Embryonic axis formation is essential for patterning and morphogenesis in vertebrates and is tightly regulated by the dorsal organizer. Previously, we demonstrated that maternally derived Huluwa (Hwa) acts as a dorsal determinant, dictating axis formation by activating β-catenin signaling in zebrafish and Xenopus. However, the mechanism of activation and fine regulation of the Hwa protein remains unclear. Through candidate screening we identified a mutation at Ser168 in the PPNSP motif of Hwa that dramatically abolishes its axis-inducing activity. Mechanistically, mutating the Ser168 residue reduced its binding affinity to Tankyrase 1/2 and the degradation of the Axin protein, weakening β-catenin signaling activation. We confirmed that Ser168 is phosphorylated and that phosphorylation increases Hwa activity in β-catenin signaling and axis induction. Several kinases including Cdk16, Cdk2, and GSK3β, were found to enhance Ser168 phosphorylation in vitro and in vivo. Both dominant-negative Cdk16 expression and pHwa (Ser168) antibody treatment reduce Hwa function. Lastly, a knock-in allele mutating Ser168 to alanine resulted in embryos lacking body axes, demonstrating that Ser168 is essential to axis formation. In summary, Ser168 acts as a phosphorylation switch in Hwa/β-catenin signaling for embryonic axis induction, regulated by multiple kinases.

胚胎轴的形成对于脊椎动物的模式化和形态发生至关重要，并且受到背组织者的严格调节。以前，我们证明了母系来源的 Huluwa （Hwa） 作为背侧决定因素，通过激活斑马鱼和非洲爪蟾中的 β-catenin 信号传导来决定轴的形成。然而，Hwa 蛋白的激活和精细调节机制仍不清楚。通过候选筛选，我们在 Hwa 的 PPNSP 基序中发现了 Ser168 的突变，该突变显着消除了其轴诱导活性。从机制上讲，突变 Ser168 残基降低了其与 Tankyrase 1/2 的结合亲和力和 Axin 蛋白的降解，从而削弱了 β-catenin 信号激活。我们证实 Ser168 被磷酸化，磷酸化增加了 β-catenin 信号传导和轴诱导中的 Hwa 活性。发现包括 Cdk16、Cdk2 和 GSK3β 在内的几种激酶在体外和体内可增强 Ser168 磷酸化。显性阴性 Cdk16 表达和 pHwa （Ser168） 抗体处理都会降低 Hwa 功能。最后，将 Ser168 突变为丙氨酸的敲入等位基因导致胚胎缺乏体轴，表明 Ser168 对轴的形成至关重要。总之，Ser168 在 Hwa/β-catenin 信号转导中充当胚胎轴诱导的磷酸化开关，受多种激酶调节。