MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
Open Biol. 2012 May;2(5):120080. doi: 10.1098/rsob.120080.
Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser(65). We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser(65). We further show that phosphorylation of Parkin at Ser(65) leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser(65) or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr(257), which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser(65) and/or PINK1 at Thr(257) represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.
PTEN 诱导的激酶 1（PINK1）中的错义突变导致常染色体隐性遗传帕金森病（PD）。我们利用最近发现的重组昆虫 PINK1 具有催化活性，来测试 PINK1 是否直接磷酸化 15 种与 PD 相关的基因编码的蛋白质，以及报道与 PINK1 结合的蛋白质。我们发现昆虫 PINK1 仅能有效地磷酸化其中一种蛋白质，即 E3 连接酶 Parkin。我们已经将磷酸化位点定位到 Parkin 的 Ubl 结构域中的一个高度保守的残基，即丝氨酸（Ser）65。我们表明，人 PINK1 可以被线粒体膜电位（Δψm）去极化特异性激活，从而使其在 Ser（65）处磷酸化 Parkin。我们进一步表明，Parkin 在 Ser（65）处的磷酸化导致其 E3 连接酶活性的显著激活，而 Ser（65）的突变或 PINK1 的失活会阻止这种激活。我们提供的证据表明，一旦被激活，PINK1 会在几个残基上进行自身磷酸化，包括 Thr（257），伴随着电泳迁移率的带位移。这些结果首次证明 PINK1 在 Δψm 去极化后被激活，并表明 PINK1 直接磷酸化并激活 Parkin。我们的研究结果表明，监测 Parkin 在 Ser（65）处的磷酸化和/或 PINK1 在 Thr（257）处的磷酸化可能代表体内检查 PINK1-Parkin 信号通路活性的首个生物标志物。我们的研究结果还表明，模拟 PINK1 磷酸化作用的 Parkin 小分子激活剂可能为 PD 提供治疗益处。
