Mayr B M, Canettieri G, Montminy M R
Peptide Biology Laboratories, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10936-41. doi: 10.1073/pnas.191152098. Epub 2001 Sep 4.
Ser-133 phosphorylation of the cAMP-responsive element-binding protein (CREB) is sufficient to induce cellular gene expression in response to cAMP, but additional promoter-bound factors are required for target gene activation by CREB in response to mitogen/stress signals. To compare the relative effects of different signals on recruitment of the coactivator CREB-binding protein (CBP) to CREB in living cells, we developed a fluorescence resonance energy transfer (FRET) assay. cAMP promoted the interaction of CREB with CBP in a phosphorylation-dependent manner by FRET analysis, but mitogen/stress signals were far less effective in stimulating complex formation even though they induced comparable levels of Ser-133 phosphorylation. cAMP and non-cAMP stimuli were comparably active in promoting this interaction in the cytosol; the formation of CREB x CBP complexes in response to non-cAMP signals was specifically inhibited in the nucleus. Non-cAMP signals had no effect on intrinsic CREB- or CBP-binding activities by Far Western blot assay, thereby supporting the presence of a distinct CREB x CBP antagonist. Our studies indicate that the relative effects of cAMP and mitogen/stress signals on CREB x CBP complex formation impart selectivity to gene activation through CREB phosphorylated at Ser-133.
环磷酸腺苷反应元件结合蛋白（CREB）的133位丝氨酸磷酸化足以诱导细胞基因表达以响应环磷酸腺苷，但在有丝分裂原/应激信号作用下，CREB激活靶基因还需要其他启动子结合因子。为了比较不同信号对活细胞中共激活因子CREB结合蛋白（CBP）与CREB募集的相对影响，我们开发了一种荧光共振能量转移（FRET）分析方法。通过FRET分析，环磷酸腺苷以磷酸化依赖的方式促进CREB与CBP的相互作用，但有丝分裂原/应激信号在刺激复合物形成方面的效果要差得多，尽管它们诱导了相当水平的133位丝氨酸磷酸化。环磷酸腺苷和非环磷酸腺苷刺激在促进细胞质中的这种相互作用方面具有相当的活性；在细胞核中，非环磷酸腺苷信号诱导的CREB×CBP复合物的形成受到特异性抑制。通过Far Western印迹分析，非环磷酸腺苷信号对内在的CREB或CBP结合活性没有影响，从而支持存在一种独特的CREB×CBP拮抗剂。我们的研究表明，环磷酸腺苷和有丝分裂原/应激信号对CREB×CBP复合物形成的相对影响，赋予了通过133位丝氨酸磷酸化的CREB进行基因激活的选择性。
