蛋白質酪氨酸去磷酸酶MEG交互作用蛋白之分子分析

Abstract

蛋白質酪氨酸磷酸酶 (Protein tyrosine kinases；PTKs)與蛋白質酪氨酸去磷酸酶 (protein tyrosine phosphatases；PTPs)相互調節酪氨酸磷酸化與去磷酸化作用，並且在細胞週期、細胞分化與細胞之移動中扮演重要的角色。而典型蛋白質酪氨酸去磷酸酶中又分為受器型 (receptor-like PTPs；RPTPs) 與非受器型 (non-transmembrane PTPs；NTPTPs) 兩種，雖然在果蠅研究中已確定許多受器型酪氨酸去磷酸酶在神經發育之功能，但多數的非受器型酪氨酸去磷酸酶之功能仍不清楚。在本論文中我將探討一個非受器型的酪氨酸去磷酸酶－dPTPMEG。根據先前研究結果已知dPTPMEG對果蠅腦中蕈狀體(mushroombody)之軸突伸展(axon projection)的維持有關。我主要利用酵母菌雙雜交系統 (yeast two-hybrid)之方法篩選出在果蠅胚胎基因庫(Drosophila embryonic cDNA library)中與dPTPMEG有交互作用的基因。經過進一步實驗確認後從中挑選出兩個可能與dPTPMEG交互作用之基因做後續研究。我利用GST 融合蛋白沉澱法(GST pull-down assays)與共同免疫沉澱法 (co-immunoprecitipation) 更進一步證實dPTPMEG與此兩蛋白間之交互作用，此外也利用果蠅的翅膀觀察其遺傳性狀交互作用。雖然在本篇論文中對dPTPMEG及其交互作用蛋白之關係有了初步的研究成果，這些蛋白之間的交互作用在果蠅發育上扮演的角色則需要更進一步的實驗研究。

Protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) reciprocally regulate protein tyrosine phosphorylation, and is involved in various cellular processes, including cell cycle, proliferation, differentiation and cell movement. Classical PTPs can be classified into receptor-like PTPs (RPTPs) and non-transmembrane PTPs (NTPTPs). Although many Drosophila RPTPs have been found to act during neurogenesis, the roles of NTPTPs in development remain unclear. In this study, we focused on the dPTPMEG, one of the Drosophila classical non-transmembrane PTPs (NTPTPs), which has been reported to play a role in the maintenance of axon projection of Drosophila mushroom body. To further explore the function of dPTPMEG, the yeast two-hybrid screen was performed to isolate dPTPMEG-interacting proteins from Drosophila embryonic cDNA library. Two proteins have been identified that can potentially interact with dPTPMEG. The interaction between dPTPMEG and these two proteins was further confirmed by in vitro GST pull-down assays and in vivo co-immunoprecipitation experiments. We further investigated their genetic interactions in Drosophila developing wings. The developmental roles of these interactions will be further investigated.