Wang Guannan, Matsuura Isao, He Dongming, Liu Fang
Center for Advanced Biotechnology and Medicine.
J Biol Chem. 2009 Apr 10;284(15):9663-73. doi: 10.1074/jbc.M809281200. Epub 2009 Feb 13.
Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity.
Smad蛋白将细胞表面的转化生长因子-β（TGF-β）信号转导至细胞核内的基因调控过程。在TGF-β处理后，高度同源的Smad2和Smad3在C末端尾巴的SSXS基序处被TGF-β受体磷酸化。在此我们表明，除了C末端尾巴外，Smad3连接区的三个（丝氨酸/苏氨酸）-脯氨酸位点，即Ser(208)、Ser(204)和Thr(179)，在TGF-β刺激下也会发生磷酸化。连接区磷酸化在TGF-β处理后1小时达到峰值，滞后于C末端尾巴磷酸化的峰值。我们提供的证据表明，TGF-β受体介导的C末端尾巴磷酸化对于TGF-β诱导的连接区磷酸化是必需的。尽管TGF-β受体对于连接区磷酸化是必需的，但受体本身并不磷酸化这些位点。我们进一步表明，ERK并不负责这三个位点的TGF-β依赖性磷酸化。我们发现GSK3参与了TGF-β诱导的Ser(204)磷酸化。泛CDK抑制剂黄酮哌啶醇可消除TGF-β诱导的Thr(179)和Ser(208)磷酸化，这表明CDK家族负责响应TGF-β时Thr(179)和Ser(208)的磷酸化。将连接区磷酸化位点突变为不可磷酸化的残基会增强Smad3激活TGF-β/Smad靶基因的能力以及Smad3的生长抑制功能。因此，这些观察结果表明，TGF-β诱导的Smad3连接区位点磷酸化会抑制其抗增殖活性。
