Cytokines are secreted proteins or glycoproteins that are responsible for mediating cell to cell signals and orchestrating numerous biological events including the activation, proliferation and differentation of the cells of the immune system. Cytokine signals are mediated through specific transmembrane receptors on the surface of their target cells. The binding of a hematopoietic cytokine to its receptor triggers the activation of receptor-associated Janus kinases (JAKs). This is followed by the activation of their downstream targets, Signal transducers and activators of transcription (STATs) through tyrosine phosphorylation. Activated STATs dimerize and translocate to the nucleus, where they modulate the transcription of their target genes. The activity of this signaling cascade is under the control of regulatory proteins, such as Protein inhibitors of activated STAT (PIAS), Suppressors of cytokine signaling (SOCS) and various protein tyrosine phosphatases (PTPs). Moreover, JAKs and STATs are regulated by different post-translational modifications, like in the case of STAT1 and STAT5 through the covalent conjugation of a small ubiquitin-like modifier (SUMO).
The JAK/STAT pathway has been conserved throughout evolution. In the fruit fly, Drosophila melanogaster, the JAK/STAT cascade plays a crucial role in various developmental events as well as in the immune response, and it is activated by a set of secreted mediators called Unpaired (Upd). In this thesis Drosophila melanogaster was used as a model organism to study the regulation of the JAK/STAT pathway. The only known Drosophila STAT transcription factor, Stat92E, was shown to be modified with SUMO at a single lysine, K187. Mutating this SUMO conjugation site increased the Upd-induced transcriptional activity of Stat92E, suggesting that the sumoylation of Stat92E negatively regulates the Drosophila JAK/STAT pathway. This indicates that sumoylation is an evolutionary conserved regulatory mechanism of STAT-mediated signal transduction.
RNA interference (RNAi) has been found to function exceptionally well in Drosophila cells, and also in vivo in adult flies. A genome-wide RNAi screen was conducted in Drosophila S2 cells to reveal novel JAK/STAT pathway associated genes. As a result six potential JAK/STAT pathway regulating genes were found, of which two; Eye transformer (ET) and Not4 were studied in more detail. ET encodes a gp130-related transmembrane protein that was found to inhibit Upd-induced Stat92E tyrosine phosphorylation, thus it functions as a negative regulator of the Drosophila JAK/STAT pathway. RNAi of Not4 was found to repress the Drosophila JAK/STAT pathway target gene expression in Drosophila cells, indicating that Not4 is needed for the JAK/STAT pathway to function properly. Furthermore, overexpression of Not4 resulted in enhanced Stat92E-mediated gene responses, confirming Not4 as a positive regulator of the Drosophila JAK/STAT pathway. We found that Not4 is able to interact with Stat92E, but does not affect Stat92E tyrosine phosphorylation. Finally, our experiments indicated that Not4 is needed for Stat92E to properly bind to its target DNA sequence at the promoter of the stress gene TurandotM (TotM). The Not4 mammalian homologue CNOT4 was also shown to participate in STAT1- and STAT6-mediated gene expression in human cells, indicating that Not4/CNOT4 is an evolutionary conserved regulator of JAK/STAT signaling.
