Polo-Like Kinase 1(PLK1) Phosphorylates p97/VCP Thr76 on Centrosome for Mitosis Progression

Polo-Like Kinase 1(PLK1) 於中心體上磷酸化 p97/VCP第76位蘇氨酸以推動有絲分裂進行

Student thesis: Doctoral Thesis

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Award date29 Jul 2019

Abstract

Cell cycle (or cell-division cycle) is divided into interphase and M phase: cells conduct DNA replication and prepare for the division during interphase; while in M phase, cells undergo chromosome segregation and cell division. Unfaithful chromosome segregation is a leading cause for tumorigenesis. Polo-like kinase 1 (PLK1) is a key protein kinase that regulates a variety of mitotic processes, such as centrosome maturation, chromosome alignment, kinetochore-spindle attachment, Golgi fragmentation, and cytokinesis. However, how PLK1 regulates these processes remains unclear, especially considering that these processes happen in different cellular locations at different cell cycle stage. To find novel PLK1 interacting proteins, tandem affinity purification and mass spectrometry analysis were performed, and a total of 87 potential PLK1 binding proteins were identified. Among them, valosin-containing protein (VCP) or p97 was chosen for further study since it has an optimal PLK1 docking site and a conserved PLK1 phosphorylation motif. Notably, VCP/p97 was firstly identified in Yeast as a cell division protein, named cdc48. But in mammalian cells, the study of VCP/p97’s function during mitosis is very limited. Interestingly, VCP/p97 is a very abundant protein (1% of total cytosolic proteins) and is highly conserved from bacteria to human cells. Currently, the best-known function of VCP/p97 is as an ubiquitin-selective chaperone in proteasome- or/and lysosome-mediated protein degradation. However, the role of VCP in mitosis is not well characterized.

My study revealed that PLK1 interacted with VCP during mitosis, and PLK1 also phosphorylated VCP at Thr76 as shown by both in vitro kinase assay and phospho-specific antibody immunoblotting. VCP was found to be accumulated on centrosome, spindle, and midbody at different stages of mitosis, yet the VCP Thr76 phosphorylation only occurred on centrosome from late G2 phase to early anaphase, which was well correlated with PLK1’s activity on centrosome during mitosis. Further experiments, such as proximity ligation assay (PLA) and Bimolecular fluorescence complementation (BiFC), had confirmed the interaction between VCP and PLK1 on centrosome during mitosis. PLK1 was also found to target VCP through docking to Thr14 and/or Thr613 in VCP. To study the role of this phosphorylation site on mitosis progression, point mutations were induced on VCP Thr76. I found that VCPT76A mutant HeLa cells dramatically delayed cell cycle progression as shown by PI staining and FACS analysis. Moreover, live-cell imaging data revealed that metaphase-anaphase transition was delayed in VCPT76A mutant cells, resulting in unfaithful chromosome segregation. Moreover, spindle assembly defects and abnormal separation of centrosomes were found in VCPT76A mutant cells. Centrosomes in VCPT76A mutant cells spent longer time to move to a bipolar position as compared to that in control cells. Mass spectrometry analysis was performed to identify the interacting proteins of mitotic VCP, and Eg5, a kinesin protein responsible for centrosome separation, was found in the VCP complex. The interaction between VCP and Eg5 was confirmed by IP and PLA assay. Moreover, less Eg5 was recruited to the mitotic spindle in VCPT76A mutant cells, and this explained the defects of centrosome movement. Furthermore, I found that unlike in WT VCP cells, VCP did not accumulate on centrosome and the mitotic spindle in VCPT76A mutant cells. Thus, these data not only indicate that PLK1 phosphorylates VCP Thr76 during mitosis to recruit it to the centrosome, but also suggest that the dephosphorylation of VCP Thr76 is requried for VCP enrichment at the mitotic spindle. Moreover, PTEN, a phosphatase, was found to be responsible for the dephosphorylation of T76 in VCP, and PTEN inhibition or knockdown abolished the spread of VCP from the centrosome to the spindle. Collectively, our findings elucidate a dynamic switch of phosphorylation and dephosphorylation of VCP-T76, mediate by PLK1 and PTEN, respectively, and this switch participates in the centrosome and spindle orientation for faithful chromosome segregation.