The cardiovascular system develops through two distinct processes in embryogenesis: vasculogenesis, whereby the primary plexus in the heart is formed along with embryonic and extraembryonic vasculature, and angiogenesis, which begins after vasculogenesis and results in the refinement and maturation of the branched vessel system. In pathological angiogenesis, tumors expand by releasing pro-angiogenic factors in response to hypoxic conditions. The Tie receptors, Tie1 and Tie2, are receptor tyrosine kinases that are integral to angiogenic pathways. A family of Angiopoietins, Ang1-4, have been shown to act as ligands for Tie2, of which Ang1 and Ang2 are best characterized. Activation of the receptor causes dimerization and autophosphorylation, whereby adaptor proteins recognizing the phosphorylated tyrosine activate downstream signaling via their Src homology 2 (SH2) domains. Currently there are no phosphospecific antibodies for Tie2, therefore, identifying critical residues responsible for certain pathways remains difficult. In our study, we aim to use purified SH2 domains of known binding partners to Tie2 to assess the phosphorylation state of the receptor under various cellular conditions and settings, utilizing immunoprecipitation and western blotting. Unexpectedly, we found that Tie2 can bind non-specifically to nickel sepharose when the SH2 proteins were used as antibody mimetics, and was unable to be consistently precipitated in Protein A sepharose when used in conjunction with a monoclonal YFP antibody. Under the latter conditions however we were able to precipitate the SH2 protein itself. When immunoprecipitations were used with cobalt activated IMAC beads, we were able to precipitate Tie2 in overexpressed systems using the SH2 domains of Shp2 N-C and Grb2. As expected, phosphorylation of Tie2 in the presence of its orphan receptor Tie1 was attenuated compared to wild-type levels. Based upon available data, we anticipate this method as a useful tool to assess the phosphorylation state of Tie2 and its signaling pathways in the near future.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-1274 |
| Date | 02 December 2011 |
| Creators | Yuth, Kenneth |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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