Rapid progress has been made toward understanding the significance of CDK inhibitor proteins (CKIs) in the regulation of cell cycle progression. The overall goal of this study has been targeted to further expand our knowledge of CKI function through the investigation of a previously uncharacterized CKI named cki-2 during development in C. elegans. The characterization of cki-2 using a reverse genetic approach called co-suppression has revealed a novel mechanism that cki-2 and its related cell cycle regulators are required for the appropriate elimination of centrioles during oogenesis. Loss of cki-2 in the germ line caused perdurance of centrioles into the one-cell embryo, resulting in supernumerary centrosomes and aberrant cell divisions in the first cell cycle. This was significantly suppressed by reduction of cyclin E and a Cdk2 homologue, indicating that these cell cycle regulators are involved in this critical developmental process. In order to further understand the function of cki-2, a yeast two-hybrid screen was conducted which allowed us to identify three CKI-2 interacting proteins: orthologues of PCNA (PCN-1), SUMO (SMO-1), and a RING finger protein called RNF-1. CKI-2 has functionally separable domains in its amino (Cyclin/Cdk binding)- and carboxy (PCNA binding)-terminus and they exert distinct roles in cell cycle progression. It was observed that CKI-2 is covalently modified by SUMO on its N-terminus and this causes CKI-2 to relocalize to thr nucleolus, which is associated with its rapid degradation. Since many RING finger proteins act as components of the multi-subunit E3 ubquitin ligases, we speculated that RNF-1 might be involved in the CKI-2 degradation. This possibility was tested by co-expression of RNF-1 with CKI-2, revealing that co-expression of RNF-1 suppresses the embryonic lethality caused by the CKI-2 overexpression and moreover, this is correlated with an increased rate of CKI-2 degradation. In addition, western blot analyses performed on different genetic backgrounds suggested that the CKI-2 degradation occurs in an ubiquitin-dependent manner through the proteasome-mediated proteolysis pathway. Furthermore, a yeast-based assay developed to test a possible role of SUMO in modulating the CKI-2/RNF-1 interaction demonstrated that SUMO may antagonize the interaction between CKI-2 and RNF-l, these highlighting an intriguing model that appropriate levels of CKI-2 are regulated through ubiquitin-dependent proteolysis mediated by RNF-l, and which maybe modulated by SUMO.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.102991 |
| Date | January 2007 |
| Creators | Kim, Dae Young, 1968- |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biology.) |
| Rights | © Dae Young Kim, 2007 |
| Relation | alephsysno: 002599380, proquestno: AAINR32199, Theses scanned by UMI/ProQuest. |
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