Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia.
Proteomics. 2011 Aug;11(16):3390-401. doi: 10.1002/pmic.201100036.
The transforming growth factor-β (TGF-β) signaling pathway progresses through a series of protein phosphorylation regulated steps. Smad4 is a key mediator of the classical TGF-β signaling pathway; however, reports suggest that TGF-β can activate other cellular pathways independent of Smad4. By investigating the TGF-β-regulated phosphoproteome, we aimed to uncover new functions controlled by TGF-β. We applied titanium dioxide to enrich phosphopeptides from stable isotope labeling with amino acids in cell culture (SILAC)-labeled SW480 cells stably expressing Smad4 and profiled them by mass spectrometry. TGF-β stimulation for 30 min resulted in the induction of 17 phosphopeptides and the repression of 8 from a total of 149 unique phosphopeptides. Proteins previously not known to be phosphorylated by TGF-β including programmed cell death protein 4, nuclear ubiquitous casein and cyclin-dependent kinases substrate, hepatoma-derived growth factor and cell division kinases amongst others were induced following TGF-β stimulation, while the phosphorylation of TRAF2 and NCK-interacting protein kinase are examples of proteins whose phosphorylation status was repressed. This phosphoproteomic screen has identified new TGF-β-modulated phosphorylation responses in colon carcinoma cells.
转化生长因子-β（TGF-β）信号通路通过一系列受蛋白磷酸化调节的步骤进行。Smad4 是经典 TGF-β信号通路的关键介质；然而，有报道表明，TGF-β可以激活独立于 Smad4 的其他细胞途径。通过研究 TGF-β 调节的磷酸蛋白质组，我们旨在揭示 TGF-β 控制的新功能。我们应用二氧化钛从稳定同位素标记的氨基酸在细胞培养（SILAC）标记的稳定表达 Smad4 的 SW480 细胞中富集磷酸肽，并通过质谱进行分析。TGF-β 刺激 30 分钟后，诱导了 17 个磷酸肽，抑制了 8 个磷酸肽，总共有 149 个独特的磷酸肽。先前未知的被 TGF-β磷酸化的蛋白质，包括程序性细胞死亡蛋白 4、核普遍存在的酪蛋白和细胞周期依赖性激酶底物、肝癌衍生生长因子和细胞分裂激酶等，在 TGF-β刺激后被诱导，而 TRAF2 和 NCK 相互作用蛋白激酶的磷酸化状态则是被抑制的蛋白质的例子。这个磷酸蛋白质组学筛选已经鉴定出结肠癌细胞中 TGF-β 调节的新磷酸化反应。
