Effect of novel phosphorylation sites on the function of the tumor suppressor Merlin. Sophia Yip, Angela Effa, Sarah Hughes. University of Alberta, Edmonton, Canada.

   Using Drosophila as a model, we are analyzing the role and mechanism of action of 4.1 family protein member Merlin in Neurofibromatosis Type II (NF2), a disorder associated with development of nervous system tumors. Merlin is a tumor suppressor that is also involved in adhesion, and its activity is deactivated by phosphorylation. Merlin can be multiply-phosphorylated, and we hypothesize that the multiple phosphorylation sites are involved in fine-tuned control of Merlin activity. Our lab has identified 14 potential novel phosphorylation sites for regulating Merlin activity. Mutations of these specific residues to non-phosphorylatable residues are hypothesized to reduce the tumor-suppressor activity of Merlin. To test this hypothesis, potential phosphorylatable serines and threonines were mutated to either a phosphor-mimic or non-phosphorylatable residue and the effects of the mutations on the location and function of Merlin were examined. As the cellular location of Merlin is related to its activity state, a pulse-chase assay was used to test the effect of the mutations on Merlin localization over time in Drosophila Schneider 2 cells. Mutations leading to a different localization pattern over time are likely to be potential phosphorylation sites affecting Merlin activity. Using this method, serine 371 and threonine 18 were identified as potential Merlin phosphorylation sites. To further test the functional effect of these mutations on Merlin activity, transgenic flies carrying the mutant Merlin genes were crossed to wing-specific drivers using the UAS/GAL4 system. The effect of the mutations on proliferation, adhesion and changes in morphology of adult wings and wing imaginal discs were determined. Staining of the wing imaginal discs showed differences in localization of actin and E-cadherin when Merlin^S371D is overexpressed, suggesting that serine 371 is a key phosphorylation site affecting Merlin function. By identifying the mechanism regulating Merlin activity, we can begin to move toward possible approaches that will allow for future treatment and diagnosis of NF2 patients.