Date of Graduation

5-2010

Document Type

Dissertation (PhD)

Program Affiliation

Genes and Development

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Jill M. Schumacher, Ph.D

Committee Member

Jeffrey Frost, Ph.D

Committee Member

Mong-Hong Lee, Ph.D.

Committee Member

Sharon Dent, Ph.D

Committee Member

Gregory S. May, Ph.D.

Abstract

A hallmark of tumorigenesis and certain birth defect syndromes is the loss of ploidy that can result from incorrect chromosome segregation. Chromosomes that are not partitioned properly during mitosis are often fragmented, changing the genetic makeup of daughter cells. Inheriting extrachromosomal fragments that contain cell survival genes or losing chromosomal loci that encode tumor suppressors can promote tumor development. Thus, it is essential to elucidate molecular mechanisms required for correct chromosome segregation. Chromosomes are connected to mitotic spindle microtubules by way of a proteinacous, chromosome-bound organelle called the kinetochore. Two decades of research have confirmed that the conserved Aurora B/AIR-2 kinase is required for multiple mitotic events including the proper attachment of microtubules to kinetochores, activation of a checkpoint that monitors kinetochore-microtubule attachment, and separation of daughter cells during cytokinesis. Our previous work identified the C. elegans Tousled-like kinase (TLK-1) as an interactor and substrate activator of AIR-2. However, the role of TLK-1 kinase activity in TLK-1-promoted AIR-2 activation remained enigmatic. The research presented herein reveals that the Aurora B-activating role of TLK-1 is independent of TLK-1 kinase activity in vivo and suggests that this mechanistic function is conserved. Moreover, we hypothesized that phosphorylation influences TLK-1 protein-protein interactions. Towards this goal, the execution of a tethered-catalysis yeast two-hybrid screen to discover proteins that bind TLK-1 in a phosphorylation-specific manner was performed. This screen identified cyclin B3 (CYB-3) as a phospho-TLK-1 interactor. The mitotic defects caused by the loss of CYB-3 are profound and suggest that CYB-3 has crucial roles in promoting kinetochore activity necessary for a critical point during mitosis: the irreversible metaphase-toanaphase transition. Altogether, these results suggest that CYB-3 has an important role in kinetochore function and predict that CYB-3 influences TLK-1-mediated AIR-2 activation.

Keywords

Chromosome segregation, kinetochore, kinetochore-microtubule attachments, Tousled-like kinase, C. elegans B-type cyclins, mitotic Chk1 function, tethered-catalysis yeast two-hybrid screen