[{"key":"dc.contributor.author","value":"Felberg, Jackie","language":null},{"key":"dc.date.accessioned","value":"2009-09-14T18:50:00Z","language":null},{"key":"dc.date.available","value":"2009-09-14T18:50:00Z","language":null},{"key":"dc.date.issued","value":"2001","language":null},{"key":"dc.identifier.uri","value":"http:\/\/hdl.handle.net\/2429\/12717","language":null},{"key":"dc.description.abstract","value":"CD45 is a transmembrane two-domain protein tyrosine phosphatase (PTP) essential for thymocyte development and B cell maturation. Its tyrosine phosphatase activity is required for efficient signal transduction initiated by lymphocyte antigen receptors. As with most transmembrane two-domain\r\nphosphatases, the role of the second, C-terminal domain, D2, is unclear. To\r\ninvestigate the role of D2 and to determine if it plays a role in regulating CD45\r\nactivity, substrate specificity, or molecular interactions, recombinant proteins\r\ncontaining the first, N-terminal PTP domain, D1, without D2, and D2 without\r\nD1, were expressed and purified from E. coli and analyzed.\r\nIt was found that a purified D1 recombinant protein dimerized, and was\r\nenzymatically active in the absence of PTP-D2, whereas no phosphatase activity\r\nwas detected for D2 which was a monomer in solution. D2 enhanced activity of\r\nDl in the monomeric full length CD45 cytoplasmic domain protein (D1\/D2), and\r\nthis was shown to be specific to a PTP domain, as the Lck SH2 domain could not\r\nsubstitute for D2. Specific binding of the recombinant 6His-Dl to a recombinant\r\nGST-D2 protein, and the association of N - and C- terminal tryptic fragments of\r\n6His-Dl\/D2 provided evidence for an intramolecular interaction occurring in the\r\nCD45 cytoplasmic domain. Co-purification of 6His-Dl and GST-D2 from E. coli\r\nindicated that this is a stable interaction resulting in increased stability of Dl.\r\nThis interaction did not require the acidic region unique to D2. It was, however,\r\naffected by a destabilizing point mutation, Q1180G, in D2.\r\nNo evidence was obtained for a role for D2 in determining substrate\r\nspecificity using a panel of peptide substrates or between full length Src family kinase protein substrates. However in in vitro binding assays, 6His-D2\r\npreferentially bound to its proposed physiological substrate, the Src family\r\nkinase p56lck, expressed as a GST fusion protein, compared to a related, non-Src\r\nfamily tyrosine kinase, GST-Csk. Using in vitro binding assays, it was\r\ndetermined that CD45-D2 bound to the kinase domain of Lck, and this\r\ninteraction did not require the acidic region in D2. The interaction was\r\nindependent of the tyrosine phosphorylation state of Lck, and 6His-D2 did not\r\nbind phosphotyrosine.","language":"en"},{"key":"dc.format.extent","value":"9755927 bytes","language":null},{"key":"dc.format.mimetype","value":"application\/pdf","language":null},{"key":"dc.language.iso","value":"eng","language":"en"},{"key":"dc.publisher","value":"University of British Columbia","language":null},{"key":"dc.rights","value":"For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https:\/\/open.library.ubc.ca\/terms_of_use.","language":null},{"key":"dc.title","value":"Characterization of the C-terminal protein tyrosine phosphatase-like domain of CD45","language":"en"},{"key":"dc.type","value":"Text","language":null},{"key":"dc.degree.name","value":"Doctor of Philosophy - PhD","language":"en"},{"key":"dc.degree.discipline","value":"Microbiology and Immunology","language":"en"},{"key":"dc.degree.grantor","value":"University of British Columbia","language":null},{"key":"dc.date.graduation","value":"2001-05","language":"en"},{"key":"dc.type.text","value":"Thesis\/Dissertation","language":"en"},{"key":"dc.description.affiliation","value":"Science, Faculty of","language":null},{"key":"dc.description.affiliation","value":"Microbiology and Immunology, Department of","language":null},{"key":"dc.degree.campus","value":"UBCV","language":"en"},{"key":"dc.description.scholarlevel","value":"Graduate","language":"en"}]