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Title page for ETD etd-03202018-160050


Type of Document Dissertation
Author Elmore, Zachary Cole
Author's Email Address zachary.c.elmore@vanderbilt.edu
URN etd-03202018-160050
Title Stay on target: mechanisms of casein kinase 1 targeting to control mitotic checkpoint function
Degree PhD
Department Cell and Developmental Biology
Advisory Committee
Advisor Name Title
Ethan Lee Committee Chair
Dave Cortez Committee Member
Kathleen L. Gould Committee Member
Todd Graham Committee Member
Keywords
  • Schizosaccharomyces pombe
  • casein kinase 1
  • spindle pole body
  • centrosome
  • mitotic checkpoint
  • cytokinesis
Date of Defense 2018-02-23
Availability unrestricted
Abstract
Post-translational modification of proteins are events involved in many cellular processes, including the cell cycle. During mitosis, the metaphase to anaphase transition is regulated by the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C). While the mitotic substrates of the APC/C have been well characterized, it is not clear what role deubiquitinating proteases (DUBs) play in antagonizing APC/C function. Here we performed a genetic screen to determine what DUB, if any, antagonizes the function of the APC/C in the fission yeast Schizosaccharomyces pombe. Our evidence demonstrates that deletion of the SAGA associated DUB Ubp8 (ubp8Δ) suppresses the temperature sensitive phenotypes of APC/C mutants and this suppression is specific to the SAGA DUB module. Furthermore, mutations abolishing histone H2B ubiquitylation were not able to suppress the APC/C temperature sensitive phenotype. On the basis of these data, we conclude that Ubp8 antagonizes APC/C function utilizing a mechanism dependent on H2B ubiquitination.

Hhp1/2 are the soluble casein kinase 1 (CK1) family members in S. pombe. One of their functions is to inhibit the septation initiation network (SIN) during a mitotic checkpoint arrest. Hhp1/2 phospho-prime the SIN scaffold protein Sid4 for ubiquitination by the E3 ligase Dma1; Sid4 ubiquitination delays SIN activation and thus cell division. The SIN is assembled at spindle pole bodies (SPBs), and though Hhp1/2 also localize to SPBs, it is not known if their SPB localization is required for SIN inhibition. Here, we establish that Hhp1/2 localize constitutively to SPBs, the nucleus, cell tips, and division site. We find that their catalytic domains but not enzymatic function are used for SPB targeting and that this targeting strategy is conserved in human CK1δ/ε localization to centrosomes. Further, we pinpoint amino acids in the Hhp1 catalytic domain required for SPB interaction; mutation of these residues disrupts Hhp1 association with the core SPB protein Ppc89, and the inhibition of cytokinesis in the presence of spindle stress. Taken together, we have defined a molecular mechanism used by CK1 enzymes to target to a specific cellular locale for compartmentalized signaling.

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