The Cdc25 phosphatases are highly conserved from yeast through humans and play pivotal roles in regulating the activities of cyclin-dependent kinases (Cdks). Cdc25A is one of three human Cdc25 family members, and has previously been shown to be overexpressed in numerous cancers and to transform rodent fibroblasts. Cdc25A therefore represents a rational target for chemotherapeutic development. Further, a thorough understanding of its biology and regulation in normal and transformed cells may facilitate the development of strategies to specifically interfere with the proliferation of cancerous cells. In this work I describe experiments which demonstrate that bisperoxoVanadium compounds, and specifically bpV(Me2Phen), inhibit Cdc25A phosphatase in vitro and in vivo. Further, these compounds cause cell-cycle arrest, are cytotoxic to cancer cells, and slow the growth of tumours in mouse models. With respect to the fundamental biology of Cdc25A, I have identified a sequence element (NLS) responsible for nuclear localization of Cdc25A phosphatase. An analysis of this sequence demonstrated high conservation of flanking phosphoacceptor sites, notably Serine 292. S292 was predicted to be a consensus PKA or CamKII substrate. Using site-directed mutagenesis I have shown that S292 is the sole site of PKA phosphorylation in vitro. The functional importance of S292 phosphorylation was investigated via transfections of phospho-mimetic mutants of S292 (S292E) expressed as GFP-fusion proteins; these studies indicate that S292 phosphorylation may promote nuclear localization. Studies by other groups have indicated that S292 is a phosphorylation site for inhibitory kinases, namely Chk1 and Chk2 (4). I generated a phospho-specific antibody to this site and demonstrate by immunofluorescence and western blotting an unexpected pattern of S292 phosphorylation associated with nuclear bodies and the mitotic apparatus. I provide evidence to suggest that these sites represent local fine-tuning of Cdc25A, allowing Cdk activity to be controlled at the level of specific subcellular structures. These studies highlight the complexity of Cdc25 regulation and indicate a previously unappreciated degree of control of their activity such that these enzymes exist in multiple discrete pools within a given cell.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.103293 |
| Date | January 2007 |
| Creators | Scrivens, Paul James. |
| 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 (Division of Experimental Medicine.) |
| Rights | © Paul James Scrivens, 2007 |
| Relation | alephsysno: 002651471, proquestno: AAINR38643, Theses scanned by UMI/ProQuest. |
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