Activated receptor tyrosine kinase (RTK), such as the epidermal growth factor (EGF) receptor (EGFR) and the platelet-derived growth factor (PDGF) receptor (PDGFR), recruit downstream signalling proteins, including phosphatidylinositol 3-kinase (PI3K). PI3K, composed of a regulatory p85 subunit and a catalytic p110 subunit, phosphorylates phosphatidylinositol 4,5-bisphosphate at the 3 position to generate phosphatidylinositol 3,4,5-trisphosphate. This lipid second messenger activates Akt, which promotes cell growth, cell cycle entry and progression, as well as cell survival and cellular migration. PTEN, a tumor suppressor protein, dephosphorylates phosphatidylinositol 3,4,5-trisphosphate at the 3 position, turning off Akt signalling. PTEN contains a C-terminal PDZ binding motif that binds to the PDZ2 domain of MAGI-2, a scaffolding protein that localizes signalling molecules to the plasma membrane. MAGI-2 has ten domains that potentially mediate multiple protein-protein interactions simultaneously. A PTEN associated-complex (PAC) has been described and may contain MAGI-2, PTEN and p85. The PAC is hypothesized to form at the plasma membrane at appropriate sites for PTEN to gain access to its lipid substrates, since the binding of PTEN to MAGI-2 has been shown to enhance the suppression of PI3K-mediated Akt signalling. In order to better understand the role of the PAC in attenuation of the Akt signalling pathway, regions of the MAGI-2 scaffolding protein were mapped to identify the interactions taking place in the PAC. MAGI-2, and its individual domains, were expressed as GST fusion proteins. These were immobilized onto beads and allowed to bind to cellular proteins including PTEN, p85, PDGFR and EGFR using a GST pull-down experiment. The proteins bound to GST-MAGI-2 were identified using an immunoblot analysis. In vitro pull-down experiments revealed that MAGI-2 PDZ2 and PDZ5 domains bind to PTEN, and both MAGI-2 WW domains were shown to bind to p85. EGFR and PDGFR did not bind to the PDZ domains of MAGI-2 under the conditions studied. In order to study protein-protein interactions in cells, immunoprecipitation assays were also performed. Full length MAGI-2 was expressed tagged to a Myc epitope. This was used in immunoprecipitation assays to determine if MAGI-2 could co-immunoprecipitate with proteins involved in the Akt signalling pathway, such as PTEN, p85, PDGFR and EGFR. MAGI-2 can co-immunoprecipitate with PTEN upon 5 min EGF stimulation however, this result was inconclusive because replicate experiments did not verify this initial observation. MAGI-2 does not co-immunoprecipitate with the EGFR nor p85, under the conditions tested. We examined for these interactions after 5 min of growth factor stimulation and more experiments that test different time points after growth factor stimulation would reveal if these interactions are present at shorter time points. MAGI-2 has been shown to bind to PTEN and p85 in vitro and therefore has the potential to regulate the attenuation of the PI3K/Akt signalling pathway in response to activated EGFR and/or PDGFR.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-09172009-110738 |
| Date | 24 September 2009 |
| Creators | Poland, Sharon Franceska |
| Contributors | Anderson, Deborah H., Khandelwal, Ramji L., Moore, Stan A., Desautels, Michel, Roesler, William J. |
| 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-09172009-110738/ |
| 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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