Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-β signaling
Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-β signaling
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DOI：
10.1074/jbc.m700186200
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发表时间：
2007-04-13
影响因子：
4.8
通讯作者：
Lin, Kai
中科院分区：
文献类型：
--
作者：
Chen, Weijun;Lam, Suvana S.;Lin, Kai
Smad 蛋白家族介导细胞生长和分化中的转化生长因子-β (TGF-β) 信号传导。 Smad 分别通过与辅抑制因子（例如 Ski）或共激活因子（例如 CREB 结合蛋白（CBP））相互作用来抑制或激活 TGF-β 信号传导。具体来说，Ski 已被证明会干扰 Smad3 和 CBP 之间的相互作用。然而，目前尚不清楚Ski是否与CBP竞争结合Smads，以及它们是否可以在Smad3的同一结合表面上与Smad3相互作用。我们通过尺寸排阻色谱、等温滴定量热法和突变研究在体外研究了 Smad、Ski 和 CBP 的纯化构建体之间的相互作用。在这里，我们表明 Ski-(16 - 192) 直接与受体调节 Smad 蛋白 (R-Smad) 的同源三聚体相互作用，例如 R-Smad。 g。 Smad2或Smad3，形成六聚体； Ski-(16 - 192) 与 R-Smad 相互作用。 Smad4 异源三聚体形成五聚体。还发现 CBP-(1941 - 1992) 直接与 R-Smad 同源三聚体相互作用形成六聚体，并与 R-Smad 相互作用。 Smad4 异源三聚体形成五聚体。此外，Ski 和 CBP 的这些结构域相互竞争与 Smad3 的结合。我们的突变研究表明，Ski 和 CBP 的结构域在 Smad 锚定结合表面的一部分与 Smad3 相互作用，从而激活受体。我们的结果表明，Ski 通过取代 R-Smad 复合物中的 CBP 来负调节 TGF-β 信号传导。我们的工作模型表明，Smad 蛋白活性通过 TGF-β 途径中的 Ski 和 CBP 进行微妙的平衡。
The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors ( e. g. Ski) or coactivators ( e. g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16 - 192) interacted directly with a homotrimer of receptor-regulated Smad protein ( R-Smad), e. g. Smad2 or Smad3, to form a hexamer; Ski-( 16 - 192) interacted with an R- Smad . Smad4 heterotrimer to form a pentamer. CBP-(1941 - 1992) was also found to interact directly with an R- Smad homotrimer to form a hexamer and with an R- Smad . Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.