Integration of JAK/STAT receptor–ligand trafficking, signalling and gene expression in Drosophila melanogaster cells

ABSTRACTThe JAK/STAT pathway is an essential signalling cascade required for multiple processes during development and for adult homeostasis. A key question in understanding this pathway is how it is regulated in different cell contexts. Here, we have examined how endocytic processing contributes to signalling by the single cytokine receptor in Drosophila melanogaster cells, Domeless. We identify an evolutionarily conserved di-leucine (di-Leu) motif that is required for Domeless internalisation and show that endocytosis is required for activation of a subset of Domeless targets. Our data indicate that endocytosis both qualitatively and quantitatively regulates Domeless signalling. STAT92E, the single STAT transcription factor in Drosophila, appears to be the target of endocytic regulation, and our studies show that phosphorylation of STAT92E on Tyr704, although necessary, is not always sufficient for target transcription. Finally, we identify a conserved residue, Thr702, which is essential for Tyr704 phosphorylation. Taken together, our findings identify previously unknown aspects of JAK/STAT pathway regulation likely to play key roles in the spatial and temporal regulation of signalling in vivo.

A Sensitized Genetic Screen to Identify Novel Regulators and Components of the Drosophila Janus Kinase/Signal Transducer and Activator of Transcription Pathway

The JAK/STAT pathway exerts pleiotropic effects on a wide range of developmental processes in Drosophila. Four key components have been identified: Unpaired, a secreted ligand; Domeless, a cytokine-like receptor; Hopscotch, a JAK kinase; and Stat92E, a STAT transcription factor. The identification of additional components and regulators of this pathway remains an important issue. To this end, we have generated a transgenic line where we misexpress the upd ligand in the developing Drosophila eye. GMR-upd transgenic animals have dramatically enlarged eye-imaginal discs and compound eyes that are normally patterned. We demonstrate that the enlarged-eye phenotype is a result of an increase in cell number, and not cell volume, and arises from additional mitoses in larval eye discs. Thus, the GMR-upd line represents a system in which the proliferation and differentiation of eye precursor cells are separable. Removal of one copy of stat92E substantially reduces the enlarged-eye phenotype. We performed an F1 deficiency screen to identify dominant modifiers of the GMR-upd phenotype. We have identified 9 regions that enhance this eye phenotype and two specific enhancers: C-terminal binding protein and Daughters against dpp. We also identified 20 regions that suppress GMR-upd and 13 specific suppressors: zeste-white 13, pineapple eye, Dichaete, histone 2A variant, headcase, plexus, kohtalo, crumbs, hedgehog, decapentaplegic, thickveins, saxophone, and Mothers against dpp.

Inhibition of ornithine decarboxylase induces STAT3 tyrosine phosphorylation and DNA binding in IEC-6 cells

Polyamines are required for the proliferation of the rat intestinal mucosal IEC-6 cell line. Ornithine decarboxylase (ODC) is the enzyme that catalyzes the first step in polyamine synthesis. ODC inhibition not only leads to polyamine depletion but also leads to inhibition of cell proliferation and regulates the expression of the immediate-early genes c- fos, c- myc, and c- jun. Members of the signal transducers and activators of transcription (STAT) transcription factor family bind to the sis-inducible element (SIE) present in the promoters to regulate the expression of a variety of important genes. In the present study, we tested the hypothesis that the STAT3 transcription factor, which is responsible for activation of the acute phase response genes, is activated after inhibition of ODC. We found that inhibition of ODC rapidly induces STAT3 activation as determined by STAT3 tyrosine phosphorylation, translocation of STAT3 from the cytoplasm into the nucleus, and the presence of STAT3 in SIE-dependent DNA-protein complexes. STAT3 activation upon inhibition of ODC was accompanied by the activation of a STAT3-dependent reporter construct. Moreover, prolonged polyamine depletion resulted in downregulation of cellular STAT3 levels.