Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore.
Department of Biological Sciences, National University of Singapore, Singapore.
Sci Rep. 2017 Jan 4;7:39998. doi: 10.1038/srep39998.
Yersinia bacteria cause a range of human diseases, including yersiniosis, Far East scarlet-like fever and the plague. Yersiniae modulate and evade host immune defences through injection of Yersinia outer proteins (Yops) into phagocytic cells. One of the Yops, YopO (also known as YpkA) obstructs phagocytosis through disrupting actin filament regulation processes - inhibiting polymerization-promoting signaling through sequestration of Rac/Rho family GTPases and by using monomeric actin as bait to recruit and phosphorylate host actin-regulating proteins. Here we set out to identify mechanisms of specificity in protein phosphorylation by YopO that would clarify its effects on cytoskeleton disruption. We report the MgADP structure of Yersinia enterocolitica YopO in complex with actin, which reveals its active site architecture. Using a proteome-wide kinase-interacting substrate screening (KISS) method, we identified that YopO phosphorylates a wide range of actin-modulating proteins and located their phosphorylation sites by mass spectrometry. Using artificial substrates we clarified YopO's substrate length requirements and its phosphorylation consensus sequence. These findings provide fresh insight into the mechanism of the YopO kinase and demonstrate that YopO executes a specific strategy targeting actin-modulating proteins, across multiple functionalities, to compete for control of their native phospho-signaling, thus hampering the cytoskeletal processes required for macrophage phagocytosis.
耶尔森氏菌会引起多种人类疾病，包括耶尔森菌病、远东猩红热样热和鼠疫。耶尔森氏菌通过向吞噬细胞中注射耶尔森氏外蛋白（Yops）来调节和逃避宿主的免疫防御。其中一种 Yops，YopO（也称为 YpkA）通过扰乱肌动蛋白丝调节过程来阻碍吞噬作用——通过隔离 Rac/Rho 家族 GTPases 来抑制聚合促进信号，并利用单体肌动蛋白作为诱饵招募和磷酸化宿主肌动蛋白调节蛋白。在这里，我们着手确定 YopO 蛋白磷酸化的特异性机制，这将阐明其对细胞骨架破坏的影响。我们报告了与肌动蛋白复合物的肠致病性大肠杆菌 YopO 的 MgADP 结构，揭示了其活性位点结构。使用全蛋白质组激酶相互作用底物筛选（KISS）方法，我们鉴定了 YopO 磷酸化广泛的肌动蛋白调节蛋白，并通过质谱法确定了它们的磷酸化位点。使用人工底物，我们澄清了 YopO 的底物长度要求及其磷酸化的共有序列。这些发现为 YopO 激酶的机制提供了新的见解，并表明 YopO 执行了一种针对肌动蛋白调节蛋白的特定策略，针对多种功能，争夺对其天然磷酸化信号的控制，从而阻碍巨噬细胞吞噬作用所需的细胞骨架过程。
