The p85 subunit of phosphatidylinositol 3-kinase (PI3K) has long been thought of as a regulatory subunit that has no other function than the regulation of the p110 catalytic subunit. Our laboratory is studying other roles of the p85 subunit, in particular determining the role of the p85 BH domain. The BH domain has homology to GTPase activating protein (GAP) domains that are involved in the stimulation of monomeric G proteins to hydrolyze their bound GTP to GDP. This converts the G protein from its active conformation to its inactive conformation. We have determined that p85 interacts with Rab proteins, monomeric G proteins that regulate vesicle fusion during the endocytosis of receptors. We have shown that p85 binds to Rab5 regardless of nucleotide-bound state of Rab5. The p85 subunit of PI3K has in vitro GAP activity towards Rab5. It was determined that p85 also has in vitro GAP activity towards Rab4, Rab7, Rab6 as well as the Rho-family G proteins, Rac1 and Cdc42. This GAP activity was localized to the BH domain of p85 and mutation of Arg 274 to Ala abolishes the GAP activity of p85. When this p85R274A mutant was expressed in cells, PDGFR degradation was severely inhibited and there was a corresponding increase in the duration of MAPK and Akt signalling. This increase in cell signalling caused a transformed phenotype in cells expressing the p85 protein with the Arg 274 mutation. These cells have lost contact inhibition for growth, are able to grow independent of attachment as well as in the presence of limited growth factors. They also form tumours in nude mice. These cellular effects seem to be due to an increase in receptor recycling because of the loss of the GAP activity of p85. This increase in receptor recycling may interfere with receptor targeting to the late endosome, which would cause the decrease in receptor degradation that is seen in the p85R274A cells.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-06272007-145442 |
| Date | 28 June 2007 |
| Creators | Chamberlain, Michael Dean |
| Contributors | Sulakhe, Prakash, Roesler, William J., Laferte, Suzanne, Khandelwal, Ramji L., Forsyth, George W., Anderson, Deborah |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-06272007-145442/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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