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標題: | 蛋白激酶SIK2和蛋白磷酸水解酶PP2A和PP1的交互調控 Functional interaction between SIK2 and PP2A or PP1 phosphatase |
作者: | Hsin-Yun Chang 張馨勻 |
指導教授: | 呂勝春(Sheng-Chung Lee) |
關鍵字: | SIK2,PP2A,PP1,p97/VCP,RVxF motif,MEF2C調控轉錄機制, SIK2,PP2A,PP1,p97/VCP,RVxF motif,MEF2C-mediated transcription, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 蛋白激酶SIK2屬於AMPK家族中的一員。目前已知SIK2在胰島素和葡萄醣代謝扮演重要角色,但其餘相關作用都還未知。實驗室目前已知SIK2的激酶活性會受到p300/CBP的乙醯化和HDAC的去乙醯化所調控。為研究SIK2之功能,從其交互作用的蛋白質著手是一常用的研究法;SIK2是AMPK家族中唯一會和p97/VCP和蛋白磷酸水解酶PP2A有交互作用的成員。另外,SIK2和p97/VCP的交互作用會促進內質網蛋白質降解。本研究主要針對SIK2和PP2A的功能及交互作用探討,同時也會討論SIK2和PP1的交互作用。我的主要目標是研究調控SIK2-PP2A複合體的形成的相關機制。SIK2只會和完整三個次單元組成之具活性之PP2A結合且PP2A依然保有磷酸水解酶的活性。由於SIK2上的羧丁胺酸Thr175受到磷酸化也顯示在SIK2-PP2A複合體中的SIK2可能保有活性。此外,我觀察到當利用高濃度的okadaic acid (OA)處理的HEK293T細胞,在蛋白質電泳當中,SIK2會出現泳動遲至的現象。而磷酸化的SIK2/T175在高濃度OA (大於0.3 μΜ)處理下可以被偵測到。這個結果說明PP1可能會影響SIK2相關的磷酸化機制。實驗結果發現SIK2和其結合的p97/VCP都會與PP1有交互作用。PP1與SIK2蛋白質的結合點位在RVGF (胺基酸位置16-19), 但與p97/VCP產生交互作用結合點尙待鑑定。最後,我也證明SIK2可能參與在MEF2C所調控的基因表現。 Salt-inducible kinase 2 (SIK2) is a member of AMPK family. Except for its roles in insulin signaling and glucose metabolism, the functions of SIK2 remain largely unknown. Our laboratory has demonstrated that the SIK2 kinase activity may be regulated by p300/CBP-mediated acetylation and HDAC6-induced deacetylation. SIK2 is the only member of the AMPK family capable of interacting with p97/VCP and protein phosphatase 2A (PP2A). Furthermore, interaction between SIK2 and p97/VCP was shown to facilitate ER-associated protein degradation (ERAD). In this thesis, I present results of physical and functional interactions between SIK2 and PP2A as well as protein phosphatase 1 (PP1). My major aim is to study the physiological cues responsible for regulation of SIK2 and PP2A complex formation. One of the major findings was that SIK2 interacts with PP2A holoenzyme. The elevated phosphorylation of SIK2 at Thr175 in SIK2-PP2A complex suggested that SIK2 activity was preserved in the complex. The kinase activity of SIK2-PP2A complex was further demonstrated by phosphorylation of a GST-syntide-2 substrate. In addition to PP2A, I have observed that when HEK293T cells were treated with high, but not low, concentration of okadaic acid (OA, ~0.3 μΜ vs. 0.1 μΜ), the mobility of SIK2 in SDS gel was retarded. The phosphorylated SIK2/T175 level of OA-treated sample is higher than that of control. These results suggest that PP1 inactivation may contribute to the mobility shift and hyper-phosphorylation of SIK2. Further experiments have uncovered that SIK2 and its associated p97/VCP protein both interact with PP1. The docking site of PP1 essential for its binding to SIK2 has been identified in the RVGF (amino acid 16-19) region while the docking site(s) in p97/VCP remains to be determined. Finally, I have demonstrated that SIK2 plays regulatory functions in MEF2C-mediated gene expression. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5907 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 分子醫學研究所 |
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