Mutation of potential MAPK phosphorylation sites in the Notch antagonist Hairless

Cellular differentiation during eumetazoan development is based on highly conserved signalling pathways. Two of them, the Notch and the EGFR signalling pathways, are closely intertwined. We have identified two potential target sites of the Mitogen activated kinase (MAPK), the downstream effector kinase of EGFR, within Hairless (H), the major antagonist of Notch signalling in Drosophila. Assuming that phosphorylation of these sites modulates H activity, a direct influence of EGFR signalling on Notch pathway regulation might be possible. This hypothesis was tested by generating a phospho-deficient and a phospho-mimetic H isoform and by assaying for their biological activity. We first addressed the binding of known H interaction partners Su(H), Gro, CtBP and Pros26.4 which was similar between mutant and wild type H. Next we assayed eye, wing and bristle development which are strongly affected by the overexpression of H due to the inhibition of Notch signalling. Overexpression of the mutant constructs resulted in phenotypes similar to wildtype H overexpression, yet with subtle differences in phenotypic severity. However, large variations suggest that the mutated residues may be critical for the overall structure or stability of H. Albeit of minor impact, EGFR may fine tune Notch signalling via MAPK dependent phosphorylation of H.