Phosphorylation Of A Noval Centrosomal Protein TACP1 By PLK1 And Its Regulation Of Centrsome

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.