Mutations in protein-binding hot-spots on the hub protein Smad3 differentially affect its protein interactions and Smad3-regulated gene expression.
Mutations in protein-binding hot-spots on the hub protein Smad3 differentially affect its protein interactions and Smad3-regulated gene expression.
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DOI：
10.1371/journal.pone.0025021
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Hoffmann FM
中科院分区：
文献类型：
--
作者：
Schiro MM;Stauber SE;Peterson TL;Krueger C;Darnell SJ;Satyshur KA;Drinkwater NR;Newton MA;Hoffmann FM
枢纽蛋白通过结合相互作用与许多其他蛋白质连接。Smad 3是由转化生长因子β（TGF-β）诱导的信号转导介质，作为超过50种蛋白质-蛋白质相互作用的中心蛋白。由Smad 3介导的不同细胞反应是细胞类型和环境依赖性Smad 3-有核蛋白复合物协同作用的产物。我们的假设是，这一光谱的蛋白质复合物的扰动突变的单一蛋白质结合的热点Smad 3将有不同的后果Smad 3介导的反应。我们突变了Smad 3 MH 2结构域表面上的28个氨基酸，并鉴定了22种Smad 3变体，其与17种Smad 3结合蛋白（包括Smad 4、SARA、Ski、Smurf 2和SIP 1）的亚群结合减少。与Smad 4结合缺陷的突变，例如，D408 H，或核质穿梭缺陷，例如，W 406 A在调节Smad 3依赖性报告基因或六个内源性Smad 3响应基因Mmp 9、IL 11、Tnfaip 6、Fermt 1、Olfm 2和Wnt 11的表达水平方面受到损害。然而，Smad 3突变体Y226 A、Y297 A、W326 A、K341 A和E267 A在TGF-β信号转导方面存在明显差异。例如，K341 A和Y226 A都将Smad 3介导的报告基因的激活降低了约50%，但K341 A仅降低了Olfm 2的TGF-β诱导性，而Y226 A与W 406 A突变一样严重地降低了所有六种内源基因的TGF-β诱导性。E267 A增加了蛋白结合，但降低了TGF-β诱导，因为它引起了更高的基础表达水平。Y297 A增加了TGF-β的诱导作用，因为它降低了Smad 3诱导的基础基因表达水平。Smad 3上蛋白结合热点的突变减少了与相互作用蛋白质的不同子集的结合，并引起Smad 3诱导的基因表达的一系列定量变化。这种方法应该是有用的解开哪些Smad 3蛋白复合物是关键的特定的生物反应。
Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses.
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