Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23874
標題: 轉錄共活化因子CBP之KIX domain 與轉錄因子CREB、MLL及SREBP結合位置之探討
作者: 劉雅萍
Liu, Ya-Ping
關鍵字: 轉錄因子
CREB
轉錄共活化因子
CBP
MLL
SREBP
出版社: 生物化學研究所
摘要: cAMP response element binding protein (CREB)為一轉錄因子,在訊息cAMP的刺激下會誘發含有cAMP response element(CRE)啟動子序列之基因的轉錄機制啟動。此種轉錄機制啟動仰賴CREB之kinase-induciable activation domain (KID)的Serine 133位置被protein kinase A (PKA)磷酸化,進而與CREB binding protein (CBP)之KIX domain產生交互作用,並使transcriptional machinery被聚集在基因上游的啟動子區域,最後導致下游基因的轉錄活化機制啟動。 已知CBP除了與CREB產生交互作用外,還發現也能與其他轉錄因子做結合,如: mixed-lineage leukemia (MLL)、sterol-response element-binding protein (SREBP)。經胺基酸序列比對發現,這些轉錄因子間並沒有同源性。於是為了解KIX要如何去辨識這些不同的轉錄因子,我們在CBP的KIX domain上設計了二十個不同位置胺基酸的定點突變,探討這些點突變是否影響了KIX與不同轉錄因子間的結合。 從GST pulldown實驗結果顯示,KIX上的某些胺基酸若被置換,會嚴重地破壞與Serine 133磷酸化之KID的結合。將這部分的結果對照已利用NMR方法分析出的KIX-pKID複合物結構,發現KIX上這些嚴重影響與pKID結合的胺基酸都是直接與pKID接觸並且參與了交互作用。因此我們認為,利用KIX的定點突變再結合GST pulldown的方法,可以幫助我們了解KIX與轉錄因子間的分子辨識。所以我們以相同方法進一步地分析KIX與MLL或與SREBP之間的分子辨識。結果發現,會影響與MLL或SREBP結合的KIX定點突變與影響Serine 133磷酸化KID結合的定點突變差異很大。因此我們推測,這種現象很可能是因為KIX以不同區域分別參與了pKID、MLL或SREBP的結合所導致。
cAMP response element binding protein (CREB) is a transcription factor capable of activating the cAMP response element (CRE) upstream of cAMP responsive genes. In this cAMP-dependent signaling pathway, cAMP activates protein kinase A (PKA) to phosphorylate the Ser-133 in the kinase-induciable activation domain (KID) of CREB and facilitate the binding of phorylated KID to the KIX domain of CREB binding protein (CBP) .Eventually, the components of basal transcriptional machinery will be assembled on the promoter regions of target gene for transcription activation. In addition to the interaction between CREB and the KIX domain of CBP, KIX can also interact with other transcription factors, such as mixed-lineage leukemia (MLL) and sterol-response element-binding protein (SREBP). Because there is no sequence similarity among pKID, MLL and SREBP, we are interested in studying how KIX interact with different transcription factors. In order to map the pKID binding site on KIX, I designed 20 site-directed mutagenesis on KIX and investigated the interaction between mutant KIX and pKID by GST pulldown. The results of GST pulldown experiment reveal that some KIX mutations can reduce the binding affinity between KIX and pKID. This data also agree with the NMR structure of KIX-pKID complex and suggesting these residues contact pKID directly. We also use GST pulldown assay to examine the binding of KIX to MLL and SREBP and to map the MLL and SREBP binding sites on KIX. The GST pulldown data also indicate that the binding site of MLL and SREBP on KIX might be different from that of pKID. This data imply that KIX interact with different proteins by using distinct binding surfaces.
URI: http://hdl.handle.net/11455/23874
Appears in Collections:生物化學研究所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.