A phosphorylation switch of Huluwa receptor for embryonic axis induction

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 receptor remain unclear. In a systematic mutation screen, a mutation at the 168 ^th serine (Ser168) in the PPNSP motif of Hwa dramatically abolished 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. Furthermore, the Ser168 site was confirmed to be phosphorylated via LC-MS/MS and immunoblotting with a phosphorylation-specific antibody. Additionally, several kinases, including Cdk16, Cdk2, and GSK3β, were found to enhance Ser168 phosphorylation in vitro and in vivo , enhancing Hwa's activity in β-catenin signaling and axis induction. Meanwhile, the use of dominant-negative Cdk16 and pHwa (Ser168) antibodies reduced Hwa's function. In summary, Ser168 acts as a phosphorylation switch in Hwa/β-catenin signaling for embryonic axis induction, involving multiple kinases to ensure developmental robustness.