Williams J S, Andrisani O M
Department of Physiology and Pharmacology, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA.
Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3819-23. doi: 10.1073/pnas.92.9.3819.
The X gene product encoded by the hepatitis B virus, termed pX, is a promiscuous transactivator of a variety of viral and cellular genes under the control of diverse cis-acting elements. Although pX does not appear to directly bind DNA, pX-responsive elements include the NF-kappa B, AP-1, and CRE (cAMP response element) sites. Direct protein-protein interactions occur between viral pX and the CRE-binding transcription factors CREB and ATF. Here we examine the mechanism of the protein-protein interactions occurring between CREB and pX by using recombinant proteins and in vitro DNA-binding assays. We demonstrate that pX interacts with the basic region-leucine zipper domain of CREB but not with the DNA-binding domain of the yeast transactivator protein Gal4. The interaction between CREB and pX increases the affinity of CREB for the CRE site by an order of magnitude, although pX does not alter the rate of CREB dimerization. Methylation interference footprinting reveals differences between the CREB DNA and CREB-pX DNA complexes. These experiments demonstrate that pX titers the way CREB interacts with the CRE DNA and suggest that the basic, DNA-binding region of CREB is the target of pX. Transfection assays in PC12 cells with the CREB-dependent somatostatin promoter demonstrate a nearly 15-fold transcriptional induction after forskolin stimulation in the presence of pX. These results support the significance of the CREB-pX protein-protein interactions in vivo.
乙型肝炎病毒编码的X基因产物，称为pX，是一种在多种顺式作用元件控制下对多种病毒和细胞基因具有广泛作用的反式激活因子。尽管pX似乎不直接结合DNA，但pX反应元件包括核因子κB、激活蛋白-1和环磷酸腺苷反应元件（CRE）位点。病毒pX与CRE结合转录因子CREB和激活转录因子（ATF）之间存在直接的蛋白质-蛋白质相互作用。在此，我们通过使用重组蛋白和体外DNA结合试验来研究CREB与pX之间发生的蛋白质-蛋白质相互作用的机制。我们证明pX与CREB的碱性区域-亮氨酸拉链结构域相互作用，但不与酵母反式激活蛋白Gal4的DNA结合结构域相互作用。CREB与pX之间的相互作用使CREB对CRE位点的亲和力提高了一个数量级，尽管pX不会改变CREB二聚化的速率。甲基化干扰足迹法揭示了CREB-DNA复合物与CREB-pX-DNA复合物之间的差异。这些实验表明pX改变了CREB与CRE DNA相互作用的方式，并提示CREB的碱性DNA结合区域是pX的作用靶点。在PC12细胞中用依赖CREB的生长抑素启动子进行的转染试验表明，在存在pX的情况下，福斯高林刺激后转录诱导增加了近15倍。这些结果支持了CREB-pX蛋白质-蛋白质相互作用在体内的重要性。
