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標題: 轉錄活化作用之非結構性蛋白的分子辨識
Study on protein-protein rcognition of unstructured protein in transcription activation.
作者: 張清文
Chang, Ching-Wen
關鍵字: 轉錄共活化因子
出版社: 生物化學研究所
CBP (CREB binding protein)為一般的轉錄共活化因子,目前已知可與不同的轉錄因子作用,例如CREB (cAMP response element binding protein) 和SREBP (sterol-response element binding protein),並調控其特定基因(target gene)的表現。而CREB為一個可以被protein kinase A磷酸化進而活化帶有cAMP response element基因表現的轉錄因子。CREB包含有一個kinase-inducible activation domain (KID),其蛋白上之Ser 133會被為cAMP活化之protein kinase A所磷酸化。CREB只有在被磷酸化之後才可與轉錄共活化因子CBP (CREB binding protein)上的KIX domain作用來促進特定基因的表現。反之,SREBP在缺乏PKA的磷酸化下仍可與CBP的KIX domain作用。有趣的是將CREB的130-135區域置換為SREBP 28-33區域的六個胺基酸後(CREBDIEDML),竟能夠在缺乏PKA的情形之下活化CREB所調控的基因,且與CBP的結合能力比正常的Ser-133磷酸化CREB還要強(Jean-Rene Cardinaux, et al.,2000)。為了研究造成上述現象的原因我們將CREB之KID domain (130-135) 胺基酸序列置換為KIDDIEDML。同時我們也選殖、純化出mixed-lineage leukemia (MLL) 2829-2861區域蛋白(此區域已知會與CBP的KIX domain作用,並增進CREB-CBP複合物的穩定性) 來探討CREB之KID domain、SREBP1-29、MLL2829-2861和突變的KIDDIEDML(101-160)等四者與CBP的KIX domain的作用之異同,並藉此瞭解不同的轉錄因子對於轉錄共活化因子之間的分子辨識。

CBP is a general transcription co-activator that can interact with different transcription activators, such as CREB (cAMP response element binding protein) and SREBP (sterol-response element binding protein) to regulate the expression of specific genes. CREB contains a kinase-inducible domain (KID) that can be phosphorylated at its Ser 133 by cAMP-activated PKA (protein kinase A) to stimulate the expression of gene with upstream CRE (cyclic AMP response element) through interaction of the KIX domain of CBP. Interaction of CREB with CBP is mediated via the Ser 133 phosphorylated KID of CREB and the KIX domain of CBP. By contrast, SREBP interacts with the KIX domain of CBP in the absence of PKA phosphorylation. Interestingly, when a six residues stretch of CREB is replaced with the corresponding region in SREBP (residues 28-33) to generate a chimeric CREB (CREBDIEDML), it is found that CREBDIEDML has a higher affinity for KIX than that of phosphory-lated-Ser 133 CREB and can stimulate the expression of target gene in the absence of PKA. In order to study the difference in the recognition of KIX-PKID and KIX-SREBP, we construct a chimeric KIDDIEDML protein to examine its ability for KIX binding. Moreover, we have also cloned and purified a MLL (mixed-lineage leukemia) domain residues 2829-2861 gene product that can interact with KIX domain in a CREB-CBP complex and stabilizes it to investigate the protein-protein interaction among the pKID101-160—KIX, KIDDIEDML(101-160)—KIX, SREBP1-29-KIX and MLL2829-2861-KIX.
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