【作者】 朱园园；

【导师】 黄河；

【作者基本信息】 浙江大学 ， 内科血液病学， 2007， 博士

【摘要】 中心体是参与细胞有丝分裂的重要细胞器，是动物细胞中主要的微管组织中心，它影响了细胞内所有与微管相关的过程，它与双极纺锤体的形成、纺锤体的定位和胞质分裂直接相关。纺锤体数目的异常直接干扰了双极纺锤体的形成以及染色体的分离过程。因此在分裂细胞中，中心体的复制和分离必须与染色体的复制和分离过程相协调。在特定的细胞周期中，中心体只能分裂一次，中心体数目过多会导致多极纺锤体形成，出现错误的有丝分裂过程，从而直接导致染色体非整倍性的发生，染色体的不稳定性通常认为是引起肿瘤发生的主要机制。中心体上结合的蛋白超过一百种，75％具有coiled-coil结构域，至今仍有许多中心体蛋白质尚未被发现，它们的功能也不完全清楚。中心体蛋白质中有不少是重要的有丝分裂激酶，与细胞周期和调定点调控高度相关。目前公认的有丝分裂调控蛋白主要有CdK家族，Polo家族，Aurora家族，NIMA家族，TTK，Cep55，Bub1，BubR1等。PLK1(Polo-like Kinase 1)就是在脊椎动物中的一种PLK，隶属于Polo家族，是一种与细胞有丝分裂高度相关的激酶。PLK1可以特异性地磷酸化蛋白质的丝氨酸／苏氨酸，它通过磷酸化它的下游底物来放大调节它们的功能活性。PLK1蛋白质与PLK的其它蛋白质在结构上可以分为两部分：N端的激酶结构域(kinase domain)和C端的Polo盒结构域(PBD，polo-box domain)，PLK1通过PBD与其他蛋白质发生相互作用。它在进化中表现得相当保守，从酵母、果蝇、爪蟾到哺乳动物都有它的同源物。如果PLK1被敲除后会引起单级纺锤体，染色体排列异常，有丝分裂阻断等表型。另外PLK1在DNA损伤和调节细胞周期的各调定点也至关重要。本课题组在前一阶段的工作中利用端粒结合蛋白TRF1特异性抗体，通过免疫共沉淀结合蛋白质肽指纹谱技术从分裂期细胞裂解液中分离鉴定了一个TRF1新的相互作用中心体蛋白，将该蛋白命名为TACP1(Telomere Associated Centrosome Protein 1)。本研究是在此基础上对TACP1这个新的中心体蛋白功能展开，以期完善中心体蛋白网络中新蛋白TACP1的分子机制。本研究分为两个部分。第一部分：中心体蛋白TACP1与有丝分裂激酶PLK1相互作用和磷酸化调控的研究。本研究发现TACP1在细胞有丝分裂期能发生特异性磷酸化，通过免疫共沉淀后用质谱分析发现Thr221、Thr457是它的潜在磷酸化位点，而它们的磷酸化激酶可能分别是Nek2A和PLK1。随即通过免疫共沉淀和pull-down实验证实TACP1在体内、体外都能与PLK1形成复合物即两者之间存在相互作用，免疫荧光实验结果也显示它们在有丝分裂期共同定位于中心体。生化实验结果还提示PLK1与TACP1结合依赖于TACP1的羧基端。TACP1-C是PLK1的结合功能区，PLK1与TACP1结合并不依赖于TACP1的中心体定位功能域(TACP1-MC)。PLK1可以在体外条件下特异性磷酸化野生型TACP1，该实验还证实了PLK1激酶活性缺失型则无法磷酸化TACP1，模拟非磷酸化状态的TACP1457A突变型也无法被PLK1磷酸化，从而证实PLK1的确是TACP1蛋白457位苏氨酸的磷酸化激酶。当siRNA基因抑制内源性的PLK1后，TACP1稳定表达的Hela细胞G2／M期的细胞明显增多。我们推测TACP1得不到PLK1的磷酸化，过量非磷酸化的TACP1蛋白造成细胞无法通过G2／M调定点从而造成阻滞。为了深入探讨TACP1被PLK1磷酸化发生的时间空间信息，利用针对TACP1的457位苏氨酸磷酸化抗体进行免疫荧光实验，结果发现PLK1磷酸化TACP1发生于有丝分裂前期、前中期、中期，空间上位于中心体上。后期开始以后，TACP1在中心体上的信号就开始弥散削弱，末期时完全消失。siRNA基因干扰抑制PLK1表达导致TACP1无法定位于中心体，但如果仅抑制PLK1激酶活性，TACP1仍定位于中心体，因此TACP1的中心体定位依赖于PLK1而非PLK1的磷酸化作用。第二部分：TACP1对中心体功能的调控。为了探讨TACP1的功能，筛选建立了TACP1稳定表达的Hela细胞株。在此细胞株上进行免疫荧光实验，结果发现TACP1在细胞周期中的定位呈现出动态性，间期分布于胞质，部分共定位于F-actin，有丝分裂期则定位于中心体上。本研究主要立足于探讨TACP1在有丝分裂期的功能研究。利用互联网上的数据库，通过蛋白质序列比对发现TACP1与裂殖酵母中心体蛋白Pcp1同源。若用TACP1 siRNA干扰抑制内源性TACP1蛋白表达后，免疫荧光实验结果显示染色体异常排列的细胞明显增多：滞后染色体以及染色体排列紊乱等，并出现多中心体表型，计数这些异常表型的细胞，数量明显高于对照组。本研究运用分子生物学、细胞生物学和蛋白质组学的方法来研究有丝分裂激酶PLK1对中心体蛋白TACP1的磷酸化修饰，TACP1磷酸化的功能，TACP1在中心体上的细胞生物学作用，从而阐明TACP1参与的细胞周期调控信号通路和分子机制。获得以下结果①TACP1在细胞有丝分裂期能发生特异性磷酸化，Thr221、Thr457是它的潜在磷酸化位点，而它们的磷酸化激酶可能分别是Nek2A和PLK1。②体外的蛋白Pull-down实验和体内的免疫共沉淀实验证实TACP1在体内、体外都能与PLK1相互作用，免疫荧光实验结果显示它们在有丝分裂期共同定位于中心体。③证明了PLK1与TACP1结合依赖于TACP1的羧基端。④PLK1可以在体外的条件下特异性磷酸化野生型TACP1，而PLK1KD(激酶活性缺失型)则无法磷酸化TACP1，模拟非磷酸化状态的TACP1457A突变型也无法被PLK1磷酸化。表明PLK1的确是TACP1蛋白457位苏氨酸的磷酸化激酶。⑤过量表达的TACP1得不到PLK1的磷酸化，大量非磷酸化的TACP1蛋白会使细胞阻滞在G2／M期。⑥PLK1磷酸化TACP1时间上发生于有丝分裂前期、前中期、中期，空间上位于中心体上。⑦TACP1定位于中心体依赖于PLK1而非PLK1的磷酸化作用。⑧TACP1在细胞周期中的定位呈现出动态性，间期分布于胞质一部分与F-actin共定位，有丝分裂期则定位于中心体上。⑨TACP1与裂殖酵母中心体蛋白Pcp1同源。⑩TACP1参与了中心体稳定性的调控。综上所述，本研究拓展了对中心体蛋白质的认识，首次证实了中心体蛋白TACP1的中心体定位依赖于有丝分裂激酶PLK1，TACP1参与了中心体稳定性的调控，是一个新的有丝分裂相关蛋白。更多还原

【Abstract】 The centrosome is a tiny organelle of surprising structural complexity and it plays a critical role during mitosis. In animal cells, the centrosome is the major microtubuleorganizing center (MTOC). Thus, it influences all microtubule (MT)-dependent processes and also contributes to control spindle bipolarity, spindle positioning and cytokinesis. Any aberration in centrosome numbers can interfere with bipolar spindle formation and chromosome segregation. Therefore, centrosome duplication and segregation need to be tightly coordinated with the