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Biochemical characterization of Drosophila receptor tyrosine phosphatases

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  • Biology


Two classes of enzymes are responsible for modulation of intracellular phosphotyrosine levels, namely protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Together these enzymes maintain the appropriate balance of phosphoproteins required for a variety of developmental processes including axon pathfinding. In Drosophila, five receptor-like protein tyrosine phosphatases (RPTPs) regulate axon pathfinding, but little is known about their downstream signaling pathways or the means by which their enzymatic activity is regulated. Chapter 2 of this thesis deals with experiments to test whether dimerization regulates the activity of these enzymes. Crystallographic data indicates that some RPTPs form dimers in which each monomer is precluded from binding substrate due to the insertion of a helix-turn-helix segment of the opposing monomer into the active site. I introduced ?tagged? RPTP constructs into Drosophila S2 tissue culture cells and tested for dimer formation using immunoprecipitation and Western blotting. I did not detect stable dimers, however. This may suggest that dimer formation requires other protein components (such as the putative RPTP ligands) that are not expressed in S2 cells. In Chapter 3 I investigated the possibility that Roundabout (Robo), a receptor mediating axonal repulsion from the embryonic midline, is a substrate for RPTPs DPTP69D and/or DPTP10D. Previous genetic studies implicate these RPTPs in participating in the Robo signaling pathway. Experiments detailed here show that Robo can be phosphorylated on tyrosine residues in S2 cells, characteristic of an RPTP substrate. However, Robo did not co-immunoprecipitate with ?substrate trap? mutants of either of these RPTPs, possibly because their interaction is dependent on co-factors not present in the cell culture system. Chapter 4 is a characterization of DPTP69D-associated proteins purified from embryos expressing a substrate trap version of DPTP69D. We identified one of the associated proteins as non-muscle myosin II heavy chain (nmm II hc). Proper regulation of nmm II hc is essential for axon patterning in mushroom bodies (MBs). I found that expression of the DPTP69D trap in MBs results in an axon retraction phenotype similar to that seen when nmm II hc activity is elevated, suggesting that this protein may be a target for DPTP69D activity.

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