Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.
Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.
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DOI：
10.15252/embr.201540352
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发表时间：
2015-08
期刊：
影响因子：
7.7
通讯作者：
Muqit MM
中科院分区：
文献类型：
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作者：
Kazlauskaite A;Martínez-Torres RJ;Wilkie S;Kumar A;Peltier J;Gonzalez A;Johnson C;Zhang J;Hope AG;Peggie M;Trost M;van Aalten DM;Alessi DR;Prescott AR;Knebel A;Walden H;Muqit MM
线粒体蛋白激酶PINK 1突变与常染色体隐性遗传帕金森病（PD）相关。我们和其他研究小组已经报道，PINK 1通过直接磷酸化帕金丝氨酸65（Ser 65）（位于其泛素样结构域（Ubl）内）和间接磷酸化泛素Ser 65来激活帕金E3连接酶活性。Ser 65磷酸化的泛素（ubiquitinPhospho-Ser 65）如何促进帕金激活目前尚不清楚。在这里，我们证明了泛素磷酸化Ser 65与帕金蛋白的结合显著增加了PINK 1在体外Ser 65处的帕金蛋白磷酸化的速率和化学计量。通过定点突变证实的Parkin结构的分析表明，保守的His 302和Lys 151残基在泛素磷酸化Ser 65的结合中起关键作用，从而促进Parkin Ser 65的磷酸化和其体外E3连接酶活性的激活。His 302的突变显著抑制线粒体处的Parkin Ser 65磷酸化，这与线粒体去极化后其E3连接酶活性的显著降低相关。我们发现，结合泛素磷酸丝氨酸65帕金破坏Ubl结构域和C-末端区域之间的相互作用，从而增加帕金丝氨酸65的可及性。最后，纯化的帕金蛋白在体外Ser 65处最大程度磷酸化，不能通过加入泛素磷酸化Ser 65进一步激活。因此，我们的研究结果表明，泛素磷酸化Ser 65的一个主要作用是促进PINK 1介导的磷酸化帕金在Ser 65，导致最大激活帕金E3连接酶活性。His 302和Lys 151可能在Parkin表面排列一个磷酸化Ser 65结合口袋，这对泛素磷酸化Ser 65相互作用至关重要。这项研究提供了新的机制的见解通过ubiquitinPhospho-Ser 65激活帕金，这可能有助于开发模仿ubiquitinPhospho-Ser 65作用的帕金激活剂。
Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho-Ser65.