2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643390摘要：SIK2是唯ㄧ之AMPK family 成員中可與 p97/VCP 以及 PP2A產生蛋白質複合體 的蛋白激酶.延續 SIK2, p97/VCP 以及 PP2A 交互作用 對 ER stress response 和 autophagy 調控的研究. SIK2 在 ubiquitin proteasome system (UPS) 和 autophagy 二個系統具互補調和者(compensatory coordinator) 之 角色, 但SIK2必須和p97/VCP 以2A共同作用.及PPSIK2在何種條件下可被活化 (經由 T175 的磷酸化) 和 抑制已在今年之研究中釐清: 它在 ER stress 狀態下 受到 JNK的磷酸化繼而產生 autophosphorylation at T175. SIK2 活性之抑制則經由K53被 p300/CBP乙醯化 (acetylation), 其再活化則經HDAC6去乙醯化 (deacetylation)達成. 此外, 過度表現SIK2 kinase dead mutants, SIK2‐K49M 和 SIK2‐K53Q 均可造成 aggresomes之產生, 而SIK2‐WT則否! SIK2 用siRNA knockdown 會造成細胞在ER stress時之凋亡. 處以MG132, ER lumen misfolded or aggregated protein 無法外送到ER外, autophagy也被抑制! 因此, SIK2在 ERAD 和 hagy 均具關autop鍵功能. SIK2 和其他九個AMPK family 成員 (SIK1, SIK3, BRSK1, BRSK2, MARK1, MARK2, MARK3, MARK4, MELK)含UBA domain. 在所有之 kinome中, 就這十個 kinases 含 UBA domain. 從我的結果 (SIK2‐K49M and SIK2‐K53Q 均可造成 aggresomes之產生)ad!看來, SIK2 有aptor之功能PP2A在UPS 和 autophagy 均不可或缺; 它由A, B, 和 C subunits 組 成之heterotrimers, 其catalytic activity 調控除已知之B 和 A subunits 外, catalytic C subunit 之C‐terminal Leucine 可被 LCMT‐1作methylation. Methylated 之PP2A C subunit 活性較好. PME‐1 是catalytic C subunit 之 demethylase, Demethylated PP2A 和 PME‐1PME‐1 不存在SIK2VC白質複合體內.之結合強！我們發現‐PP2A‐p97/P蛋我將針對 SIK2‐PP2A‐p97/VCP蛋白質複合體如何在autophagy 和UPS之調控作生化和 細胞生物方面之研究! 與此有關之HDAC6, CaMK1, p62/SQSTM1, Bcl‐2 and HSP90 也會深入探討!預期成果本計畫在 autophagy 和 ERAD 之基礎研究 以及Life style diseases 和 neurodegenerative disease之探討十分重要!<br> Abstract: SIK2 belongs to the AMPK protein kinase family, SIK subfamily. SIK2 and nine other members of the AMPK family (i.e., SIK1, SIK3, MARK1, MARK2, MARK3, MARK4, MELK1, BRSK1 and BRSK2) are the only kinases contain UBA domain in the entire kinome (Fig. 1). The importance and functions of these UBA domains remain completely unclear. When UBA domain is deleted, SIK2 kinase activity becomes constitutively activated. This result may suggest that UBA domain is part of the intramolecular autoinhibitory region or when UBA domain binds to polyubiquitin the autoinhibition is relieved. SIK2 is the only AMPK family member that can associate with PP2A and p97/VCP. The mechanism underlying this tripartite complex formation is likely to involve the hitherto unknown adaptor function of SIK2, i.e., recruits PP2A and p97/VCP for complex formation. The key question is whether UBA domain of SIK2 may bind to polyubiquitinated proteins. The UBA domain of SIK2 may bind to polyubiquitinated proteins thereby sequestering them to the PP2A and p97/VCP complex‐containing autophagosome or p97/VCP‐mediated ERAD complex for degradation. Indeed, our results suggest that SIK2 could serve as adaptor for sequestering proteins into p97/VCP‐mediated aggresome formation under stress conditions. Clearly, understand how SIK2 is activated and inactivated is key to unravel its roles in compensatory intersection of UPS and autophagy (Fig. 2). SIK2 is activated by autoactivation after JNK‐mediatedphosphorylation of S545/550 and S670 during ER stress. Our initial observation on the aggresome formation of overexpressed recombinant SIK2‐KD, but not SIK2‐WT, suggests that inactive SIK2 may facilitate aggresome formation. Active SIK2 may facilitate removal of aggresome. The key question is what physiological signal(s) could attenuate the SIK2 kinase activity transiently and facilitate aggresome formation. We have discovered that SIK2 kinase activity can be down regulated by acetylation at K53 by p300/CBP. K53 of SIK2 is located in the ATP‐binding pocket. Its acetylation results in decreased ATP binding, similar to the K49M kinase‐dead mutant. When acetylated at K53, SIK2 facilitates the aggresome formation! SIK2‐K53Ac may be deacetylated by HDAC6 and reactivated its kinase activity, thus promotes the maturation of autophagosomes. HDAC6 is a key player for aggresome formation and autophagy. Autophagy acts as a compensatory degradation system when the UPS is impaired in Drosophila melanogaster, HDAC6 is an essential mechanistic link in this compensatory interaction.We have demonstrated in cancer cells that SIK2 is important for both protein degradation by both UPS and autophagy. The interaction between SIK2, P97/VCP and PP2A coordinates autophagy when the function of proteasome is impaired. The phosphatase activity of PP2A in the complex is crucial for regulating the level of JNK-phosphorylated BCL-2 and activated AKT. The activity of PP2A is regulated at the heterotrimer formation in which regulatory B subunit plays crucial roles. The catalytic subunit of PP2A is also activated by carboxymethylation at its C-terminal Leucine residue by LCMT1. PME-1 is a PP2A catalytic (C) subunit-specific carboxymethylesterase. When PME-1 is complexed with C subunit, PP2A is inactivated (Fig. 3).The specific aims of this proposal are (1) To address whether SIK2 can serve as an adaptor for assembly of SIK2-PP2A-p97/VCP complex for facilitating ERAD and autophagy, (2) To investigate the important regulators of autophagy or UPS targeted by SIK2 and PP2A in the context of SIK2-PP2A-p97/VCP complex.