Regulation of ER-associated Degradation by Akt and Biochemical Analysis of SIK2

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.Master Thesis……………………………………………………………………………i謝...................................................................................................................................ii中文摘要………………………………………………………………………..………iiiBSTRACT…….……...……………………………………………..………………..ivONTENTS……………………………………………………………………….……vNTRODUCTION……………………………………………………………….……. 1ATERIALS AND METHODS…….……………………………………...…………7lasmids and antibodies………………………….………...…………….……….7ite-directed mutagenesis…….……..……………………………….………...….8ell culture and transfection.………...……………………………………….….9iochemical fractionation…….……………………...…………………………...9mmunofluorescence staining…………………………………………………...10n vitro kinase assay……………………………………………………………...10rotein purification and in vitro binding assay………………………………...11ESULTS…...…………………….…………………………………………………...13kt negatively regulates ER-associated degradation.........................................13IK2 kinase activity is increased after Akt inactivation....................................14kt phosphorylates SIK2 at Ser358.....................................................................15 verexpression of mutated SIK2 (S358A) facilitates ER-associated degradation.............................................................................................................164-3-3 associates in SIK2’s complex and directly interacts with SIK2.............164-3-3 binds to Ser358 and Ser587 of SIK2.........................................................17omparison between subcellular localizations of wild-type, and mutated forms of SIK2.........................................................................................................18 IK2 kinase activity is slightly increased after 14-3-3 association....................18ISCUSSION………………………………………………………………………….20EFERENCES………………………………………………………………………..26IGURES.........................……………………………………………………………..33ig 1. Akt inactivation facilitates CD3δ degradation in 293T cells...................33ig 2. Overexpression of dominant negative Akt facilitates CD3δ degradation.………………………………………………………………………………….34 Fig 3. SIK2 kinase activity is increased when Akt is inhibited or over- expression of dominant negative Akt in 293T cells.............................................35ig 4. cells expressing wild-type Flag-SIK2 shows the accelerated degradation rate on CD3δ after Akt inhibitor treatment........................................................36ig 5. Akt phosphorylates SIK2 at Ser358 in vitro..............................................38ig 6. Comparison between the degradation rate of CD3δ in cells expressing ild-type SIK2 or mutated SIK2 (S358A)...........................................................39ig 7. Comparison between the kinase activity of wild-type SIK2 and mutated IK2 (S358A)..........................................................................................................40ig 8. SIK2 directly interacts with 14-3-3ε and 14-3-3ζ in vitro........................41ig 9. SIK2 co-localizes with 14-3-3 in 293T cells...............................................42ig 10. Mutations of Ser358 and Ser587 prevent SIK2 from interacting with 14-3-3.......................................................................................................................43ig 11. Subcellular localizations of wild-type and mutated forms of SIK2......44ig 12. Binding of 14-3-3 to SIK2 slightly enhances its kinase activity.……....45ig 13. SIK2 phosphorylates 14-3-3 in vitro........................................................4