Analysis of Developmental Relevance of Cdk1 Inhibitory Phosphorylation in Drosophila

  • Author / Creator
    Ayeni, Joseph O
  • Cyclin dependent kinase 1, Cdk1, inhibitory phosphorylation is at the core of conserved checkpoint mechanisms that prevent mitosis from interfering with DNA replication or repair. It is also crucial for coordinating cell cycle progression with morphogenetic processes during organismal development. During interphase, Wee1 and Myt1 function as Cdk1 inhibitory kinases. Both kinases phosphorylate Cdk1 on a conserved tyrosine, Y15, however the dual specificity Myt1 kinase also phosphorylates an adjacent threonine, T14. In spite of the functional redundancy with Wee1 for Y15 phosphorylation, Myt1 serves specialized developmental functions that may reflect its unique capabilities as a T14 or dual specificity Cdk1 inhibitor. To define distinct developmental requirements for T14 and Y15 as well as dual phosphorylation of Cdk1, new transgenic strains expressing Gal4-inducible VFP-tagged wild-type Cdk1 and three Cdk1 phospho-acceptor mutants: Cdk1(T14A)-VFP, Cdk1(Y15F)-VFP and Cdk1(T14AY15F)-VFP, were engineered. Genetic and biochemical evidence revealed that T14 and Y15 inhibitory phosphorylation are functionally distinct mechanisms for regulating Cdk1 activity. Y15 inhibitory phosphorylation was shown to be necessary and sufficient for developmentally regulated G2 phase arrest, while the T14 phosphorylation of Cdk1 may have evolved as a mechanism for accumulating dually inhibited Cdk1-Cyclin B complexes. The Myt1-mediated dual phosphorylation of Cdk1 was implicated as a genetic innovation evolved in metazoans for allowing cells to remain stably arrested in G2 phase for prolonged periods. The new transgenic tools were also used to define how the temporal regulation of Cdk1 and the timing of G2-phase quiescence are linked with the developmentally regulated signal crucial for specifying neuronal cell fate during sensory organ development in Drosophila. Genetic evidence demonstrated that forced mitosis in G2 quiescent SOP cells conferred self-renewal potential on the cells that would normally terminally differentiate. Thus, linking the timing of G2 quiescence in SOP cells to the critically important developmental choice of self-renewal versus terminal differentiation. Further analysis of the transgenic Cdk1 fusion proteins in cdc25twe mutant spermatocytes, lacking active endogenous Cdk1 proteins, suggested the existence of both Cdc25Twe-dependent and –independent regulation of Cdk1 activity in male meiosis.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Biological Sciences
  • Specialization
    • Molecular Biology and Genetics
  • Supervisor / co-supervisor and their department(s)
    • Dr S.D. Campbell (Biological Sciences)
  • Examining committee members and their departments
    • Dr. John Locke (Biological Sciences)
    • Dr Esther Verheyen (External Examiner)
    • Dr. Frank Nargang (Biological Sciences)
    • Dr. Lisa Stein (Biological Sciences)
    • Dr. Marty Srayko (Biological Sciences)
    • Dr. S.D. Campbell (Biological Sciences)