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研究生:黃貞琪
研究生(外文):Jen-Chi Huang
論文名稱:磷酸化p62/SQSTM1對其功能的影響
論文名稱(外文):Functional analysis of phosphorylated SQSTM1/p62
指導教授:呂勝春
指導教授(外文):Sheng-Chung Lee
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:42
中文關鍵詞:磷酸化
外文關鍵詞:phosphorylated SQSTM1/p62
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p62/SQSTM1是一個可與泛素結合的鷹架蛋白(scaffold protein),可參與許多訊息傳遞途徑。它可藉由IL-1或是TNF-α的活化以調控NF-κB的表現,也可經由泛素鏈結(ubiquitinantion),調控訊息傳遞。p62是一個高度磷酸化蛋白質,且至少有18個氨基酸已被鑑定出有磷酸化。但是造成這些後修飾作用的生理意義、訊息傳遞路徑,以及對於p62的功能有何影響,皆還不清楚。p62可藉由PB1、ZZ、TB、LIR及UBA區域作為一個接引蛋白(adaptor protein),而有些已被鑑定出的磷酸化氨基酸坐落在這些特殊區域裡,所以這些磷酸化氨基酸對於p62的接引功能可能有所影響。在這篇論文中,我們發現到S24這個氨基酸是受到PKA所調控,且這個氨基酸的磷酸化會調控p62與PKCζ的結合。而且我們也發現S24、S272、S332、S355及S366會影響LC3B的脂化作用(lipidation)和自我吞食機制(autophagy)。且從脂化作用結果,可知S272是作為一個負向的調控因子,而S355則相反為一個正向調控因子。而在功能上的研究,在經過RNAi降低內生(endogenous)的p62,再補回不被RNAi影響的野生型(WT)及突變型(mutant)的p62,可發現S24的磷酸化在MEK5-ERK5-Mef2C 訊息傳遞路徑上扮演一個正向調控的角色。

p62/SQSTM1 is a ubiquitin-binding, scaffold protein which involves in diverse signaling pathways. It may regulate NF-κB activation by IL-1, TNF-αand signaling cascades through ubiquitination. It is a highly phosphorylated protein with at least eighteen phosphorylation sites has been identified. The physiological cues and the signaling pathways leading to these posttranslational modifications and the functional consequences remain poorly understood. p62/SQSTM1 serves as an adapter protein through PB1, ZZ, TB, LIR and UBA domains. Some of the identified phosphorylation sites fall into or in the vicinity of these domains. It is likely that some of these phosphorylations may have important consequence for the adapter functions of p62. In this study, we have found that S24 is a target of PKA. Phosphorylation of p62/S24 could regulate its interaction with PKCζ. Further, phosphorylations at S24, S272, S332, S355 and S366 can affect the lipidation of LC3B and autophagy. And phosphorylation of S272 serves as a negative while S355 as a positive regulatory functions. Functional studies by RNAi knockdown of endogenous and complemented with RNAi-resistant wild-type or mutant p62 showed that S24 phosphorylation is a positive regulator in MEK5-ERK5-Mef2C signaling pathway.



目錄
誌謝 I
摘要 II
Abstract III
Introduction 1
Materials and Methods 4
Plasmid constructs 4
Cell culture and transfeciton 6
Autophagy induction 7
Preparation of Whole Cell Extraction 7
Luciferase assay 7
Western blot analysis 8
Antibodies 10
Immunoprecipitation 10
RNA interference 11
Immunofluorescence 11
Results 13
Antibodies to p62/SQSTM1 13
Identification of the kinases involved in the phosphorylation of p62 13
Phosphorylation mimetics of p62/S24A, 24D, S272A, S272DS332A and S332D display lipidation defects of LC3B 14
The effect of p62/S24 phosphorylation on its interaction with PKCζ or NBR1 15
Regulation of MEK5-ERK5-Mef2C signaling by p62 16
Discussion 17
References 21
List of figures 27
Figure 1. Structure of p62/SQSTM1 and phosphorylation sites on its structure. 27
Figure 2.The specificityof p62/SQSTM1 antibodies. 28
Figure 3. Upstream kinase(s) that phosphorylates p62. 29
Figure 4. Phosphorylation mutants of p62/SQSTM1 display lipidation defects of LC3B. 32
Figure 5. Heterodimerization of p62 and S24 mutants with PKCzeta and NBR1. 33
Figure 6. The effect of p62 on MEK5-ERK5-Mef2c signaling. 34
Figure 7. The ubiquitination of transfected wild-type p62, S24A, and S24D. 35
Figure 8. Wild-type p62/SQSTM1 forms aggregates when overexpressed. 36
Figure 9. MG132 treatment has no effect on the turnover of LC3B. 37
Figure 10. Three subtypes of PB1 domain, type A (NBR1 or MEK5), type B (MEKK2 or MEKK3), and mixed type (i e., type A/B, p62 and aPKC). 38
Figure 11. Polyubiquitination of transfected p62/wild-type, S24A, and S24D. 39
Figure 12. The phosphorylation pattern of endogenous p62 in HEK 293T cells in the absence of presence of autophagy induction. 40
List of tables 41
Table 1. Phosphorylation sites and their localizations to p62 domains. 41
Table 2. Mutants of p62, their localizations and functions. 42



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