Akt對內質網蛋白質降解路徑的調控以及SIK2之生化性質分析

Abstract

本論文主要探討蛋白質激酶B (Akt/PKB) 在調控內質網蛋白質降解路徑中所扮演之角色，以及鹽誘導激酶2 (SIK2) 相關分子性質。本實驗室近年研究成果顯示，SIK2 會促進內質網蛋白質降解。論文中實驗結果發現，Akt 會負向調控內質網蛋白質降解。藉由基因轉殖後細胞中 Akt 蛋白之顯性抑制作用，以及給予細胞Akt抑制劑處理，發現由內質網蛋白降解路徑而降解 CD3δ 蛋白速率增加，並伴隨 SIK2 激酶活性提升。活體外實驗發現，Akt 能磷酸化 SIK2 上 358 位置的絲胺酸，並伴隨 Akt 與 SIK2 一同免疫沈澱。將 SIK2 上 358 位置的絲胺酸突變為丙胺酸，SIK2 激酶活性顯著提升，細胞內 CD3δ 具更高降解效率。以上證據提供一個可能之假設: Akt 藉由磷酸化 SIK2 降低其激酶活性，達成內質網蛋白質降解路徑之負向調控。另一方面，SIK2 與 14-3-3 具交互作用，將SIK2 上的兩個絲胺酸 (358 與 587 號位置胺基酸) 個別或共同突變成丙胺酸，與 14-3-3 結合能力降低，結合後 SIK2 之激酶活性些微提昇，本論文並發現，14-3-3在活體外實驗中為 SIK2 之基質。

The purpose of this study is to investigate the regulatory role for Akt in ER-associated degradation, and other molecular characters of SIK2. SIK2 has been demonstrated to promote ER-associated degradation (ERAD) in our lab. Here we found that Akt negatively regulates ERAD. Treatment with Akt inhibitor VIII or overexpression of dominant negative Akt resulted in the accelerated degradation rate on CD3δ, a known substrate for ERAD, and enhanced SIK2 kinase activity in 293T cells. SIK2 was phosphorylated by Akt at Ser358 in vitro, and was associated with Akt in cells. The mutated SIK2 (S358A) comprised stronger kinase activity, and faster degradation rate on CD3δ was observed in cells expressing mutated SIK2 (S358A). These results suggest that Akt negatively regulates ERAD via phosphorylating SIK2 to repress its kinase activity. In addition, we found SIK2 physically interacts with 14-3-3. Mutations of Ser358 and Ser587 to Ala resulted in the decreased association of SIK2 with 14-3-3. We also found 14-3-3 is an in vitro substrate of SIK2, and binding of 14-3-3 to SIK2 slightly enhanced its kinase activity.