Parkin-catalyzed Ubiquitin-Ester Transfer Is Triggered by PINK1-dependent Phosphorylation
Parkin-catalyzed Ubiquitin-Ester Transfer Is Triggered by PINK1-dependent Phosphorylation
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DOI：
10.1074/jbc.m113.467530
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发表时间：
2013-07-26
影响因子：
4.8
通讯作者：
Matsuda, Noriyuki
中科院分区：
文献类型：
--
作者：
Iguchi, Masahiro;Kujuro, Yuki;Matsuda, Noriyuki
PINK1 和 PARKIN 是常染色体隐性遗传家族性帕金森病的致病基因。 PINK1 是一种线粒体 Ser/Thr 激酶，而 Parkin 则充当 E3 泛素连接酶。在稳态条件下，Parkin 定位于细胞质，其 E3 活性受到抑制。线粒体膜电位的降低会触发 Parkin E3 活性，并将其募集至去极化线粒体，以实现线粒体底物的泛素化。 Parkin E3 活性如何通过线粒体损伤重建的分子基础尚未确定。在这里，我们提供了重组 Parkin 的 Cys-431 上泛素硫酯形成的体外生化证据。我们还报告说，Parkin 在细胞线粒体膜电位降低后形成泛素酯，并且该事件对于底物泛素化至关重要。重要的是，Parkin RING2 结构域充当转硫基或酰基转移结构域，而不是 E2 招募结构域。此外，泛素酯的形成取决于 Parkin Ser-65 的 PINK1 磷酸化。磷酸化缺陷突变完全抑制 Parkin 泛素酯中间体的形成，而磷酸化模拟物（例如 Ser 到 Glu 的取代）能够部分形成中间体，而与 Ser-65 磷酸化无关。我们认为 Parkin 的 PINK1 依赖性磷酸化会导致 RING2 结构域的泛素酯转移反应，这是 Parkin 激活的重要步骤。
PINK1 and PARKIN are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation.