Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila.
Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila.
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DOI：
10.1371/journal.pbio.1001239
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发表时间：
2012-01
期刊：
影响因子：
9.8
通讯作者：
Jiang J
中科院分区：
文献类型：
--
作者：
Li S;Chen Y;Shi Q;Yue T;Wang B;Jiang J
Hedgehog通过促进果蝇细胞表面七跨膜蛋白Smoothened（Smo）的表达来传递信号，但其潜在机制尚不清楚。在这里，我们证明，Smo是下调泛素介导的内吞作用和降解，并Hh增加Smo细胞表面表达抑制其泛素化。我们发现Smo在多个赖氨酸残基处被泛素化，包括在其自身抑制结构域（SAID）中的那些，导致Smo通过溶酶体和蛋白酶体依赖性机制的内吞和降解。Hh通过PKA/CK 1介导的SAID磷酸化抑制Smo泛素化，导致Smo细胞表面积累。泛素活化酶Uba 1的失活或内吞机制的多个组分的扰动导致Smo积累和Hh途径活化。此外，我们发现非视觉β-arrestin Kurtz（Krz）与Smo相互作用，并与泛素化平行作用，下调Smo。最后，我们表明，Smo泛素化是由去泛素化酶UBPY/USP 8抵消。UBPY的获得和丢失导致Smo细胞表面表达的相互变化。两者合计，我们的研究结果表明，泛素化起着关键作用，在下调Smo保持Hh通路活性关闭在配体的情况下，和Hh诱导的磷酸化促进Smo细胞表面积累通过抑制其泛素化，这有助于Hh通路激活。Hedgehog（Hh）家族的分泌蛋白在从果蝇到人类的不同物种中控制细胞生长和图案化。Hh通过调节称为Smoothened（Smo）的膜蛋白的亚细胞位置和构象来跨越细胞表面膜发出信号。在果蝇中，Smo响应于Hh而在细胞表面上积累，而在Hh不存在的情况下，Smo被内化和降解。控制这种细胞内运输和Smo降解的分子机制是未知的，但在这里，我们表明，Smo是通过连接几个分子的小蛋白质称为泛素，标记它的内化和细胞内的降解进行修改。Hh通过两种类型的蛋白激酶诱导Smo的细胞内尾的另一种修饰（磷酸化）来抑制Smo的这种泛素化。这种泛素化的丧失和磷酸化的获得导致Smo在细胞表面的积累。此外，我们发现另一种名为Kurtz的蛋白与Smo相互作用，并与泛素化过程平行，以促进Smo的内化，去泛素化酶UBPY/USP 8抵消Smo的泛素化，以促进其细胞表面积累。我们的研究表明，可逆的泛素化在调节Smo进出细胞表面的运输中起着关键作用，因此它为Hh信号从细胞外到细胞内的机制提供了新的见解。
Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. Here we demonstrate that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. We find that Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, we find that the non-visual β-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, we show that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, our results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation. The Hedgehog (Hh) family of secreted proteins governs cell growth and patterning in diverse species ranging from Drosophila to human. Hh signals across the cell surface membrane by regulating the subcellular location and conformation of a membrane protein called Smoothened (Smo). In Drosophila, Smo accumulates on the cell surface in response to Hh, whereas in the absence of Hh it is internalized and degraded. The molecular mechanisms that control this intracellular trafficking and degradation of Smo were unknown, but here we show that Smo is modified by attachment of several molecules of a small protein called ubiquitin, which tags it for internalization and degradation within the cell. Hh inhibits this ubiquitination of Smo by inducing another modification, phosphorylation, of its intracellular tail by two types of protein kinase enzymes. This loss of ubiquitination and gain of phosphorylation causes the accumulation of Smo at the cell surface. What's more, we find that another protein called Kurtz interacts with Smo and acts in parallel with the ubiquitination process to promote internalization of Smo, and that the deubiquitinating enzyme UBPY/USP8 counteracts ubiquitination of Smo to promote its cell surface accumulation. Our study demonstrates that reversible ubiquitination plays a key role in regulating Smo trafficking to and from the cell surface and thus it provides novel insights into the mechanism of Hh signaling from the outside to the inside of the cell.
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