Regulation of Smoothened ubiquitylation and cell surface expression through a Cul4-DDB1-Gβ E3 ubiquitin ligase complex
Regulation of Smoothened ubiquitylation and cell surface expression through a Cul4-DDB1-Gβ E3 ubiquitin ligase complex
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DOI：
10.1242/jcs.218016
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发表时间：
2018-08-01
影响因子：
4
通讯作者：
Jiang, Jin
中科院分区：
文献类型：
--
作者：
Li, Shuang;Cho, Yong Suk;Jiang, Jin
Hedgehog (Hh) 通过促进果蝇细胞表面积累和 G 蛋白偶联受体 (GPCR) 家族蛋白 Smoothened (Smo) 的激活来转导信号，但调节 Smo 运输的分子机制仍知之甚少。在这里，我们确定 Cul4-DDB1 E3 泛素连接酶复合物对于 Smo 泛素化和细胞表面清除至关重要。我们发现Smo的C端胞内结构域通过三聚体G蛋白（G beta）的β亚基招募Cul4-DDB1，并且Cul4-DDB1-G beta促进Smo及其结合伙伴G蛋白偶联受体激酶2（Gprk2）的泛素化并诱导Smo的内化和降解。 Hh 通过招募蛋白激酶 A (PKA) 的催化亚基磷酸化 DDB1，从而破坏其与 G beta 的相互作用，从而将 Cul4-DDB1 与 Smo 分离。 Cul4-DDB1 复合物的失活导致 Smo 细胞表面表达升高，而过量的 Cul4-DDB1 会阻止 Smo 积累并减弱 Hh 通路激活。总而言之，我们的研究确定了一种针对 Smo 进行泛素化的 E3 泛素连接酶复合物，并为 Hh 信号如何调节 Smo 运输和细胞表面表达提供了新的见解。
Hedgehog (Hh) transduces signals by promoting cell surface accumulation and activation of the G-protein-coupled receptor (GPCR)-family protein Smoothened (Smo) in Drosophila, but the molecular mechanism underlying the regulation of Smo trafficking remains poorly understood. Here, we identified the Cul4-DDB1 E3 ubiquitin ligase complex as being essential for Smo ubiquitylation and cell surface clearance. We found that the C-terminal intracellular domain of Smo recruits Cul4-DDB1 through the beta subunit of trimeric G protein (G beta), and that Cul4-DDB1-G beta promotes the ubiquitylation of both Smo and its binding partner G-protein-coupled-receptor kinase 2 (Gprk2) and induces the internalization and degradation of Smo. Hh dissociates Cul4-DDB1 from Smo by recruiting the catalytic subunit of protein kinase A (PKA) to phosphorylate DDB1, which disrupts its interaction with G beta. Inactivation of the Cul4-DDB1 complex resulted in elevated Smo cell surface expression, whereas an excessive amount of Cul4-DDB1 blocked Smo accumulation and attenuated Hh pathway activation. Taken together, our study identifies an E3 ubiquitin ligase complex targeting Smo for ubiquitylation and provides new insight into how Hh signaling regulates Smo trafficking and cell surface expression.