Hedgehog (Hh) pathway plays multiple roles in many physiological processes and its dysregulation leads to congenital disorders and cancers. Hh regulates the cellular localization of Smoothened (Smo) and the stability of Cubitus interruptus (Ci) to fine-tune the signal outputs. However, the underlying mechanisms are still unclear. Here, we show that the scaffold protein Rack1 plays dual roles in Hh signaling. In the absence of Hh, Rack1 promotes Ci and Cos2 to form a Ci–Rack1–Cos2 complex, culminating in Slimb-mediated Ci proteolysis. In the presence of Hh, Rack1 dissociates from Ci–Rack1–Cos2 complex and forms a trimeric complex with Smo and Usp8, leading to Smo deubiquitination and cell surface accumulation. Furthermore, we find the regulation of Rack1 on Hh pathway is conserved from Drosophila to mammalian cells. Our findings demonstrate that Rack1 plays dual roles during Hh signal transduction and provide Rack1 as a potential drug target for Hh-related diseases.

Hedgehog (Hh) 通路在许多生理过程中发挥多重作用，其失调会导致先天性疾病和癌症。Hh 调节平滑 (Smo) 的细胞定位和肘部中断 (Ci) 的稳定性，以微调信号输出。然而，其潜在机制仍不清楚。在这里，我们展示了支架蛋白 Rack1 在 Hh 信号传导中的双重作用。在没有 Hh 的情况下，Rack1 促进 Ci 和 Cos2 形成 Ci-Rack1-Cos2 复合物，最终导致 Slimb 介导的 Ci 蛋白水解。在 Hh 存在下，Rack1 从 Ci-Rack1-Cos2 复合物解离并与 Smo 和 Usp8 形成三聚体复合物，导致 Smo 去泛素化和细胞表面积累。此外，我们发现 Rack1 对 Hh 通路的调节在果蝇中是保守的到哺乳动物细胞。我们的研究结果表明 Rack1 在 Hh 信号转导过程中发挥双重作用，并提供 Rack1 作为 Hh 相关疾病的潜在药物靶点。