Kwast-Welfeld J, Walker P R, Whitfield J F, Sikorska M
Institute for Biological Sciences, National Research Council, Ottawa, Canada.
Cell Mol Biol Res. 1993;39(3):231-42.
We analyzed the ability of cyclic AMP-response element binding proteins (CREBs) to interact with the CRE sequences derived from different genes and examined the role of sequences flanking the core CRE element in rendering cAMP-responsiveness to the enhancer. We were able to detect reproducibly, sing the Southwestern blotting technique, five major CREB factors of molecular weights 56, 47, 40, and 36-34 kDa which were present in various rat tissues and cultured cells. The 34-40 kDa proteins (CREB-327/341) were able to bind to the CRE of cAMP-inducible genes (somatostatin, c-fos, E2A), but not to genes whose expression is not controlled by cAMP (glucagon, parathyroid hormone). The novel 47 kDa CREB had a high specificity for the core octameric CRE sequence and it bound equally well to the consensus CRE of cAMP-inducible and noninducible genes. On the other hand, the 47 kDa CREB did not bind at all to the phorbol ester response element (TRE), whereas the 56 kDa protein, reminiscent of the CRE-BP1 protein, could bind to both elements. A computer aided sequence analysis of cAMP-inducible gene promoters revealed the presence of an additional conserved element starting 4-6 nucleotides 3' to the octomer with the consensus C/GAGA/C. We have shown this element to be essential for maximal cAMP-responsiveness of the enhancer in transient expression assays of CRE-CAT plasmid constructs indicating that the functional interaction of CREB proteins with the cAMP-inducible enhancer involves an additional 8-10 base pairs immediately downstream from the CRE core element.
我们分析了环磷酸腺苷反应元件结合蛋白（CREBs）与源自不同基因的CRE序列相互作用的能力，并研究了核心CRE元件侧翼序列在赋予增强子cAMP反应性方面的作用。我们能够通过蛋白质印迹技术可重复地检测到分子量为56、47、40和36 - 34 kDa的五种主要CREB因子，它们存在于各种大鼠组织和培养细胞中。34 - 40 kDa的蛋白质（CREB - 327/341）能够结合到cAMP诱导型基因（生长抑素、c - fos、E2A）的CRE上，但不能结合到其表达不受cAMP控制的基因（胰高血糖素、甲状旁腺激素）的CRE上。新发现的47 kDa CREB对核心八聚体CRE序列具有高度特异性，并且它与cAMP诱导型和非诱导型基因的共有CRE结合得同样好。另一方面，47 kDa CREB根本不与佛波酯反应元件（TRE）结合，而56 kDa的蛋白质类似于CRE - BP1蛋白，能够结合这两种元件。对cAMP诱导型基因启动子的计算机辅助序列分析揭示了在八聚体3'端4 - 6个核苷酸处存在一个额外的保守元件，其共有序列为C/GAGA/C。我们已经表明，在CRE - CAT质粒构建体的瞬时表达测定中，该元件对于增强子的最大cAMP反应性至关重要，这表明CREB蛋白与cAMP诱导型增强子的功能相互作用涉及CRE核心元件下游紧邻的另外8 - 10个碱基对。
