Fibroblast Growth Factor 2 (FGF2) is a potent mitogen with a prominent role in tumor-induced angiogenesis. In addition, FGF2 exerts both direct and indirect effects at the interface between angiogenesis and inflammatory processes as well as functions as a survival factor of tumor cells by inhibiting apoptosis. These biological activities of FGF2 are mediated by a ternary complex consisting of FGF high affinity receptors, heparan sulfate proteoglycans (HSPGs) and FGF2, a signaling module that is formed on the surface of target cells. Despite these defined extracellular functions, FGF2 does not contain a signal peptide for translocation into the ER and, therefore, is exported by an unconventional mechanism of protein secretion from cells. This process depends on phosphatidylinositol-4,5-bisphosphate-mediated recruitment and oligomerization of FGF2 at the inner leaflet of the plasma membrane concomitant with the formation of a lipidic membrane pore. Subsequently, cell surface HSPGs act as an extracellular trap mediating translocation of FGF2 into the extracellular space. This thesis started from the previous observation that Tec kinase, a gene product identified in a genome-wide RNAi screen, supports efficient secretion of FGF2 from cells. It aimed at i.) the validation of the role of Tec kinase in unconventional secretion of FGF2 using independent tools and a physiologically relevant read-out system and ii.) to obtain insight into the mechanism by which Tec kinase supports secretion of FGF2 from cells. Employing a pharmacological inhibitor of Tec kinase in combination with a cell proliferation assay as a read-out of FGF2 secretion, a requirement for the enzymatic activity of Tec kinase in unconventional secretion of FGF2 could be established. These experiments confirmed the results from the RNAi screening procedure and, along with other findings from our laboratory, led to discovery of FGF2 being a substrate for tyrosine phosphorylation catalyzed by Tec kinase. In the second part of this thesis, oligomerization-dependent membrane pore formation was found stimulated by tyrosine phosphorylation of FGF2, providing a potential mechanism by which Tec kinase may increase the efficiency of FGF2 secretion from cells. Furthermore, cell-based assays analyzing the tyrosine-phosphorylated pools of FGF2 inside cells and following secretion suggest that tyrosine phosphorylation of FGF2 occurs transiently during membrane translocation as FGF2 did not accumulate on cell surfaces in a phosphorylated form. In order to investigate the localization of a metabolically stable, phosphomimetic FGF2 variant form in cells, microinjection experiments were conducted using a recombinant FGF2 variant that contains the phosphomimetic unnatural amino acid pCMF in the position of the naturally occurring phospho-tyrosine residue. Employing both immuno-electron microscopy and fluorescence microscopy, phosphomimetic FGF2 was found to accumulate in the cell periphery. This localization depended on microtubules as treatment of cells with nocodazole caused perinuclear localization of phosphomimetic FGF2. Finally, to test the relevance of these observations for the overall process of FGF2 export from cells, secretion assays in the absence and presence of nocodazole were performed suggesting a role for microtubules in intracellular transport of FGF2 into the cell periphery and, therefore, for efficient secretion of FGF2 into the extracellular space. Taken together, in this thesis, i.) a role for Tec kinase in the overall process of FGF2 secretion could be validated based on pharmacological evidence, ii.) a role of tyrosine phosphorylation of FGF2 in membrane pore formation was supported by reconstitution experiments, iii.) evidence for transient phosphorylation of FGF2 during membrane translocation was provided by cell-based experiments and iv.) a role for microtubules in FGF2 transport into the cell periphery as part of the overall process of FGF2 secretion was supported by a combination of microinjection and cell imaging.
|Supervisor:||Nickel, Prof. Dr. Walter|
|Date of thesis defense:||28 June 2013|
|Date Deposited:||09 Jul 2013 07:37|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences|
|Subjects:||570 Life sciences|