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      The interaction of the p85 subunit of PI3K with rab proteins

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      Date
      2007-06-28
      Author
      Chamberlain, Michael Dean
      Type
      Thesis
      Degree Level
      Doctoral
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      Abstract
      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.
      Degree
      Doctor of Philosophy (Ph.D.)
      Department
      Biochemistry
      Program
      Biochemistry
      Supervisor
      Anderson, Deborah
      Committee
      Sulakhe, Prakash; Roesler, William J.; Laferte, Suzanne; Khandelwal, Ramji L.; Forsyth, George W.
      Copyright Date
      June 2007
      URI
      http://hdl.handle.net/10388/etd-06272007-145442
      Subject
      endocytosis
      PDGFR
      p85
      PI3K
      Rab
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