UBC Theses and Dissertations
An analysis of the structure and function of protein tyrosine phosphatase ⍺ Harder, Kenneth W.
The molecular basis for the control of cell growth, proliferation, and differentiation involves the coordinated activities of a diverse collection of molecules. Many, if not all, developmental processes are influenced by growth factors, extracellular matrix components, and/or adhesion molecules, which elicit changes in cytoskeletal organization, metabolism, and gene transcription. Signal transduction often involves the activation of tyrosine kinase activity associated with specific receptors. Receptor, or receptor-linked tyrosine kinases, then orchestrate the assembly of multimeric signaling complexes and regulate the activities of various downstream enzymes. Thus, tyrosine kinases form a critical link between the extracellular environment and the signal transduction machinery of the cytoplasm. The protein tyrosine phosphatases (PTPs), by dephosphorylating phosphotyrosine residues, play a critical, albeit less understood role in the regulation of tyrosine phosphorylation. A search for additional members of the PTP family, which now includes over 40 enzymes, led to the identification of PTP⍺, a widelyexpressed receptor-like PTP. In studies aimed at characterizing this PTP, we expressed PTP⍺ in both eukaryotic and bacterial cells. To assess the potential substrate specificity of these enzymes, select bacterially expressed PTPs were assayed with a group of synthetic phosphopeptide substrates using a modified colorimetric assay. These studies suggested that the substrate specificity of PTPs such as PTPβ may be influenced by the context of amino acids surrounding a given phosphotyrosine residue. Studies of the in vivo activity of PTP⍺, investigated by its overexpression in human epidermoid cells, revealed a role for PTP⍺ in the activation and/or dephosphorylation of specific Src family kinases. Moreover, PTP⍺ overexpression dramatically increased the cell-substratum adhesion of these cells and altered the tyrosine phosphorylation and associations of specific focal adhesion molecules, suggesting a role for this enzyme in cell-adhesion. Furthermore, an SH3 domain-binding motif was identified in the membrane-proximal region of PTP⍺, which like similar proline-rich regions in various retroviral oncoproteins and cytokine receptors, was able to bind specific SH3 domains in vitro.
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