Regulation of Human PINK1 ubiquitin kinase by Serine167, Serine228 and Cysteine412 phosphorylation
Regulation of Human PINK1 ubiquitin kinase by Serine167, Serine228 and Cysteine412 phosphorylation
复制标题
DOI：
10.1101/2023.03.31.534916
复制
发表时间：
2023-04
期刊：
影响因子：
--
通讯作者：
Andrew D. Waddell;Hina Ojha;Shalini Agarwal;C. Clarke;Ana Terriente-Félix;Houjiang Zhou;Poonam S. Kakade;A. Knebel;A. Shaw;Robert Gourlay;Joby Varghese;Renata Soares;R. Toth;Thomas J. Macartney;P. Eyers;N. Morrice;R. Bayliss;Alexander J. Whitworth;C. Eyers;M. Muqit
中科院分区：
文献类型：
--
作者：
Andrew D. Waddell;Hina Ojha;Shalini Agarwal;C. Clarke;Ana Terriente-Félix;Houjiang Zhou;Poonam S. Kakade;A. Knebel;A. Shaw;Robert Gourlay;Joby Varghese;Renata Soares;R. Toth;Thomas J. Macartney;P. Eyers;N. Morrice;R. Bayliss;Alexander J. Whitworth;C. Eyers;M. Muqit
人类PINK 1激酶（hPINK 1）的功能缺失突变是早发性帕金森病（PD）的病因。hPINK 1的激活诱导磷酸化泛素，以启动通过自噬去除受损的线粒体。之前，我们解决了昆虫PINK 1直向同源物赤拟谷盗PINK 1的结构，并表明Ser 205的自磷酸化对于泛素相互作用和磷酸化是关键的（Kumar，Tamjar，Waddell等人，2017年）。在这里，我们报告的磷酸化hPINK 1的调节的新发现。我们重建了E.在pSer 228位点直接掺入磷酸丝氨酸，表达了hPINK 1的活性，为丝氨酸228磷酸化在hPINK 1活化中的作用提供了直接证据。此外，使用质谱法，我们确定了六个新的丝氨酸/苏氨酸自磷酸化位点，包括调节丝氨酸167磷酸化（pSer 167），除了pSer 228所需的泛素识别和磷酸化。引人注目的是，我们还检测到在hPINK 1激活片段中保守的半胱氨酸412（pCys 412）残基的磷酸化。结构模型表明，pCys 412抑制泛素识别，我们证明，突变的Cys 412丙氨酸使hPINK 1更积极地对泛素在人类细胞中表达时。这些结果概述了pSer 167和pSer 228对hPINK 1激活的新见解以及pCys 412介导的新抑制机制。这些发现将有助于开发hPINK 1的小分子激活剂。
Loss-of-function mutations in the human PINK1 kinase (hPINK1) are causative of early-onset Parkinson’s disease (PD). Activation of hPINK1 induces phosphorylated ubiquitin to initiate removal of damaged mitochondria by autophagy. Previously we solved the structure of the insect PINK1 orthologue, Tribolium castaneum PINK1, and showed that autophosphorylation of Ser205 was critical for ubiquitin interaction and phosphorylation (Kumar, Tamjar, Waddell et al., 2017). Here we report new findings on the regulation of hPINK1 by phosphorylation. We reconstitute E. coli expressed hPINK1 activity in vitro by direct incorporation of phosphoserine at the equivalent site Serine 228 (pSer228), providing direct evidence for a role for Ser228 phosphorylation in hPINK1 activation. Furthermore, using mass spectrometry, we identify six novel Ser/Thr autophosphorylation sites including regulatory Serine167 phosphorylation (pSer167), which in addition to pSer228 is required for ubiquitin recognition and phosphorylation. Strikingly, we also detect phosphorylation of a conserved Cysteine412 (pCys412) residue in the hPINK1 activation segment. Structural modelling suggests that pCys412 inhibits ubiquitin recognition and we demonstrate that mutation of Cys412 to Ala renders hPINK1 more active towards ubiquitin when expressed in human cells. These results outline new insights into hPINK1 activation by pSer167 and pSer228 and a novel inhibitory mechanism mediated by pCys412. These findings will aid in the development of small molecule activators of hPINK1.