Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
EMBO J. 2011 Nov 1;30(23):4777-89. doi: 10.1038/emboj.2011.393.
TGF-β signalling is regulated by post-translational modifications of Smad proteins to translate quantitative difference in ligand concentration into proportional transcriptional output. Previous studies in cell culture systems suggested that Smad ubiquitination regulatory factors (Smurfs) act in this regulation by targeting Smads for proteasomal degradation, but whether this mechanism operates under physiological conditions is not clear. Here, we generated mice harbouring a target-disrupted Smurf2 allele. Using primary mouse embryonic fibroblasts and dermal fibroblasts, we show that TGF-β-mediated, Smad-dependent transcriptional responses are elevated in the absence of Smurf2. Instead of promoting poly-ubiquitination and degradation, we show that Smurf2 actually induces multiple mono-ubiquitination of Smad3 in vivo. Phosphorylation of T179, immediately upstream of the Smad3 PY motif, enhances Smurf2 and Smad3 interaction and Smad3 ubiquitination. We have mapped Smurf2-induced Smad3 ubiquitination sites to lysine residues at the MH2 domain, and demonstrate that Smad3 ubiquitination inhibits the formation of Smad3 complexes. Thus, our data support a model in which Smurf2 negatively regulates TGF-β signalling by attenuating the activity of Smad3 rather than promoting its degradation.
TGF-β 信号转导受 Smad 蛋白的翻译后修饰调节，以将配体浓度的定量差异转化为比例转录输出。细胞培养系统中的先前研究表明，Smad 泛素化调节因子（Smurfs）通过靶向 Smads 进行蛋白酶体降解来在这种调节中发挥作用，但该机制在生理条件下是否起作用尚不清楚。在这里，我们生成了携带靶基因缺失 Smurf2 等位基因的小鼠。使用原代小鼠胚胎成纤维细胞和真皮成纤维细胞，我们表明 TGF-β 介导的 Smad 依赖性转录反应在缺乏 Smurf2 的情况下升高。我们表明 Smurf2 实际上在体内诱导 Smad3 的多种单泛素化，而不是促进多泛素化和降解。Smad3 PY 基序上游的 T179 磷酸化增强了 Smurf2 和 Smad3 的相互作用和 Smad3 泛素化。我们已经将 Smurf2 诱导的 Smad3 泛素化位点映射到 MH2 结构域的赖氨酸残基上，并证明 Smad3 泛素化抑制 Smad3 复合物的形成。因此，我们的数据支持这样一种模型，即 Smurf2 通过减弱 Smad3 的活性而不是促进其降解来负调控 TGF-β 信号转导。
