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The role for the p85 subunit of PI3kinase in the regulation of rab proteins

dc.contributor.advisorAnderson, Deborahen_US
dc.contributor.committeeMemberMousseau, Darrell D.en_US
dc.contributor.committeeMemberKhandelwal, Ramji L.en_US
dc.contributor.committeeMemberDesautels, Michelen_US
dc.contributor.committeeMemberWarrington, Rob C.en_US
dc.creatorKing, Jennifer Cen_US
dc.date.accessioned2008-11-17T09:37:07Zen_US
dc.date.accessioned2013-01-04T05:08:41Z
dc.date.available2010-01-26T08:00:00Zen_US
dc.date.available2013-01-04T05:08:41Z
dc.date.created2008en_US
dc.date.issued2008en_US
dc.date.submitted2008en_US
dc.description.abstractUpon activation by the platelet-derived growth factor receptor (PDGFR), phosphatidylinositol 3'-kinase (PI3K) converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate to activate the PI3K/Akt cellular survival signalling pathway within cells. The p85 subunit of PI3K has also been shown to have GTPase activating protein (GAP) activity towards Rab proteins involved in receptor endocytosis and trafficking, specifically Rab5 and Rab4. Rab5 is responsible for regulating the fusion of vesicles containing activated receptors to traffic them to intracellular early/sorting endosomes. Rab4 is responsible for regulating the exit of receptors to a recycling pathway back to the plasma membrane. The p85 RabGAP activity is responsible for deactivating Rab5 and Rab4 function by accelerating their GTPase activity, resulting in the inactive conformation of Rab5 and Rab4, and decreased vesicle fusion events during receptor trafficking. The work in this thesis was performed to understand how p85 interacts with, and regulates, Rab5 and Rab4. Glutathione S-transferase pulldown experiments showed the p85 protein was able to interact with Rab5 through its BH domain and another unidentified domain. Cells expressing a p85-R274A mutant defective for RabGAP activity displayed increased PDGFR activation and decreased degradation. To understand the mechanism of decreased PDGFR degradation, PDGFR immunoprecipitation experiments showed the PDGFR was ubiquitinated, a signal needed for multi-vesicular body sorting. Biotinylation experiments showed the PDGFR was being more rapidly endocytosed and then sequestered within the cell. Immunofluorescence experiments showed cells expressing the p85-R274A mutant clearly altered PDGFR trafficking during receptor endocytosis. These results suggest the PDGFR was not spending longer periods of time on the cell surface to continue signalling and was not lacking the modification needed to be sorted to a degradative pathway. The defective trafficking observed in p85-R274A expressing cells, over time, may block PDGFR trafficking, which prevents normal PDGFR dephosphorylation and degradation, and could be attributed to a lack of sufficient cytosolic Rab5-GDP and Rab4-GDP required to associate with new membranes and facilitate additional vesicle fusion events. The lack of lysosomal targeting allows the receptor to be sequestered in cells, but still have the ability to signal as the receptor would not be targeted to multi-vesicular bodies where signalling is abolished.en_US
dc.identifier.urihttp://hdl.handle.net/10388/etd-11172008-093707en_US
dc.language.isoen_USen_US
dc.subjectPDGFR Traffickingen_US
dc.subjectEndocytosisen_US
dc.subjectRab5en_US
dc.subjectRab4en_US
dc.subjectPI3Ken_US
dc.subjectGAP activityen_US
dc.subjectGTPasesen_US
dc.titleThe role for the p85 subunit of PI3kinase in the regulation of rab proteinsen_US
dc.type.genreThesisen_US
dc.type.materialtexten_US
thesis.degree.departmentBiochemistryen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelMastersen_US
thesis.degree.nameMaster of Science (M.Sc.)en_US

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