- 學位論文
蛋白激酶SIK2的調控及其功能之研究
Regulation and Function of Protein Kinase SIK2
摘要
SIK2是屬於AMPK家族的絲胺酸/蘇胺酸蛋白激酶(serine/threonine protein kinase)。過去的研究顯示，SIK2在脂肪細胞分化過程中具有調控早期胰島素訊息傳遞的功能，並可透過CREB調節反應賀爾蒙或營養狀態的基因表現。然而，SIK2活性的調控機制尚待研究釐清。在本研究中，我們發現當SIK2在第53位置的賴胺酸(Lys53)被p300/CBP乙醯化，會導致SIK2激酶活性受到抑制；而HDAC6可將SIK2的Lys53去乙醯化，並恢復其活性。此外，過量表現以麩醯胺酸取代Lys53(K53Q)的乙醯化擬態突變型(acetylation-mimetic mutant)，會造成SIK2聚集在自噬體(autophagosome)內；反之，以精胺酸取代Lys53(K53R)的抗乙醯化突變型(non-acetylatable mutant)則不會造成此現象。再者，我觀察到SIK2基因剔除(knockdown)會癱瘓自噬體和溶酶體的融合，更證實了SIK2及其活性為清除TDP-43Δ包涵體(inclusion body)所必需。值得注意的是在MG132處理的細胞中，內生(endogenous)SIK2聚集在自噬體內，且伴隨著SIK2乙醯化的上升；然而，同樣的現象並沒有發生在無血清培養的細胞中(serum-starved cells)。此結果說明蛋白酶體功能障礙(proteasome dysfunction)可能引發SIK2的乙醯化，並藉此清除隨之而生的蛋白質堆積。另一方面，我發現內質網蛋白質降解路徑的受質(ERAD substrate)由內質網到細胞質的逆向轉傳(retrotranslocation)，依然需要SIK2的激酶活性才能完成。不只SIK2，另一個SIK蛋白家族成員SIK3也同樣可被CBP和HDAC6所控制的乙醯化修飾。除了SIK家族的激酶，我更進一步地觀察到MARK1的活性如同SIK2一般，受控於由CBP和HDAC6所調節的乙醯化修飾。此現象暗示著MARK1在神經退化疾病中可能也受到類似的機制所調控。總結上述，本研究證明SIK2為調控內質網蛋白質降解路徑和自噬作用的關鍵因子，並可透過激酶與去乙醯酶(deacetylase)的交互作用共同調節細胞內蛋白質的平衡。
並列摘要
Salt-inducible kinase 2 (SIK2) is a serine/threonine protein kinase belonging to the AMP-activated protein kinase (AMPK) family. SIK2 has been shown to function in the insulin-signaling pathway during adipocyte differentiation and to modulate CREB-mediated gene expression in response to hormones and nutrients. However, molecular mechanism underlying the regulation of SIK2 kinase activity remains largely elusive. In this study, I report a dynamic, post-translational regulation of its kinase activity that is coordinated by an acetylation-deaceytlation switch – p300/CBP-mediated Lys53-acetylation inhibits SIK2 kinase activity, while HDAC6-mediated deacetylation restores the activity. Interestingly, overexpression of acetylation-mimetic mutant of SIK2 (SIK2-K53Q), but not the non-acetylatable K53R variant, resulted in sequestration of SIK2 in autophagosomes. Further consistent with a role in autophagy, knockdown of SIK2 abrogated autophagosome and lysosome fusion. Consequently, SIK2 and its kinase activity are indispensable for the removal of TDP-43Δ inclusion bodies. Remarkably, the accumulation of endogenous SIK2 in autophagosomes and the corresponding elevation of its acetylation were also observed in cells treated with MG132, yet not in serum-starved cells, revealing that this acetylation-based regulation may be induced by proteasome dysfunction and required for disposal of the resultant protein aggregates. Moreover, the requirement of SIK2 activity for retrotranslocation of ERAD substrate from ER to cytosol was evident. On the other hand, another SIK subfamily member, SIK3, was also modified reciprocally by CBP and HDAC6. In addition to SIK subfamily, the activity of MARK1 was under the control of CBP and HDAC6-mediated acetylation, implying its possible regulation in neurodegeneration diseases. Collectively, our findings uncover the critical roles of SIK2 in ERAD as well as autophagy progression and further suggest a mechanism in which the interplay among kinase and deacetylase activities coordinates cellular protein homeostasis.
參考文獻
延伸閱讀
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