Affiliations: 1) Université de Montréal; 2) Institut de Recherche en Immunologie et Cancérologie (IRIC)
Keywords: q. cellular remodeling; j. other signaling pathways
Connector eNhancer of KSR (CNK) is an evolutionarily conserved scaffold protein acting as a key regulator of the RAS-MAPK signaling pathway in Drosophila. To discover new functions of CNK, we captured the proximal interactome of CNK in Drosophila S2 cells using an improved version of the BioID technique called miniTurboID. This identified components of the RAS-MAPK pathway known to interact with CNK such as KSR, MEK, RAF and HYP. In addition, we identified the Ste20-related kinase Misshapen (MSN), which is involved in several biological events related to cell migration and planar cell polarity. To decipher the connection between CNK and MSN, we first tested whether CNK and MSN could physically interact in S2 cells. Co-immunoprecipitation experiments demonstrated their ability to associate in S2 cells. This interaction depends on the N-terminal part of CNK (containing the SAM, CRIC and PDZ domains) and the C-terminal part of MSN (containing the CNH domain). In another set of experiments, we obtained the common proximal interactors of CNK and MSN in S2 cells using miniTurboID. Strikingly, we identified 72 common interactors to CNK and MSN, which are largely associated to vesicular trafficking, cytoskeletal organization, cell polarity and migration. Finally, we used the UAS/GAL4 system to look for a genetic link between CNK and MSN. We found that knockdown of MSN during eye development had no effect on the level of phosphorylated MAPK and photoreceptor differentiation in third instar larva suggesting that the function of MSN is not related to RAS-MAPK signalling. Interestingly, we found that depletion of MSN during wing development produced wing blisters in ~40% adult Drosophila analyzed while depletion of CNK had no effect. However, co-depletion of MSN and CNK produced wing blisters in ~70% of adult flies, thus providing the first evidence of a genetic link between CNK and MSN. Blister formation in adult Drosophila wing generally results from improper apposition/adhesion of the wing epithelial bilayer. Imbalance in integrin signalling, vesicular trafficking and cytoskeletal organization have been linked to blister formation. We are now investigating the role of CNK and MSN in those biological events. Our findings suggest that CNK is not exclusively devoted to RAS-MAPK signaling, but that it might work in other signaling events that play critical roles during vesicular trafficking, cytoskeletal organization and cell migration.