The role for the p85 subunit of PI3kinase in the regulation of rab proteins
Date
2008
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Degree Level
Masters
Abstract
Upon 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.
Description
Keywords
PDGFR Trafficking, Endocytosis, Rab5, Rab4, PI3K, GAP activity, GTPases
Citation
Degree
Master of Science (M.Sc.)
Department
Biochemistry
Program
Biochemistry