Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.
Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.
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DOI：
10.15252/embj.201591593
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发表时间：
2015-11-12
期刊：
影响因子：
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通讯作者：
Muqit MM
中科院分区：
文献类型：
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作者：
Lai YC;Kondapalli C;Lehneck R;Procter JB;Dill BD;Woodroof HI;Gourlay R;Peggie M;Macartney TJ;Corti O;Corvol JC;Campbell DG;Itzen A;Trost M;Muqit MM
PTEN诱导的激酶1（PINK 1）的突变是常染色体隐性遗传帕金森病（PD）的病因。我们以前曾报道，PINK 1被激活的线粒体去极化和磷酸化的丝氨酸65（Ser 65）的泛素连接酶帕金和泛素刺激帕金E3连接酶的活性。在这里，我们采用定量磷酸化蛋白质组学在HEK（人胚肾）293细胞中寻找新的PINK 1依赖性磷酸化靶点，该细胞受线粒体去极化刺激。这导致从4,499个基因产物中鉴定出14,213个磷酸化位点。虽然大多数磷酸化位点不受影响，但我们惊人地观察到Rab GTP酶亚家族的三个成员，即Rab 8A，8B和13，它们都在高度保守的丝氨酸111（Ser 111）残基处磷酸化，以响应PINK 1激活。使用针对每种Rab的Ser 111的磷酸化特异性抗体，我们证明Rab Ser 111磷酸化特异性地响应于PINK 1激活而发生，并且在HeLa PINK 1敲除细胞和突变PINK 1 PD患者来源的成纤维细胞中被线粒体去极化刺激而消除。我们提供的证据表明，Rab 8A GT3 Ser 111磷酸化在体外不受PINK 1的直接调节，并证明在细胞中Rab 8A，8B和13的Ser 111磷酸化的时间过程相比，Parkin在Ser 65的磷酸化明显延迟。我们进一步表明，Ser 111磷酸化显着损害Rab 8A激活其同源鸟嘌呤核苷酸交换因子（GEF），Rabin 8（通过使用Ser 111 Glu磷酸化模拟）的机制。这些发现提供了PINK 1能够调节Rab GTP酶磷酸化的第一个证据，并表明监测Rab 8A/8B/13在Ser 111的磷酸化可能代表PINK 1体内活性的新生物标志物。我们的研究结果还表明，Rab GTPase介导的信号传导的破坏可能是帕金森病神经退行性级联反应的主要机制。
Mutations in the PTEN‐induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser65) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser111) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser111 of each of the Rabs, we demonstrate that Rab Ser111 phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser111 phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser65. We further show mechanistically that phosphorylation at Ser111 significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser111 may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.