛋白激酶 1α (CK1α) 作为 Wnt 信号传导的负调节剂，在丝氨酸 45 (P-S45) 处磷酸化 β-连环蛋白以启动其最终的泛素介导的降解。我们之前表明，经过重新利用的 FDA 批准的驱虫药物 pyrvinium 在体外和体内有效地抑制 Wnt 信号传导。此外，我们提出pyrvinium的Wnt抑制活性是其作为CK1α激活剂的结果。了解 pyrvinium 激活 CK1α 的机制很重要，因为 pyrvinium 被 FDA 指定用于治疗家族性腺瘤性息肉病，这是一种由组成性 Wnt 信号驱动的癌前病变。在目前的研究中，我们表明pyrvinium 刺激S45 β-连环蛋白（一种已知的CK1α 底物）的磷酸化，在基于细胞的测定中，并且以剂量和时间依赖性方式进行。CK1α 的可变剪接产生了四种具有不同生物学特性的蛋白质。我们评估了这些剪接产物，并确定了 CK1α 剪接变体 CK1αS 是对细胞中pyrvinium 反应最强烈的形式。动力学研究表明，pyrvinium 还在体外刺激纯化的重组 CK1αS 的激酶活性，提高其对底物的催化效率 (kcat/Km)。这些研究提供了强有力和明确的机制证据，表明pyrvinium增强CK1α激酶活性。CK1αS，作为对细胞中pyrvinium表现出最强烈反应的形式。动力学研究表明，pyrvinium 还在体外刺激纯化的重组 CK1αS 的激酶活性，提高其对底物的催化效率 (kcat/Km)。这些研究提供了强有力和明确的机制证据，表明pyrvinium增强CK1α激酶活性。CK1αS，作为对细胞中pyrvinium表现出最强烈反应的形式。动力学研究表明，pyrvinium 还在体外刺激纯化的重组 CK1αS 的激酶活性，提高其对底物的催化效率 (kcat/Km)。这些研究提供了强有力和明确的机制证据，表明pyrvinium增强CK1α激酶活性。