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Ubiquitin is phosphorylated by PINK1 to activate parkin

机译:泛素被PINK1磷酸化以激活Parkin

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PINK1 (PTEN induced putative kinase 1) and PARKIN (also known as PARK2) have been identified as the causal genes responsible for hereditary recessive early-onset Parkinsonism. PINK1 is a Ser/Thr kinase that specifically accumulates on depolarized mitochondria, whereas parkin is an E3 ubiquitin ligase that catalyses ubiquitin transfer to mitochondrial substrates. PINK1 acts as an upstream factor for parkin and is essential both for the activation of latent E3 parkin activity and for recruiting parkin onto depolarized mitochondria. Recently, mechanistic insights into mitochondrial quality control mediated by PINK1 and parkin have been revealed, and PINK1 -dependent phosphorylation of parkin has been reported. However, the requirement of PINK1 for parkin activation was not bypassed by phospho-mimetic parkin mutation, and how PINK1 accelerates the E3 activity of parkin on damaged mitochondria is still obscure. Here we report that ubiquitin is the genuine substrate of PINK1. PINK1 phosphorylated ubiquitin at Ser 65 both in vitro and in cells, and a Ser 65 phos-phopeptide derived from endogenous ubiquitin was only detected in cells in the presence of PINK1 and following a decrease in mitochondrial membrane potential. Unexpectedly, phosphomimetic ubiquitin bypassed PINK1 -dependent activation of a phosphomimetic parkin mutant in cells. Furthermore, phosphomimetic ubiquitin accelerates discharge of the thioester conjugate formed by UBCH7 (also known as UBE2L3) and ubiquitin (UBCH7~ubiquitin) in the presence of parkin in vitro, indicating that it acts allosterically. The phosphorylation-dependent interaction between ubiquitin and parkin suggests that phosphorylated ubiquitin unlocks autoinhibition of the catalytic cysteine. Our results show that PINK1-dependent phosphorylation of both parkin and ubiquitin is sufficient for full activation of parkin E3 activity. These findings demonstrate that phosphorylated ubiquitin is a parkin activator.
机译:PINK1(PTEN诱导的假定激酶1)和PARKIN(也称为PARK2)已被确定为遗传性隐性早发性帕金森病的病因基因。 PINK1是一种Ser / Thr激酶,专门积聚在去极化的线粒体上,而parkin是一种E3泛素连接酶,可催化泛素转移至线粒体底物。 PINK1充当派克蛋白的上游因子,对于激活潜在的E3派克蛋白和将派克蛋白募集到去极化的线粒体中都是必不可少的。最近,已经揭示了对由PINK1和Parkin介导的线粒体质量控制的机理的见解,并且已经报道了PINK1依赖性的Parkin磷酸化。然而,磷酸化模拟的帕金突变并没有绕过PINK1对帕金激活的要求,而PINK1如何加速帕金对受损线粒体的E3活性仍然不清楚。在这里,我们报道泛素是PINK1的真正底物。 PINK1在体外和细胞中均在Ser 65处磷酸化泛素,仅在存在PINK1且线粒体膜电位降低之后,才在细胞中检测到源自内源性泛素的Ser 65磷酸肽。出乎意料的是,拟膦素泛素绕过了细胞中拟磷酸酯化帕金突变体的PINK1依赖性激活。此外,模拟磷酸泛素在体外存在parkin的情况下加速了UBCH7(也称为UBE2L3)和泛素(UBCH7〜泛素)形成的硫酯偶联物的释放,表明它具有变构作用。泛素和帕金蛋白之间的磷酸化依赖性相互作用表明,磷酸化的泛素可解除对半胱氨酸催化的自动抑制作用。我们的结果表明,Parkin和泛素的PINK1依赖性磷酸化足以完全激活Parkin E3活性。这些发现表明磷酸化的遍在蛋白是一种帕金激活剂。

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  • 来源
    《Nature》 |2014年第7503期|162-166|共5页
  • 作者单位

    Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan,Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8561, Japan;

    Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan,Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8561, Japan;

    Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, The University of Tokushima, Tokushima 770-8503, Japan;

    Research Center for Materials Science, Nagoya University, Nagoya, Aichi 464-8602, Japan;

    Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan;

    Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan;

    Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan,Graduate School of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan;

    Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan;

    Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan;

    Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan;

    JST-CREST/Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan,JST-CREST/Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto 603-8555, Japan;

    McGill Parkinson Program, Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec H3A2B4, Canada;

    Department of Pharmacology &Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada;

    Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan;

    Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan;

    Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan,Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan;

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