Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity.
Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity.
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DOI：
10.1098/rsob.130213
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发表时间：
2014-03-19
期刊：
影响因子：
5.8
通讯作者：
Muqit MM
中科院分区：
文献类型：
--
作者：
Kazlauskaite A;Kelly V;Johnson C;Baillie C;Hastie CJ;Peggie M;Macartney T;Woodroof HI;Alessi DR;Pedrioli PG;Muqit MM
PINK1和Parkin基因突变与早发性帕金森病有关。我们最近发现PINK1在其Ubl结构域中磷酸化Parkin的丝氨酸65 (Ser65)，导致其在无底物活性试验中激活。我们现在证明了Ser65磷酸化对底物泛素化的关键要求，通过一种新的体外E3连接酶活性测定，使用全长无标记Parkin及其假定的底物，线粒体GTPase Miro1。我们观察到Parkin在PINK1磷酸化后有效地使Miro1在高度保守的赖氨酸残基153、230、235、330和572处泛素化。我们进一步建立了E2-泛素释放实验来评估Parkin的活性，并观察到野生型（而不是激酶失活的PINK1或Parkin Ser65Ala突变体）在Parkin的Ser65位点磷酸化时，携带泛素的UbcH7 E2连接酶的强大释放，这提示了Ser65磷酸化如何激活Parkin E3连接酶活性的可能机制。据我们所知，我们首次报道了使用全长无标记重组Parkin的底物检测中帕金病相关突变的影响。我们的突变分析表明了催化半胱氨酸Cys431的重要作用，并揭示了突变如何通过破坏Miro1泛素化、自由泛素链形成或影响Parkin从负载E2中释放泛素的能力来赋予致病性的基本新知识。这项研究提供了进一步的证据，表明Parkin的Ser65位点磷酸化对其激活至关重要。这也提供了证据，证明mir1是一个直接的Parkin底物。本研究中开发的检测和试剂对于揭示帕金森氏生物学的新见解以及帮助开发用于治疗帕金森氏病的小分子帕金森氏激活剂的筛选非常重要。
Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser65) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser65 phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser65 by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser65 phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin's ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser65 is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson's disease.
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DOI：
10.1111/gtc.12066
发表时间：
2013-08
期刊：
Genes to cells : devoted to molecular & cellular mechanisms
影响因子：
--
作者：
Koyano F;Okatsu K;Ishigaki S;Fujioka Y;Kimura M;Sobue G;Tanaka K;Matsuda N
通讯作者：
Matsuda N
DOI：
10.1083/jcb.201210111
发表时间：
2013-01-21
期刊：
The Journal of cell biology
影响因子：
--
作者：
Lazarou M;Narendra DP;Jin SM;Tekle E;Banerjee S;Youle RJ
通讯作者：
Youle RJ
影响因子：
8.6
作者：
Dawson, Ted M.;Dawson, Valina L.
通讯作者：
Dawson, Valina L.
影响因子：
5.3
作者：
Ko, HS;von Coelln, R;Dawson, TM
通讯作者：
Dawson, TM
Parkin对泛素 - 蛋白酶体系统的广泛激活对于线粒体至关重要。
影响因子：
3.5
作者：
Chan NC;Salazar AM;Pham AH;Sweredoski MJ;Kolawa NJ;Graham RL;Hess S;Chan DC
通讯作者：
Chan DC