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This dissertation, Polo-like Kinase 1 (Plk1) Phosphorylates VCP T76 During Mitosis for the Fragmentation of Golgi in Mammalian Cell by Kaiyuan, Zhu, 祝开元, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Polo-like Kinase 1(Plk1) phosphorylates T76 of VCP during mitosis for the fragmentation of Golgi in mammalian Cell Submitted by Kaiyuan Zhu For the degree of Master of Philosophy At The University of Hong Kong In August 2014 Cell cycle (or cell-division cycle) is divided into interphase and M phase: cells conduct DNA replication and prepare for division during interphase; while in M phase, cells undergo chromosome segregation and cell division. Plk1 is a key kinase that regulates a variety of mitotic processes, such as centrosome maturation, chromosome alignment, kinetochore-spindle attachment and cytokinesis. Plk1 binds to its substrates by its polo-box domain, and phosphorylates its substrates containing the optimal phosphorylation motif, D/E-X-S/T-Φ-X-D/E (X, any amino acid; Φ, a hydrophobic amino acid). VCP is a hexameric protein and wildly known as an I ubiquitin-selective chaperone. It interacts with cofactors Ufd1/Npl4 to convert energy generated by ATP hydrolysis to extract ubiquitinated substrates from the complex. However, the understanding about VCP's role during mitosis is limited in mammalian cells. Here I studied whether Plk1 phosphorylates VCP to regulate the progression of mitosis. My study revealed that VCP was a potential phosphorylation target of Plk1 by amino 32 sequence alignment, and in vitro P kinase assay and IP experiment indeed found that VCP was phosphorylated in Thr76. Accordingly; point mutations were induced on T76A VCP Thr76, and found that VCP mutants in HeLa cells dramatically delayed cell cycle progression by FACS analysis. Moreover, live cell imaging data revealed that T76A VCP mutant delayed metaphase-anaphase transition by inhibiting faithful WT T76E T76A chromosome segregation compared with VCP and VCP . Furthermore, VCP affected Golgi fragmentation, leading to accumulation of Golgi clusters in T76E prometaphase, whereasVCP led to Golgi isolated stacks dispersed in cytosol during interphase, suggesting the important role of VCP Thr76 phosphorylation in regulating Golgi structure in cell cycle. Taken together, our data suggests that VCP Thr76 is phosphorylated in M phase, and it is required for smooth cell cycle progression. Key words: Mitosis, Plk1, VCP, Phosphorylation, Golgi Fragmentation, HeLa cells II DOI: 10.5353/th_b5351051 Subjects: PhosphorylationMitosisProtein kinase

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