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Title page for ETD etd-08172012-124402


Type of Document Dissertation
Author Lu, Lucy Xiangxi
Author's Email Address xiangxi.lu@vanderbilt.edu
URN etd-08172012-124402
Title Phosphoregulation of the Cdc25 phosphatase and its effects on Schizosaccharomyces pombe mitotic entrance and exit
Degree PhD
Department Cell and Developmental Biology
Advisory Committee
Advisor Name Title
William Tansey Committee Chair
Brian E. Wadzinski Committee Member
David Cortez Committee Member
Laura A. Lee Committee Member
Keywords
  • Cdk1
  • phosphatases
  • kinases
  • mitosis
  • bistability
  • ultrasensitivity
  • schizosaccharomyces pombe
  • Cdc14
  • Clp1
  • Cdc25
  • Cdc2
Date of Defense 2012-08-07
Availability unrestricted
Abstract
Cdk1 kinase dephosphorylation and activation by Cdc25 phosphatase is essential for mitotic entry. Activated Cdk1 phosphorylates Cdc25 and other substrates, further activating Cdc25 to form a positive feedback loop that drives the abrupt G2/mitosis switch. Conversely, mitotic exit requires Cdk1 inactivation and reversal of Cdk1 substrate phosphorylation. This is mediated in part by Clp1/Cdc14, a Cdk1 antagonizing phosphatase, which reverses Cdk1 phosphorylation of itself, Cdc25, and other Cdk1 substrates. Thus, Cdc25 phosphoregulation is essential for proper G2-M transition and its contributions to cell cycle control have been modeled based on studies using Xenopus and human cell extracts. Since cell extract systems only approximate in vivo conditions where proteins interact within dynamic cellular environments, here we use Schizosaccharomyces pombe to characterize experimentally and mathematically, the in vivo contributions of Cdk1-mediated phosphorylation of Cdc25 to the mitotic transition. Through comprehensive mapping of Cdk1 phosphosites on Cdc25 and characterization of phosphomutants, we show that Cdc25 hyperphosphorylation by Cdk1 governs Cdc25 catalytic activation, the precision of mitotic entry, and unvarying cell length, but not Cdc25 localization or abundance. We propose a mathematical model that explains Cdc25 regulation by Cdk1 through a distributive and disordered phosphorylation mechanism that ultrasensitively activates Cdc25. We also show that Clp1/Cdc14 dephosphorylation of Cdk1 sites on Cdc25 controls the proper timing of cell division, a mechanism that is likely due to the double negative feedback loop between Clp1/Cdc14 and Cdc25 that controls the abruptness of the mitotic exit switch.
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