Citation
Chang, Kuang-Jung (2012) A Role for Protein Kinase Dbf2-Mob1 in Mitotic Exit. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z92F7KFT. https://resolver.caltech.edu/CaltechTHESIS:06112012-092401902
Abstract
Exit from mitosis is characterized by precise control of the cyclin-dependent kinase complex (Cdk) activity, breaking down mitotic structures, and completing cytokinesis. In Saccaromyces cerevisiae, protein phosphotase Cdc14 is involved in counteracting mitotic-Cdk activity by promoting the degradation of mitotic cyclin Clb2, and stabilizing the Cdc28 inhibitor Sic1. The activity and the cellular localization of Cdc14 are tightly regulated by the cell cycle. Cdc14 is sequestered and inhibited in the nucleolus by forming the RENT (Regulator of Nucleolar Silencing and Teleophase) complex for most of the cell cycle. It is released and distributed into the cytoplasm in anaphase and telophase, and then returns to the nucleolus in G1 phase. Activation of Cdc14 is achieved via multi-site phosphorylation of Net1 leading to the release of Cdc14. Net1 is first phosphorylated by the Fourteen Early Anaphase Release (FEAR) network, and then by the Mitotic Exit Network (MEN).
In this thesis, we show that a MEN component, protein kinase Dbf2-Mob1, plays a role in phosphorylating Net1 in late anaphase. We identified the effective Dbf2-Mob1 phosphorylation sites in the N-terminal of Net1 by in vitro kinase reaction assay. We found that cells that express mutant Net1 show growth defects and chain-like terminal morphology under restrictive temperatures. Genetic interactions suggested that the MEN kinases and Cdc14 are related to the cell cycle defects caused by the phosphosite mutated Net1. Analyzing the phosphosite mutants with Fluorecence-activated cell sorting (FACS) and immunofluorescence assay, we found that the growth defects and the abnormal cell morphology are due to a defect in releasing Cdc14 in late anaphase, leading to disruption of mitotic exit. This result is further confirmed by western blot assay and the beads releasing assay.
In summary, the regulation of Cdc14 release in late anaphase via phosphorylation of its inhibitor Net1 by Dbf2-Mob1 is demonstrated in this work. This thesis provides a crucial piece of information that furthers our understanding of the mechanism of mitotic exit. It also points to a fascinating mechanism of controlling cytokinesis and meiosis by regulating Net1 phosphorylation by the MEN.
Item Type: | Thesis (Dissertation (Ph.D.)) | ||||||||
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Subject Keywords: | cell cycle, Mitotic Exit | ||||||||
Degree Grantor: | California Institute of Technology | ||||||||
Division: | Biology | ||||||||
Major Option: | Biology | ||||||||
Thesis Availability: | Public (worldwide access) | ||||||||
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Defense Date: | 29 May 2012 | ||||||||
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Record Number: | CaltechTHESIS:06112012-092401902 | ||||||||
Persistent URL: | https://resolver.caltech.edu/CaltechTHESIS:06112012-092401902 | ||||||||
DOI: | 10.7907/Z92F7KFT | ||||||||
Default Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | ||||||||
ID Code: | 7156 | ||||||||
Collection: | CaltechTHESIS | ||||||||
Deposited By: | Kuang-Jung Chang | ||||||||
Deposited On: | 06 Mar 2017 20:01 | ||||||||
Last Modified: | 03 Oct 2019 23:56 |
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