Identification of upstream regulators of MAPK stimulated Pea3-mediated neural differentiation in PC12 cells

Pea3 transcription factor family belongs to ETS domain transcription factor super-family and act as nuclear targets of signal-transduction pathways, including cell proliferation, differentiation, development and apoptosis like the other members of ETS super-family. A wide variety of the regulatory events that involve ETS domain proteins require MAPK pathways in order to mediate the phosphorylation processes. In a previous study performed in our laboratory, it was observed that over-expression of Pea3 protein induces neuronal differentiation in PC12 cells via MAPK cascade when induced with growth factors which alone induce proliferation in the absence of Pea3. Although the phosphorylation sites for other members of Pea3 transcription family were identified and there were a few studies that investigate the post-translational modification of Pea3 protein, phosphorylation is still unknown. In this thesis project, first approach to study the upstream regulators of Pea3 mediated neuronal differentiation, is blocking suggested MAPK phosphorylation sites on Pea3, by converting suspected Serine residues into Alanine residues. MAP kinases are proline-directed Serine/Threonine kinases and phosphorylate the serine or threonine in the dipeptide S/T-P motif. Therefore, conversion of Serine to Alanine will silence phosphorylation, whereas converting Serine to Glutamate will mimic it. Comparison of the signals obtained from a mutated phosphorylation site with the wild-type will be used to identify the phosphorylation sites on Pea3. Second approach involves deletion of suggested MAPK docking sites on Pea3. MAPKs are known to interact with their targets and regulators via high affinity docking sites in order to obtain specific and efficient selectivity. To be able to phosphorylate their targets efficiently, MAPKs require the integrity of the docking site. Therefore, deletion of docking sites on Pea3 will result in decreased activity as a result of inefficient interaction with the kinases. Docking site specificity in different MAPKs will help us identify the upstream regulators of Pea3-mediated neuronal differentiation mechanism in PC12 cells. This study will permit us to better understand the regulation of ETS domain transcription factor Pea3, in neuronal differentiation. The findings from this work might be considered as a first step in the treatment of neurodegenerative disorders in the future.