阿拉伯芥Di19蛋白與14-3-3蛋白之分子間交互作用

Abstract

Di19（Dehydration-induced preotein19）蛋白在阿拉伯芥 (Arabidopsis thaliana)體內共有7個成員，分別命名為Di19-1~7，具有兩個可能會與DNA結合的類似鋅手指序列（Cys2/His2 zinc finger-like domains）。AtDi19被認為可能參與植物逆境生理與光訊息傳遞，目前已證實AtDi19蛋白會被鈣離子依賴型蛋白激酶（CDPK）磷酸化，但功能未明。根據序列分析，發現AtDi19-1、AtDi19-2與AtDi19-5具有被預測與14-3-3蛋白結合的胺基酸序列。14-3-3蛋白是普遍存在真核細胞中的鷹架蛋白（scaffold protein），會與磷酸化蛋白結合，參與植物的生長發育與逆境生理的調控。我們提出假設，認為14-3-3蛋白與AtDi19蛋白之間可能有交互作用。以14-3-3 omicron與14-3-3 omega分別與AtDi19-1、2與5進行酵母菌雙雜合之測試，初步結果顯示14-3-3 omega和Di19-1、2與5之間有交互作用，進一步以AtDi19-1、2與5的非全長蛋白與14-3-3 omega進行酵母菌雙雜合實驗，證實AtDi19-2的第98-135個胺基酸（Arg98-His135）是AtDi19-2在酵母菌中與14-3-3 omega交互作用的重要區域。另外也藉由試管內結合反應，進一步研究14-3-3 omega與AtDi19-2的交互作用是否受到AtCDPK磷酸化的影響，實驗結果顯示AtDi19-2不論是否受到AtCDPK磷酸化皆不影響與14-3-3 omega的交互作用，此結果暗示AtCDPK並不參與AtDi19-2與14-3-3的結合。

The Arabidopsis Di19 （Dehydration-induced protein 19） gene family encodes seven hydrophilic proteins, AtDi19-1~7, which contain two atypical Cys2/His2 zinc finger-like domains. It’s known that AtDi19 proteins can be involved in stress response and light signal transduction. AtDi19 proteins can be phosphorylated by CDPKs, but the function is not clear. Based on the results of protein sequence analysis, we identified putative 14-3-3 proteins binding motif in AtDi19-1, AtDi19-2 and AtDi19-5. 14-3-3 proteins are phosphoserine-binding proteins in eukaryotic cell, and may play an important role in responses to mineral nutrient deficiencies, ion transport and salt stress. We suspected that AtDi19 proteins may interact with 14-3-3 proteins. To investigate whether AtDi19 proteins can interact with 14-3-3 proteins, a yeast two-hybrid study was conducted. The result indicates that 14-3-3 omega can interact with AtDi19-1, 2 & 5.We also discovered that AtDi19-2-Arg98-His135 is the nessary region involved in the interaction with 14-3-3 omega. In addition, we tested if AtCDPK is involved in the interaction between 14-3-3 omega and AtDi19-2 by in vitro binding assay. According our results, AtCDPK is not involved in the interaction between Di19-2 and 14-3-3 omega.