阿拉伯芥14-3-3蛋白與麩氨酸接受器3.7 磷酸化依存性之交互作用的功能性研究

Abstract

在哺乳類腦神經系統當中，離子型麩氨酸接受器(iGluRs)作為配位體結合的陽離子通道，可快速調節神經的訊息傳導。而在模式植物阿拉伯芥中，麩氨酸接受器(GLR)有20個成員，並且胺基酸的序列以及預測的二級結構與動物之iGluRs非常類似。阿拉伯芥的GLR功能為非選擇型的陽離子通道，可傳送陽離子。先前的研究指出阿拉伯芥GLR在生物學上的功能是會參與在根部的延長以及非生物逆境反應。然而，在植物當中，GLR的訊息傳導路徑還不清楚。14-3-3為鷹架蛋白，它可與離子通道蛋白做交互作用並且改變離子的通透性，而14-3-3蛋白會與具有磷酸化的絲胺酸或蘇胺酸之胜肽片段作結合。先前已報導過14-3-3ω蛋白可能會與阿拉伯芥GLR3.7蛋白有交互作用。此外，阿拉伯芥GLR3.7可以被鈣離子依存性蛋白激酶(CDPK)所磷酸化。為了研究阿拉伯芥GLR3.7的磷酸化位點是否與14-3-3蛋白結合的位點相同，使用CDPK在激酶-受質磷酸化反應(kinase assay)找到磷酸化的位點。同時，利用螢光共軛焦顯微鏡觀察GLR3.7在細胞內的位置。接著使用雙分子螢光互補系統(BiFC)、pull-down、石英晶體微量平衡試驗(QCM)以及免疫共沉澱法找到14-3-3ω的結合位點，此位點與CDPK磷酸化位點重疊，表示CDPK磷酸化位點與14-3-3結合位點為同一個胺基酸。此外，結果發現阿拉伯芥GLR3.7參與在根毛的發育，並且，證據顯示阿拉伯芥GLR3.7很可能調控根毛細胞的延長。總結以上所述，推測鈣離子依存性蛋白激酶可以磷酸化阿拉伯芥GLR3.7第860號絲胺酸，並且這個位點被磷酸化後，會使得14-3-3ω蛋白與之結合，進而提高通道的活性並且影響阿拉伯芥根毛的生長發育。

Ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels that mediate fast excitatory neurotransmission in the mammalian central nervous system. In the model plant Arabidopsis thaliana, a family of 20 proteins share similarities to animal iGluRs in sequence and predicted secondary structure. AtGLRs function as non-selective cation channel to transport cations. Previous researches showed biological functions of glutamate receptor in root elongation and abotic stress response. However, the signal transduction pathway in plants is still not clear. 14-3-3 protein is a scaffold protein, which can interact with ion channel in the cytosol and change their ion permeability. 14-3-3 protein binds to phospho-serine/threonine amino acid in a sequence-specific manner. It has been reported that 14-3-3 protein may interact with GLR3.7. Besides, calcium-dependent protein kinase (CDPK) can phosphorylate GLR3.7 in virto. To confirm whether GLR3.7 phosphorylation site overlapping with the 14-3-3 binding site, we used in vitro kinase assay to identify the phosphorylation site in GLR3.7 phosphorylated by CDPK. Then we observed the subcellular localization of AtGLR3.7 using confocol microscope. Then we performed bimolecular fluorescence complementation (BiFC) assay, pull-down, quartz crystal microbalance analysis and co-immunoprecipitation to confirm 14-3-3 binding site. In addition, we found that AtGLR3.7 is involved in root hair development, and the results revealed that AtGLR3.7 may regulate root hair elongation. Overall, these findings demonstrate that CDPK phosphorylates Ser-860 of AtGLR3.7, and the phosphorylation site can be bound by 14-3-3 protein to activate the channel activity and influence root hair development in Arabidopsis thaliana.