Amelio Antonio L, Miraglia Loren J, Conkright Juliana J, Mercer Becky A, Batalov Serge, Cavett Valerie, Orth Anthony P, Busby Jennifer, Hogenesch John B, Conkright Michael D
Departments of Cancer Biology and Molecular Therapeutics and Translational Research Institute, The Scripps Research Institute, 5353 Parkside Drive, Jupiter, FL 33458, USA.
Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20314-9. doi: 10.1073/pnas.0707999105. Epub 2007 Dec 11.
Signal transduction pathways often use a transcriptional component to mediate adaptive cellular responses. Coactivator proteins function prominently in these pathways as the conduit to the basic transcriptional machinery. Here we present a high-throughput cell-based screening strategy, termed the "coactivator trap," to study the functional interactions of coactivators with transcription factors. We applied this strategy to the cAMP signaling pathway, which utilizes two families of coactivators, the cAMP response element binding protein (CREB) binding protein (CBP)/p300 family and the recently identified transducers of regulated CREB activity family (TORCs1-3). In addition to identifying numerous known interactions of these coactivators, this analysis identified NONO (p54(nrb)) as a TORC-interacting protein. RNA interference experiments demonstrate that NONO is necessary for cAMP-dependent activation of CREB target genes in vivo. Furthermore, TORC2 and NONO complex on cAMP-responsive promoters, and NONO acts as a bridge between the CREB/TORC complex and RNA polymerase II. These data demonstrate the utility of the coactivator trap by identification of a component of cAMP-mediated transcription.
信号转导通路常常利用转录成分来介导适应性细胞反应。共激活蛋白在这些通路中起着重要作用，作为通向基本转录机制的管道。在此，我们提出一种基于细胞的高通量筛选策略，称为“共激活因子捕获”，以研究共激活因子与转录因子之间的功能相互作用。我们将此策略应用于cAMP信号通路，该通路利用两类共激活因子，即cAMP反应元件结合蛋白（CREB）结合蛋白（CBP）/p300家族以及最近发现的受调控的CREB活性转导因子家族（TORCs1 - 3）。除了鉴定这些共激活因子的众多已知相互作用外，该分析还鉴定出NONO（p54(nrb)）为一种与TORC相互作用的蛋白。RNA干扰实验表明，NONO在体内对cAMP依赖的CREB靶基因激活是必需的。此外，TORC2和NONO在cAMP反应性启动子上形成复合物，并且NONO充当CREB/TORC复合物与RNA聚合酶II之间的桥梁。这些数据通过鉴定cAMP介导转录的一个成分证明了共激活因子捕获的实用性。