duplication and segregation of the genome. Throughout development and adult life, this single centrosome then needs to be duplicated once, and only once, in every cell cycle. Thus, a condition that favors the overproduction of centrosomes could contribute directly to the initiation of chromosome imbalance, through the formation of multipolar spindles and aberrant mitosis. Chromosome imbalance is the most frequent manifestation of genomic instability in human cancer cells.Centrosomes bind more than 100 regulatory proteins, whose identities suggest roles in a multitude of cellular functions. A structural analysis revealed that a high proportion (75%) contained coiled-coil regions, a common feature of centrosomal proteins. Our understanding of centrosomal proteins is still limited. Many of theseproteins are mitosis kinases. The most prominent mitotic kinase is the cyclin dependent kinase 1 (Cdk1), the founding member of the Cdk family of cell-cycle regulators. Recent studies have, however, brought to light additional mitotic kinases. These include members of the Polo family, the aurora family and the NIMA (never in mitosis A) family, as well as kinases implicated in mitotic CHECKPOINTS, mitotic exit and cytokinesis. PLK1 is one of mitotic kinases and tightly correlation with cell cycle. PLK1 regulates many proteins by phosphorylating serine/threonine of these substrates. All Plks have a similar architecture, with a canonical serine/threonine kinase domain at the amino terminus and a regulatory domain containing two signature motifs, known as polo boxes domain(PBD), at the carboxyl terminus. PLK1 is very conserved in many species, form yeast, Drosophila melanogaster, Xenopus laevis to mammals. PLK1 knock down by siRNA cause many phenotype such as monopolar spindle, chromosome misalignment, block in mitosis. PLK1 is also very critical in DNA damage and cell cycle checkpoint.In past study, a novel TRF1 interacting protein was identified from mitotic HeLa cell lysates by employing immunoaffinity isolatin and mass spectrometry (MS). We therefore refer to the protein as Telomere Associated Centrosomal Protein 1 (TACP1) since it distinguishes from other TRF1 binding proteins and locates to the centrosome. This study is based on the function of the novel centrosomal protein for the sake of indicating molecular mechanism of TACP1 in centrosome network.This thesis was divided into two parts.Part I : Study of interaction and phosphorylation between TACP1 and PLK1. In present study we noticed that TACP1 was specifically phosphorylated during mitosis, which was possibly responsible for TACP1 translocation and function in centrosome. Thr221 and Thr457 were identified as two potential phosphorylation sites by mass spectrometry. According to conserved motif analysis, the putative kinases were Nek2A and Plk1, respectively. The biochemical interaction in vitro between PLK1 and TACP1 was validated by pull-down assay and immunoprecipitation. Immunofluorescence studies revealed both PLK1 and TACP1 localized to centrosomes during mitosis. Deletion analysis indicated that the C-terminalcoiled-coil domain of TACP1 but not TACP1-MC was required for PLK1 binding. Moreover PLK1 can phosphorylate TACP1 in vitro but not TACP1457A mutant. On the other hand PLK1 kinase death mutant can not phosphorylate TACP1 in vitro either. PLK1 knocked down by