Novel level of signalling control in the JAK/STAT pathway revealed by in situ visualisation of protein-protein interaction duringDrosophiladevelopment

Abstract

It is commonly accepted that activation of most signalling pathways is induced by ligand receptor dimerisation. This belief has been challenged for some vertebrate cytokine receptors of the JAK/STAT pathway. Here we study whether DOME, the Drosophila receptor of the JAK/STAT pathway, can dimerise and if the dimerisation is ligand-dependent. To analyse DOME homo-dimerisation, we have applied a β-gal complementation technique that allows the detection of protein interactions in situ. This technique has been used previously in cell culture but this is the first time that it has been applied to whole embryos. We show that this technique, which we renameβlue-βlau technique, can be used to detect DOME homo-dimerisation in Drosophila developing embryos. Despite DOME being ubiquitously expressed, dimerisation is developmentally regulated. We investigate the state of DOME dimerisation in the presence or absence of ligand and show that DOME dimerisation is not ligand-induced, indicating that ligand independent cytokine receptor dimerisation is a conserved feature across phyla. We have further analysed the functional significance of ligand-independent receptor dimerisation by comparing the effects of ectopic ligand expression in cells in which the receptor is, or is not, dimerised. We show that ligand expression can only activate STAT downstream targets or affect embryo development in cells in which the receptor is dimerised. These results suggest a model in which ligand-independent dimerisation of the JAK/STAT receptor confers cells with competence to activate the pathway prior to ligand reception. Thus,competence to induce the JAK/STAT signalling pathway in Drosophilacan be regulated by controlling receptor dimerisation prior to ligand binding. These results reveal a novel level of JAK/STAT signalling regulation that could also apply to vertebrates.

Long‐range effect of upd, a ligand for Jak/STAT pathway, on cell cycle in Drosophila eye development

“…hop loss-of-function mutant clones are much reduced in size relative to their sister clone (Luo et al, 1999). Overexpression of the wildtype dome has been reported to give a dominant-negative effect in the embryo (Brown et al, 2003). Consistent with this dominant-negative effect, eyϾdome also caused an eye reduction phenotype (data not shown).…”

“…One of the possible regulations on the competence to response may be at the level of Dome dimerization, which was shown to precede ligand activation (Brown et al, 2003). Another possibility is that Hh sig- naling is required to collaborate with Upd signaling.…”

JAK/STAT signalling inDrosophila: insights into conserved regulatory and cellular functions

“…2D) (Hou et al, 1996;Yan et al, 1996). Although the core pathway components have been known for some time (reviewed by Castelli-Gair Hombría and Brown, 2002;, these molecules still represent an area of active research that aims to characterise their biochemical and functional activity in vivo (Brown et al, 2003;Henriksen et al, 2002;Karsten et al, 2005). However, it is the identification and characterisation of additional components and regulators of the core pathway that will be discussed here.…”

Negative regulation of Drosophila JAK–STAT signalling by endocytic trafficking

“…Finally, to ensure that the observed uptake and trapping of Upd2-GFP was not a consequence of fluid-phase uptake of conditioned medium, we also undertook experiments in which we knocked down either the JAK-STAT pathway receptor domeless (Arbouzova and Zeidler, 2006;Brown et al, 2003) or a control dsRNA not expressed in Kc 167 cells. Although intracellular Upd2-GFP was present in controls after 15 minutes, no GFP-labelled vesicles were found after knockdown of dome, suggesting that uptake is indeed receptor dependent (Fig.…”

“…Predimerised signal transducers and activators of transcription (STATs) are tyrosine phosphorylated by the JAKs, translocate to the nucleus, bind to DNA sequences present in the promoters of pathway target genes and activate transcription. This gene expression then drives a wide range of developmental, haematological and immune-related responses in many tissues (Arbouzova and Zeidler, 2006;Brown et al, 2003).…”