呂勝春臺灣大學：分子醫學研究所孫惠鈺Sun, Huei-YuHuei-YuSun2007-11-262018-07-092007-11-262018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/51343第二型鹽誘導激酶（SIK2）為一種絲胺酸/蘇胺酸蛋白質激酶（Serine/threonine protein kinase），並屬於單磷酸腺苷蛋白質激酶（AMPK）族系的其中一個成員。過去的研究顯示，此蛋白質在成熟的脂肪細胞中大量表現並且在脂肪細胞分化過程中具有調控早期胰島素傳導的功能。迄今，除了IRS-1和TOC2外，對於SIK2其他生理上的受質仍未非常明暸。第一型胰島素受器受質（IRS-1）在脂肪組織的胰島素傳導中扮演重要的功能，並且被認為在第794位置的絲胺酸上會被SIK2磷酸化。而在CREB傳導途徑中具有調控功能的TORC2，則在第171位置的絲胺酸會被磷酸化。 在本研究中，我們發現了p97/VCP，一種參與在多樣細胞活動的三磷酸腺苷水解酶（AAA-ATPase），和SIK2在物理上和功能上具有交互作用。透過免疫螢光染色和蔗糖濃度梯度分層生化實驗，SIK2和p97/VCP被證明共同分佈在內質網膜（ER membrane）上。並且SIK2和p97/VCP的交互作用可調控p97/VCP所調解的內質網相關之蛋白質降解（ERAD）。座落於SIK2三磷酸腺苷嵌合區位（ATP-binding domain）上第53位置的賴胺酸（K53）可被進行乙醯化作用（Acetylation）。在我們之前的研究中指出K53的乙醯化作用可抑制SIK2的激酶活性，而此激酶活性對於由p97/VCP所調解的ERAD是需要的。本研究更進一步證明了以股氨酸取代第53位置賴胺酸的乙醯化擬態突變型（K53Q）會降低其激酶活性並導致ERAD受質CD3δ的累積增加。另外，我們還找到SIK2 K53的乙醯化作用和去乙醯化作用（Deacetylation）是由p300/CBP和HDAC6所負責調控的。綜合上述，我們的研究結果顯示SIK2的乙醯化作用減弱了其激酶活性並且進而抑制ERAD受質的降解。Salt-inducible kinase 2 (SIK2), a serine/threonine protein kinase, belongs to members of the AMP-activated protein kinase (AMPK) family. It is highly expressed in mature adipocytes and regulates the early phase of insulin-signaling during adipocyte differentiation. So far, the physiological substrates of SIK2 are not well-understood, except for IRS-1 and TORC2. Insulin receptor substrate-1 (IRS-1), which is reported to play important roles in the insulin-signaling in the adipose tissue, is thought to be a potential endogenous substrate of SIK2 at serine 794, whereas TOR2, participating in the regulation of CREB signaling, is phosphorylated at serine 171. In the present study, we have identified that p97/VCP, an AAA-ATPase involved in a variety of cellular processes, interacts with SIK2 physically and functionally. Moreover, SIK2 and p97/VCP are co-localized to the ER membrane. The interaction between SIK2 and p97/VCP modulates p97/VCP-dependent ER-associated degradation (ERAD). Lysine 53, a site located in the kinase ATP-binding domain of SIK2, is found to be acetylated. We have previously described that lysine 53 acetylation results in the inhibition of the kinase activity of SIK2. The acetylation mimetic mutant K53Q showed accumulation of the ERAD substrate CD3δ. We further identified that p300 /CBP and HDAC6 are responsible for the acetylation and deacetylation of lysine 53 of SIK2. In conclusion, our results demonstrate that acetylation of SIK2 negatively regulates its kinase activity, thus attenuates the degradation of the ERAD substrates.ACKNOWLEDGEMENT………………………………………………….........i ABBREVIATIONS……………………………………………………….........ii ABSTRACT (CHINESE) ……………………………………………………....iii ABSTRACT (ENGLISH) ……………………………………………………….iv INTRODUCTION………………………………………………………………1 MATERIALS AND METHODS………………………………….…………........8 Cell culture and transfection…………………………………………………………....8 Plasmid constructs………………………………………………………………………9 Antibodies……………………………………………………………………………....10 SDS-PAGE and Western blot analysis……………………………………………........11 In vitro pull down assay………………………………………………………………..13 Subcellular fractionation by differential centrifugation……………………………….13 Subcellular fractionation on sucrose gradient………………………………………....15 Immunofluorenscence staining and microscopy………………………………………..16 In vitro deacetylation assay…………………………………………………………….17 Protein acetylation and deacetylation in vivo………………………………………….18 ERAD assay…………………………………………………………………………….19 RESULTS…………………………………………………………………….20 Direct interaction between SIK2 and p97/VCP…...………………...………………… 20 Co-localization of SIK2 and p97/VCP to the ER membrane…………..………………20 Membrane-bound SIK2 is a peripheral membrane protein…………………………….23 The subcellular localization of SIK2 is not dependent on lysine 53 acetylation……….23 Lysine 53 acetylation of SIK2 regulates the stability of the ERAD substrate CD3δ…..25 Acetylation of SIK2 by p300/CBP in vivo……………………………………………....26 Lysine 53 is the predominant acetylation site of p300/CBP………..…….…………….27 SIK2 is deacetylated by HDAC6 in vitro and in vivo………………………………….28 p300/CBP-mediated acetylation of SIK2 inhibits ERAD………………………………29 DISCUSSION…………………………………………………………………31 REFERENCES………………………………………………………………...37 FIGURES……………………………………………………………………. 45 Figure 1. SIK2 interacts with p97 directly…………………………………………….45 Figure 2. Subcellular localization of SIK2 and p97…………………………………...46 Figure 3. SIK2 and p97 are co-localized to the ER membrane………………………..47 Figure 4. Membrane-bound SIK2 is a peripheral membrane protein………………....48 Figure 5. Both of cytosolic and membrane-bound SIK2 are acetylated……………….49 Figure 6. The subcellular localization of SIK2 K53 mutants…………………………..50 Figure 7. K53 acetylation of SIK2 regulates the degradation of ERAD substrat……...51 Figure 8. SIK2 is acetylated by p300/CBP……………………………………………..52 Figure 9. SIK2 K53 is the predominant acetylation site by p300……………………....53 Figure 10. SIK2 K53 is the predominant acetylation site by CBP……………………..54 Figure 11. SIK2 is deacetylated by HDAC6 in vitro…………………………………....55 Figure 12. p300-mediated SIK2 acetylation is deacetylated by HDAC6 in vivo……….56 Figure 13. CBP-mediated SIK2 acetylation is deacetylated by HDAC6 in vivo………. 57 Figure 14. p300/CBP-mediated SIK2 acetylation inhibits the degradation of ERAD substrate CD3δ……………………………………………………………...58 Figure 15. SIK2 K144 is not the specific acetylation site by p300……………………..59 Figure 16. Acetylation of SIK2 regulates its kinase activity and thus inhibits ERAD…..601569815 bytesapplication/pdfen-USSIK2p97/VCP乙醯化作用p300/CBPHDAC6ERADAcetylationotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51343/1/ntu-96-R94448002-1.pdf