Regulation of JAK/STAT Signalling by Endocytosis

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.