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Tie2 receptor is a cell surface tyrosine kinase receptor expressed almost exclusively in endothelial cells, where it is mainly studied for its role in angiogenesis. Indeed, Tie2 has been related to various pathologies with vascular implication such as pulmonary hypertension, diabetes retinopathy and tumour growth. The regulation of Tie2 activation and function is complex, involving multiple factors that are still being investigated. For instance, after activation by the agonistic ligand Angiopoietin-1 (Ang1), Tie2 is internalized in cells by an endocytic mechanism that has yet to be fully characterised. As it has been shown that endocytosis of molecules can play a regulatory role in intracellular signalling in various ways I believe that the endocytosis of Tie2 may also be important in the regulation of its activity and cellular output. Therefore, I decided to characterise the endocytic mechanisms involved in the internalization of Tie2 to determine whether endocytosis can be a regulator of Tie2 signalling. To facilitate the study of Tie2 I created a HeLa cell line with inducible expression of a Tie2FLAG receptor that emulates both expression levels and characteristics of endogenous Tie2 in Human Umbilical Vein Endothelial Cells. To study the endocytosis of Tie2 receptor I developed an immunofluorescence-based assay to quantify the amount of internalized agonistic ligand Ang1 in a high throughput Screening format. I also performed complementary immunofluorescence and western blot analysis to investigate the characteristics of Tie2 internalization.

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The WASp family protein Las17 is the primary activator of the Arp2/3 complex in Saccharomyces cerevisiae and is essential for membrane invagination during endocytosis. Las17 has a similar domain structure to mammalian WASp/N-WASp with an N-terminal WH1 domain, a central proline rich region and a C-terminal WCA region. It was recently shown that the polyproline rich region of Las17 can bind to actin and promote actin filament formation in vitro. It has been hypothesised that at a site of endocytosis Las17 may provide the initial filaments necessary to allow the rapid burst of Arp2/3 mediated actin polymerisation required to achieve invagination. Proline tracts within the region were shown to influence this activity but the actin binding sites and mechanism of action were unclear. Over the course of this research we have identified the minimal component required for this Arp2/3-independent activity and characterised the key motifs involved. The effect of regions outside of this minimum component on actin polymerisation are probed and an exciting potential role in the direct regulation of filament elongation proposed. Mechanisms by which the Arp2/3 dependent and independent activities of Las17 may be regulated relative to each other are examined and the ability of the full-length protein to activate the Arp2/3 complex is found to be somewhat autoinhibited. This work provides further evidence for Las17 as a multifunctional regulator of the actin cytoskeleton in line with a growing body of work demonstrating that WASp family proteins are more than just direct activators of the Arp2/3 complex.

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The JAK/STAT pathway is a highly evolutionarily conserved signal transduction pathway, whose activation can lead to a broad range of cellular outcomes. The pathway is used repeatedly during multiple developmental stages and in adult tissue, and therefore tight regulation is required to enable accurate responses in a context specific manner. Internalisation and endocytic trafficking of signalling components provides a mechanism whereby spatial compartmentalisation can enable distinct signalling outputs. Within this study I have investigated the role of endocytosis in the regulation of the Drosophila melanogaster JAK/STAT pathway, and demonstrated that internalisation and endocytic trafficking differentially regulates target genes. Although the JAK/STAT pathway is transcriptionally competent and can regulate the expression of particular targets when the activated receptor is at the cell surface, receptor endocytosis and localisation to distinct endosomes is required for the expression of other targets. This appears to be context-dependent, as high levels of ligand stimulation overcomes endocytic regulation. STAT92E, the Drosophila JAK/STAT transcription factor, is a target of endocytic regulation. Although it is efficiently activated and undergoes nuclear translocation when endocytosis is perturbed, it is not capable of regulating a subset of target genes and therefore further STAT92E interacting partners and/or post translational modification must be required to fine-tune its transcriptional competency during endocytic trafficking. Utilising mass spectrometry I identified a novel STAT92E phosphorylation site, at threonine 702. Mutation of this threonine to prevent its phosphorylation, resulted in inhibition of STAT92E signalling and nuclear translocation, and also prevented phosphorylation of a highly conserved tyrosine residue at position 704, which is crucial for ligand activated JAK/STAT signalling outputs. Therefore, this study has enhanced our understanding of mechanisms that can modulate JAK/STAT activity. I have revealed an important role for endocytosis in fine- tuning Drosophila JAK/STAT signalling outputs and also identified a novel phosphorylation site which is crucial in the activity of STAT92E.

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