Tyrosine phosphorylation is a crucial mechanism in cellular signaling and regulates proliferation, differentiation, migration and adhesion. The phosphorylation reaction is reversible and is governed by two families of enzymes: protein tyrosine kinases and protein tyrosine phosphatases (PTPs). This thesis investigates the role of PTPs in regulating receptor protein tyrosine kinases (RTKs), and explores a mechanism for regulation of phosphatase activity. Most receptor tyrosine kinases are activated by ligand induced dimerization, which results in an increase in receptor phosphorylation. Preparations of ligand-stimulated dimeric PDGF β-receptors were shown to be less susceptible to dephosphorylation compared with unstimulated receptors. This revealed that reduced receptor dephosphorylation contributes to ligand-induced increase in RTK phosphorylation. The receptor-like phosphatase DEP-1 site-selectively dephosphorylates the PDGF β-receptor. One of the most preferred sites is the PLC-γ binding phosphotyrosine pY1021, and the autoregulatory pY857 is one of the least preferred sites. By using chimeric phospho-peptides derived from these two sites as substrate for DEP-1, it was shown that a lysine residue at position +3 acts as a negative determinant for DEP-1 and that an aspartic acid residue at position –1 is a positive determinant. The modulatory effect of TC-PTP on PDGF β-receptor signaling was explored by using mouse embryonic fibroblasts derived from TC-PTP knockout mice. PDGF β-receptors derived from knockout cells exhibited a higher level of ligand-induced phosphorylation compared to receptors from wildtype cells. The increase was unevenly distributed between different autophosphorylation sites. The PLC-γ binding site, previously implicated in chemotactic response, displayed the largest increase. Consistently, a cell migration assay revealed hyper-responsiveness to PDGF of TC-PTP knockout cells as compared to wildtype cells. Reversible oxidation of the active site cysteine in PTPs is a mechanism, which have been postulated to regulate phosphatase specific activity. An antibody-based generic method for detection of oxidized PTPs was developed. Using this method it was revealed for the first time that UV-induced inactivation of PTPs involves oxidation of the active site cysteine.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-3345 |
| Date | January 2003 |
| Creators | Persson, Camilla |
| Publisher | Uppsala universitet, Ludwiginstitutet för cancerforskning, Uppsala : Acta Universitatis Upsaliensis |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, 0282-7476 ; 1232 |
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