siRNA caused TACP1 stable expressing cells blocked in G2/M. So it is maybe caused by excessive non- phosphorylated TACP1 in cells and cells can not override G2/M checkpoint. In order to investigate the temporal and special information of phosphorylation of TACP1 by PLK1 we carried out immunofluorescence studies by using TACP1 457 phosphorylated antibody. And immunofluorescence studies also revealed that PLK1 phosphorylate TACP1 from prometaphase to anaphase in centrosome. PLK1 knock down by siRNA but not inhibiting kinase activity of PLK1 cause TACP1 disappearing in centrosome. We got the conclusion that centrosomal localization of TACP1 is regulated by polo-like kinase1 but not by its phosphorylation through immunofluorescence studies by using TACP1 457 phosphorylated antibody.Part II: TACP1’s regulation of centrosome. As TACP1 is a novel protein we have constructed a TACP1 stable expressing cell line to carry out many researches on it. Notably, immunofluorescence studies shows localization of TACP1 is highly dynamic. TACP1 bind to F-actin in interface and with a centrosomal localization during mitosis. We focused in mitosis in this study. Detailed sequence alignment reveals the fission yeast’s centrosomal protein Pcp1 is the closest ortholog of the protein. Depletion of TACP1 in HeLa cells resulted in multiplicity of spindle pole and misalignment of chromosomes in mitotic cells. The evolutionary conservancy of TACP1 in amino acid sequence and function suggested it facilitates the mitotic regulation especially in centrosme instability.In this study, we investigated the phosphorylation of TACP1 by PLK1, the function of phosphorylated TACP1 and its role in regulating of centrosome by using molecular biology, cell biology and proteomic methods. This thesis clarified the signal pathway of TACP1 in cell cycle and its molecular mechanism. The results were summed up as latter: ① TACP1 was specifically phosphorylated during mitosis, Thr221 and Thr457 were identified as two potential phosphorylation sites. The putative kinaseswere Nek2A and Plk1, respectively. ② The biochemical interaction in vitro between PLK1 and TACP1 was validated by pull-down assay and immunoprecipitation. Immunofluorescence studies revealed both PLK1 and TACP1 localized to centrosomes during mitosis. ③ The C-terminal coiled-coil domain of TACP1 but not TACP1-MC was required for PLK1 binding. ④ PLK1 can phosphorylate TACP1 in vitro but not TACP1457A mutant. On the other hand PLK1 kinase death mutant can not phosphorylate TACP1 in vitro either. ⑤ Excessive non- phosphorylated TACP1 in cells caused TACP1 stable expressing cells blocked in G2/M. ⑥ PLK1 phosphorylate TACP1 from prometaphase to anaphase in centrosome. ⑦ Centrosomal localization of TACP1 is regulated by polo-like kinase 1 but not by its phosphorylation ⑧Localization of TACP1 is highly dynamic. TACP1 bind to F-actin in interface and with a centrosomal localization during mitosis. ⑨ The fission yeast’s centrosomal protein Pcp1 is the closest ortholog of the protein.⑩ TACP1 facilitates the mitotic regulation especially in centrosme instability. So this study contributes a better understanding of centrosomal proteins and firstly validates centrosomal localization of TACP1 is depended on PLK1. TACP1 is a novel mitosis related protein and facilitates the mitotic regulation in centrosme instability.更多还原