|M.Sc Student||Agra Nataly|
|Subject||The Effect of Phosphorylation on the Activity of Dei|
Protein in Drosophila
|Department||Department of Medicine||Supervisor||Professor Adi Salzberg|
|Full Thesis text|
The Drosophila protein Delilah (Dei) is a member of the bHLH proteins and contains a serine-rich domain spanning aa 233-360. Dei expression in the embryo is restricted mainly to tendon cells, and to accessory cells of the chordotonal organs (ChO) that serve as proprioceptors in Drosophila. The expression of Dei exclusively in cells that serve as attachment sites to neighboring cells, suggests that it plays a role in regulation of adhesion processes between different cell types.
In the wing Dei is required for differentiation and adhesion of intervein cells. Dei expression is also seen in the external sensory (ES) organs of the adult, such as the thoracic bristles. Each ES organ is composed of five lineage-related daughter cells that are generated through asymmetric division of a sensory organ precursor (SOP) cell. Ectopic expression of Dei causes a prominent phenotype of SOP loss throughout the entire thorax. All of the Dei expressing cells in both the wing and the embryo express integrins. dei is required for activation of βPS expression in intervein cells and is sufficient to induce βPS expression when expressed ectopically in vein cells.
Serin 301 was identified in vitro as a putative MAPK phosphorylation site in the Dei protein (Grossman and Paroush, unpublished data). In this work we study whether Dei is indeed phosphorylated by MAPK in vivo, and how this phosphorylation affects its function. To address these questions we have generated transgenic fly strains each harboring one of three isoforms of Dei, under the regulation of an upstream activating sequence (UAS): DeiS, the native form of Dei, DeiA a non-phosphorable form of Dei and DeiD a phospho-mimetic form of Dei. We used various phenotypic assays to compare the activity of the three Dei isoforms.
Based on our results we could not determine unambiguously whether Dei is phosphorylated by MAPK in vivo and whether this phosphorylation affects Dei activity.
The results suggest that activation of βPS transcription by Dei is not affected by its phosphorylation state. On the other hand, the expression of non-phosphorable Dei caused a stronger phenotype of loss of SOPs and compensated better for the loss of the endogenous dei. Another important observation was that the DeiA protein accumulated at higher levels than the DeiS and DeiD variants, despite similar levels of transgene expression. These results indicate that phosphorylation may affect the stability of the Dei protein, causing it to be less stable and thus reduce its activity.