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Title page for ETD etd-04012013-103014


Type of Document Dissertation
Author Broadus, Matthew Ryan
Author's Email Address Matthew.R.Broadus@Vanderbilt.edu
URN etd-04012013-103014
Title Mechanisms that control localization of the Schizosaccharomyces pombe Clp1/Cdc14 phosphatase
Degree PhD
Department Cell and Developmental Biology
Advisory Committee
Advisor Name Title
Stephen Hann Committee Chair
Brian Wadzinski Committee Member
Kathleen Gould Committee Member
Ryoma Ohi Committee Member
Todd Graham Committee Member
Keywords
  • oxidative stress
  • hydrogen peroxide
  • nucleolus
  • Clp1
  • phosphorylation
  • H2O2
  • Flp1
  • phosphatase
  • Cdc14
  • cell stress
  • cell cycle
  • kinase
Date of Defense 2012-12-07
Availability unrestricted
Abstract
The Cdc14 phosphatase family antagonizes Cdk1 phosphorylation and is important for mitotic exit. Dynamic localization is a major means of regulating this phosphatase family, yet how localization changes occur, or how specific localization is achieved for this family, remains relatively uncharacterized. A major activating transition occurs when Cdc14 phosphatases are released from their interphase nucleolar sequestration during mitosis, providing them access to their substrates. Clp1/Flp1, the Schizosaccharomyces pombe Cdc14 ortholog, and Cdc14B, a mammalian ortholog, also exit the nucleolus during interphase upon DNA replication stress or damage, respectively, implicating Cdc14 phosphatases in the response to genotoxic insults. Under these conditions, I show here that relocalization of Clp1 during genotoxic stress is governed by complex phosphoregulation. Specifically, the Rad3 checkpoint effector kinases, Chk1 and Cds1, the cell wall integrity mitogen-activated protein kinase, Pmk1, and the cell cycle kinase, Cdk1, directly phosphorylate Clp1 to promote genotoxic stress-induced nucleoplasmic accumulation. However, Chk1 and Cds1 phosphorylate RxxS sites preferentially upon hydroxyurea treatment, while Pmk1 and Cdk1 preferentially phosphorylate Clp1 TP sites upon H2O2 treatment. Abolishing both Clp1 RxxS and TP phosphosites eliminates any genotoxic stress-induced redistribution. Reciprocally, preventing dephosphorylation of Clp1 TP sites shifts the distribution of the enzyme to the nucleoplasm constitutively.

Additionally, Cdc14 phosphatases localizes to both nuclear and cytoplasmic addresses throughout the cell cycle, both of which are important for their function. To better understand this distribution I determined the cis and trans factors that control Clp1 nucleocytoplasmic transport. Here I identified Sal3, a known importin-β3, as the sole importin necessary for Clp1 nuclear import. I also identified a C-terminal nuclear localization sequence within Clp1’s C-terminus necessary for its Sal3 dependent nuclear import. Conversely, I show that the exportin, Crm1, is necessary for Clp1 to exit the nucleus. Lastly, I examined how Clp1 localizes to both the nucleolus and spindle pole body utilizing fluorescence recovery after photobleaching analysis, and suggest that Clp1 likely localizes to both localizations through multiple low-affinity transient interactions. This work advances our understanding of factors influencing Clp1 localization and may provide insight into mechanisms controlling Cdc14B phosphatases in higher eukaryotes.

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