Histidine-Domain-Protein-Tyrosine-Phosphatase (HD-PTP) has been classified as a non-transmembrane protein tyrosine phosphatase (PTP), however, its catalytic activity has not been appropriately characterized. In this thesis, the tyrosine phosphatase activity of HD-PTP was characterized. To do so, the HD-PTP protein was successfully purified using the FLAG-TAG purification system and an enzymatic assay was carried out using the DiFMUP fluorogenic substrate. My results suggest that HD-PTP is an inactive PTP that can be reactivated upon the back mutation of a conserved amino acid located in its catalytic domain motif 9, which diverges from the PTP consensus sequence. Interestingly, the gene which encodes for HD-PTP is located within the tumor suppressor region on the human chromosome 3p21.3. Furthermore, we determined through colony formation assays that the active mutation does not affect the tumor suppressor potential of HD-PTP. Although wild type HD-PTP is an inactive tyrosine phosphatase, it may act as a natural trapping mutant, thus preserving its strong binding potential for phosphorylated signaling proteins. Since the active HD-PTP mutant should have lost its ability to bind phosphorylated signaling proteins, it was used in a substrate trapping experiment to identify potential binding partners. Four putative binding partners were then purified and identified through multidimensional protein identification technique (MudPIT). Lastly, cell lines that stably express HD-PTP were generated for future studies in the identification of binding partners.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.111552 |
| Date | January 2008 |
| Creators | Zhang, Yu Ling. |
| 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 | Master of Science (Department of Biochemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 003164022, proquestno: AAIMR66732, Theses scanned by UMI/ProQuest. |
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