Real‐time DNA binding measurements of the ETSl recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms
Real‐time DNA binding measurements of the ETSl recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms
复制标题
ETS1重组癌蛋白的实时DNA结合测量揭示了p42和p51亚型之间的显着动力学差异
作者：
R. Fisher;M. Fivash;J. Casas;J. Erickson;A. Kondoh;S. Bladen;Constance Fisher;D. Watson;T. Papas
我们定量检测了重组p42和p51 ETSl癌蛋白的序列特异性DNA结合，以确定p42 ETSl中外显子VII磷酸化结构域的缺失或p51 ETSl中表达的外显子VII磷酸化是否对DNA结合活性有影响。使用BIAcore（注册商标，Pharmacia Biosensor）技术，利用表面等离子体共振的实时变化来测量序列特异性DNA结合的动力学。p42和p51 ETSl的实时结合表现出显著的动力学行为差异。p51 ETSl具有快速的初始结合和转化为稳定复合物的特征，而p42 ETSl具有缓慢的初始结合和转化为稳定复合物的特征。所有的p51 ETSl DNA结合状态都具有快速转换的特征，而p42 ETSl DNA结合状态的稳定性是其4 - 20倍。提出了一个描述这些动力学步骤的模型。具有特定寡核苷酸的p42或p51 ETSl的化学计量滴定显示1:1的络合物形成。突变分析表明，p42和p51 ETSl的DNA序列特异性相似。CAM激酶II对p51 ETSl的体外磷酸化消除了其与特定DNA的结合，这表明p51 ETSl序列特异性DNA结合的调节是通过钙依赖性第二信使的磷酸化发生的。p42 ETSl缺乏这种调控结构域（外显子VII），并且与其特定的DNA序列结合对钙信号传导不敏感。
The sequence‐specific DNA binding of recombinant p42 and p51 ETSl oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETSl or the phosphorylation of expressed Exon VII in p51 ETSl had an effect on DNA binding activity. The kinetics of sequence‐specific DNA binding was measured using real‐time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real‐time binding of p42 and p51 ETSl displayed significant differences in kinetic behavior. p51 ETSl is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETSl exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETSl DNA binding states are characterized by rapid turnover, whereas the p42 ETSl DNA binding states are 4‐20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETSl with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETSl as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETSl by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETSl sequence‐specific DNA binding occurs through phosphorylation by a calcium‐dependent second messenger. The p42 ETSl lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.
登录
查看更多内容
用于独立于模型确定蛋白质-DNA 相互作用平衡结合等温线的热力学方法：监测结合的光谱方法。
影响因子：
--
作者：
Lohman,TM;Bujalowski,W
通讯作者：
Bujalowski,W
影响因子：
56.9
作者：
PONGUBALA, JMR;VANBEVEREN, C;ATCHISON, ML
通讯作者：
ATCHISON, ML
ETS 结构域蛋白对淋巴特异性免疫球蛋白 mu 重链基因增强子的调节。
DOI：
10.1126/science.8316859
发表时间：
1993
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Nelsen,B;Tian,G;Erman,B;Gregoire,J;Maki,R;Graves,B;Sen,R
通讯作者：
Sen,R
影响因子：
10.5
作者：
NYE, JA;PETERSEN, JM;GRAVES, BJ
通讯作者：
GRAVES, BJ