【作者】 张海涛；

【导师】 赵斌；

【作者基本信息】 浙江大学 ， 细胞生物学， 2020， 博士

【摘要】 蛋白质通过分选途径和翻译后修饰实现特定的亚细胞定位,形成相互作用网络,是细胞区域化的重要组成部分。阐明蛋白质组的亚细胞定位对于解析蛋白质的功能和调控,描绘信号转导网络,理解细胞生理活动机制具有重要意义。蛋白质磷酸化是一种重要的蛋白质翻译后修饰,它几乎在包括代谢、增殖、分化、迁移等在内的所有细胞生理活动中发挥调节作用。蛋白激酶是细胞中催化蛋白质磷酸化反应的生物酶。人类基因组编码538个蛋白激酶,约占整个基因组的1.7%。除了发挥重要的生理功能外,蛋白激酶的失调在人类疾病,特别是癌症中扮演着重要角色,是精准治疗的关键药物靶点。尽管已经有许多研究深入探索了特定蛋白激酶的亚细胞定位、功能、以及调控机制,但还没有研究从激酶组水平上全面阐明蛋白激酶的亚细胞定位及其与功能的关系。本研究通过构建人类蛋白激酶组表达质粒文库,在统一条件下通过免疫荧光染色检测各个激酶的亚细胞定位情况,绘制了人类蛋白激酶组亚细胞定位图谱(Kinome Atlas,KA)。图谱显示,在获得定位信息的蛋白激酶中,除了细胞质和细胞核的主要定位外,仍然有许多蛋白激酶定位在特定的细胞器,这一现象符合亚细胞定位在蛋白激酶功能和调控上发挥重要作用的论断。通过与蛋白质定位信息数据库的比对,我们发现了一批具有未知的特异性亚细胞定位的蛋白激酶。线粒体具有产生能量、调控细胞凋亡等重要生理功能。我们集中分析了线粒体定位的蛋白激酶,发现大部分已知的线粒体蛋白激酶在KA图谱中仍然表现出明确的线粒体定位,证明了本方法的可靠性。通过与线粒体蛋白数据库比对,还发现了多个新的具有线粒体定位的蛋白激酶,并确认了它们定位在特定的线粒体亚结构。通过对线粒体膜间隙激酶MOK的定位机制研究,我们发现其激酶结构域和C端非激酶结构域中的两段序列对MOK的线粒体定位发挥主要作用。虽然MOK不具有经典的线粒体定位信号,但线粒体蛋白转运复合物中的TOM20、TIM23等蛋白对于MOK的线粒体定位仍然是必要的。功能研究显示,在A375细胞中敲除MOK导致线粒体嵴数量减少、线粒体呼吸能力减弱、细胞内ATP水平下降以及在氧化剂刺激下ROS水平上升和细胞死亡增加等一系列变化。这表明MOK对线粒体功能具有调控作用。回补实验发现,缺乏激酶活性但具有更强线粒体定位的剪切体MOK2也可以回复MOK敲除造成的线粒体功能缺陷。这表明MOK对线粒体功能的调节不依赖于其激酶活性。通过串联亲和纯化鉴定了MOK的结合蛋白ATPase family AAA domain containing 3A(ATAD3A)。敲减ATAD3A也造成线粒体嵴数量减少。因此,MOK有可能通过与ATAD3A结合调控线粒体结构。本研究较为系统地描绘了人类蛋白激酶组的亚细胞定位情况,并以MOK为例初步阐明了新的蛋白激酶亚细胞定位的机制及其生理功能,为进一步研究蛋白激酶对线粒体的调控和全面研究蛋白激酶组新定位奠定了基础。更多还原

【Abstract】 As a major part of cellular compartmentalization,proteins achieve specific subcellular localization through sorting or post-translational modifications.Elucidating the subcellular localization of the proteome is of great significance for analyzing functions and regulations of proteins,which are underlying signal transduction networks governing various cellular progresses.Phosphorylation is one of the most wide-spread and well-studied post-translational modifications of proteins.It plays regulatory roles in most cellular progresses,such as metabolism,proliferation,differentiation,and motility.Protein kinases are enzymes that catalyze protein phosphorylation in cells.The human genome encodes 538 protein kinases,which are collectively referred as the kinome.They account for about 1.7% of genes encoded by the genome.In addition to important physiological functions,dysregulation of protein kinases plays important roles in human diseases,especially cancer.They have also become the most explored targets in precisional medicine.Although many studies have investigated subcellular localizations,functions and regulatory mechanisms of specific protein kinases,complete characterization of subcellular localizations of the kinome is still lacking.In this study,we constructed a plasmid library comprising more than 85% of the kinome.We then mapped the subcellular localization of each kinase by immunofluorescence staining in transfected HeLa cells under a unified condition.This collection of information is summarized in the Kinome Atlas(KA).As shown by KA,even cytoplasm and nucleus were main localizations,many kinases are organelle-specific.By alignment with protein localization databases,we found many protein kinases with previously unknown specific subcellular localization.Mitochondrion is a multifunctional organelle playing important roles such as energy metabolism and apoptosis regulation.We therefore focused on mitochondria-localized kinases.By comparing with a database of mitochondrial proteome,we found that most of the known mitochondrial protein kinases still exhibited clear mitochondrial localization in KA,confirming the reliability of our method.Furthermore,we discovered a number of kinases with previously unknown mitochondrial localizations.By super-resolution microscopy and other methods,we determined sub-mitochondrial localizations of these kinases.We next focused on a mitochondrial intermembrane space-localized kinase MOK.By mutational analysis we found that the mitochondrial localization of MOK dependents on two regions in its kinase domain and C-terminal domain,respectively.Although MOK lacks classical mitochondrial localization signal,proteins of the mitochondrial transporter complexes such as TOM20 and TIM23 are still necessary for the mitochondrial localization of MOK.Knockout of MOK in A375 cells resulted in a series of changes,including decreasing of mitochondria cristae,repression of mitochondrial respiration,decreasing of cellular ATP level,and enhanced ROS and cell death under oxidative stress.These findings demonstrate that MOK plays an important role in mitochondrial functions.Unexpectedly,MOK2,a splicing isoform with better mitochondrial localization but defective kinase activity could rescue functional defects caused by MOK knockout.This finding suggests that MOK regulates mitochondrial functions independ of its kinase activity.By tandem affinity purification,we identified ATPase family AAA domain containing 3A(ATAD3A)as a binding protein of MOK.Knockout of ATAD3 A also resulted in a decrease of mitochondrial cristae.Therefore,it is possible that MOK regulates the structure of mitochondria through ATAD3 A.Our study systematically characterized subcellular localizations of the human kinome,and further elucidated the localization mechanism and function of a mitochondria-localized kinase MOK.This study laid a foundation for further investigations of novel mitochondrial kinases and kinome functions in previously unknown subcellular localizations.更多还原