Drosophila E3 ubiquitin ligase Hyperplastic Discs interacts with Shaggy and regulates morphogen signalling in the developing eye
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The expression of the Drosophila melanogaster morphogen Hedgehog (Hh) plays a key role in co-ordinating proliferation and differentiation during animal development. Tight spatial and temporal regulation of Hh expression is essential for its correct function in these essential processes. Both mis-expression of its mammalian orthologue Sonic Hedgehog (Shh) and aberrant stimulation of the associated signalling pathway occur in a wide range of human tumours. Although there is extensive knowledge of the signal transduction pathway that is activated in a Hh-stimulated cell, very little is known about pathways governing the expression of the morphogen itself. The Drosophila tumour suppressor protein Hyperplastic Discs (Hyd), an E3 ubiquitin ligase, negatively regulates hedgehog (hh) expression and Hh pathway activity by independent mechanisms in the developing Drosophila eye. Genetically generated hyd mutant clones in the eye mis-express hh and the transcriptional activator of Hh target genes, Cubitus interruptus (Ci), and cause overgrowth of the surrounding wildtype tissue. However, the underlying molecular mechanism(s) by which Hyd regulates these morphogen regulatory pathways is not known. Hyd may be involved in ubiquitylating target proteins in these pathways, which could have degradative or non-degradative outcomes. In order to elucidate Hyd’s molecular role in potential morphogen regulatory pathways, I applied a proteomics-based approach to identify novel Hyd binding partners and ubiquitylated substrates. Tandem affinity purification in combination with mass spectrometry was used to purify and identify Hyd and its complexed binding partners from Drosophila cells. Binding and ubiquitylation assays were subsequently used to verify and characterize the interactions. In addition, a biased, literature-guided approach was applied to identify likely Hyd binding partners based on their involvement in morphogen signalling and conservation across species. Finally, to assess the functional consequences of a newly identified interaction, I used a Drosophila in vivo model to determine whether the novel binding partner was capable of modifying the hyd mutant phenotype. For this purpose, the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique was used to generate hyd mutant clones in the developing larval eye, which were expressing transgenes resulting in either the over-expression or RNAi-mediated knockdown of the gene of interest. My results indicate that Hyd is involved in regulating both Hh and Wg morphogen signalling in the Drosophila eye, and that the molecular mechanism of action may, at least in part, involve the protein kinase Shaggy (Sgg). Hyd interacts with the Hh and Wg transcriptional activator proteins Ci and Armadillo, respectively, as well as the Sgg kinase. Sgg is a negative regulator of both the Hh and Wg pathways, and acts to direct the proteolytic processing or degradation of the transcriptional effectors of these morphogen pathways. Sgg and its mammalian orthologue GSK3β were ubiquitylated in vitro, and GSK3β ubiquitylation was negatively regulated by the mammalian homologue of Hyd, EDD. Knockdown of sgg in eye disc cells mutant for hyd resulted in a dramatic rescue of the overgrowth phenotype. Loss of hyd in clones located in the anterior region of the eye disc resulted in low levels of the full-length Hh transcriptional activator protein Ci. This effect was reversed completely as a result of sgg knockdown. Furthermore, loss of hyd in eye disc clones resulted in elevated Hh and Wg morphogen expression. Mis-expression of hh in hyd mutant clones was significantly reduced upon over-expression of a constitutively active Sgg kinase. Hence sgg appears to genetically act downstream of hyd to regulate hh gene expression and Ci expression. In summary my results identify Sgg as a novel regulator of hh gene expression, whose activity may be regulated by ubiquitylation, and which may be acting downstream of Hyd in a ubiquitin-regulated manner to control both hh gene expression and Hh pathway activity in the developing Drosophila eye. Hyd may also regulate Hh pathway activity by directly interacting with Ci and affecting its activity. The results also indicate that Hyd may be a master regulator of both Hh and Wg morphogen signalling during Drosophila development.