Post-translational modifications that alter protein activity are essential for signal transduction in many signaling pathways. The Epidermal Growth Factor Receptor (EGFR) pathway uses a kinase cascade to activate Mitogen-Activated Protein Kinase (MAPK) by phosphorylation. Phosphorylated MAPK can translocate into the nucleus and activate the transcription of target genes. In the Drosophila wing disc, the effect of MAPK on its target gene argos (aos) is mediated by phosphorylation and inactivation of the transcriptional repressor Capicua (Cic). Phosphorylated Cic can be exported from the nucleus and/or degraded.
In a genetic screen for regulators of EGFR signaling, we found two mutations in the csn1b gene. Loss of CSN1b leads to ectopic activation of the EGFR target gene aos in the wing disc. csn1b encodes a subunit of the COP9 signalosome (CSN), a complex that removes Nedd8 (a Ubiquitin-like protein) from substrates. The best characterized substrates for the CSN are Cullins (scaffold subunits of E3 ubiquitin ligases), which are active in their neddylated state. aos is also misexpressed in the absence of other CSN subunits, and this misexpression requires a basal level of EGFR signaling.
We have traced the effect of the CSN on aos to its positive regulation of Cic stability. Cic levels are reduced in cells lacking CSN subunits, and the expression of other Cic target genes, including cyclin E and an artificial reporter, is increased. Interestingly, removing the MAPK docking site from Cic protects it from degradation in csn5 mutant cells, but not in csn1b mutant cells, suggesting the existence of two separate degradation mechanisms.
We are now investigating the mechanisms by which the CSN regulates Cic levels. The activity of Cullin-based ubiquitin ligases appears to be increased in CSN mutant cells, suggesting that a Cullin complex could ubiquitinate Cic and send it to 26S proteasome-mediated degradation. Alternatively, another type of ubiquitin ligase, or Cic itself, could be a substrate for deneddylation by CSN. As human Cic is a tumor suppressor gene, these results may shed light on the role of the CSN in cancer.