Stimulus-specific interaction between activator-coactivator cognates revealed with a novel complex-specific antiserum.
Stimulus-specific interaction between activator-coactivator cognates revealed with a novel complex-specific antiserum.
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新型复合物特异性抗血清揭示了激活剂-辅激活剂同源物之间的刺激特异性相互作用。
DOI：
10.1074/jbc.275.12.8263
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Montminy,M
中科院分区：
文献类型：
--
作者：
Wagner,BL;Bauer,A;Schütz,G;Montminy,M
许多第二信使途径通过磷酸化依赖性的蛋白质-蛋白质相互作用传播诱导信号。例如，第二信使 cAMP 通过蛋白激酶 A 介导的 cAMP 反应元件结合蛋白 (CREB) Ser133 磷酸化来促进细胞基因表达，而这种修饰反过来又刺激 CREB 与共激活剂 CREB 结合蛋白 (CBP) 的结合。 CREB·CBP复合物的溶液结构，使用相关的相互作用结构域、激酶诱导结构域和激酶诱导结构域相互作用结构域，分别称为KID和KIX，表明KID在与KIX结合后经历卷曲到螺旋的转变，这稳定了复合物的形成。然而，这种变化是否发生在全长 CREB 和 CBP 蛋白的背景下尚不清楚。在这里，我们描述了一种新型抗血清，它特异性结合 CREB·CBP 复合物，但不单独结合任何蛋白质。表位作图实验表明，CREB·CBP 抗血清可检测到 KID 中与 KIX 结合后发生构象变化的残基。该抗血清以磷酸（Ser133）依赖性方式识别全长 CREB·CBP 复合物的能力表明，用分离的 KID 结构域观察到的结构转变也发生在全长 CREB 蛋白的背景下。据我们所知，这是第一份记录内源性细胞蛋白质-蛋白质复合物原位形成的报告。
A number of second messenger pathways propagate inductive signals via protein-protein interactions that are phosphorylation-dependent. The second messenger, cAMP, for example, promotes cellular gene expression via the protein kinase A-mediated phosphorylation of cAMP-response element-binding protein (CREB) at Ser133, and this modification in turn stimulates the association of CREB with the co-activator, CREB-binding protein (CBP). The solution structure of the CREB·CBP complex, using relevant interaction domains, kinase inducible domain and kinase-induced domain interacting domain, referred to as KID and KIX, respectively, shows that KID undergoes a coil to helix transition, upon binding to KIX, that stabilizes complex formation. Whether such changes occur in the context of the full-length CREB and CBP proteins, however, is unclear. Here we characterize a novel antiserum that specifically binds to the CREB·CBP complex but to neither protein individually. Epitope mapping experiments demonstrate that the CREB·CBP antiserum detects residues in KID that undergo a conformational change upon binding to KIX. The ability of this antiserum to recognize full-length CREB·CBP complexes in a phospho-(Ser133)-dependent manner demonstrates that the structural transition observed with the isolated KID domain also occurs in the context of the full-length CREB protein. To our knowledge, this is the first report documenting formation of endogenous cellular protein-protein complexesin situ.