Shen Tao, Qin Lan, Lin Xia
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, 77030, USA.
Michael E. DeBakey Department of Surgery, Baylor College of Medicine, One Baylor Plaza, R711, Houston, TX, 77030, USA.
Methods Mol Biol. 2016;1344:111-9. doi: 10.1007/978-1-4939-2966-5_6.
Phosphorylation of Smad1/5/8 at the C-terminal SXS motif by BMP type I receptors is one of the most critical events in BMP signaling. Conversely, protein phosphatases that dephosphorylate phospho-Smad1/5/8 can consequently prevent or terminate BMP signaling. PPM1H is an undercharacterized phosphatase in the PPM family. We recently demonstrated that PPM1H can dephosphorylate Smad1 in the cytoplasm and block BMP signaling responses in cellular assays. Here we describe in vitro method showing that PPM1H is a bona fide phosphatase for Smad1/5/8. PPM1H is produced as GST fusion protein in E. coli, and purified against glutathione sepharose beads. Bacterially purified recombinant PPM1H possesses phosphatase activity toward artificial substrate para-nitrophenyl phosphate (pNPP). Recombinant PPM1H also dephosphorylates immuno-purified phosphorylated Smad1 in test tubes. These direct in vitro phosphatase assays provide convincing evidence demonstrating the role of PPM1H as a specific phosphatase for P-Smad1.
骨形态发生蛋白（BMP）I型受体使Smad1/5/8在C端SXS基序处发生磷酸化，这是BMP信号传导中最关键的事件之一。相反，使磷酸化的Smad1/5/8去磷酸化的蛋白磷酸酶能够阻止或终止BMP信号传导。PPM1H是PPM家族中一种研究较少的磷酸酶。我们最近证明，PPM1H能够使细胞质中的Smad1去磷酸化，并在细胞实验中阻断BMP信号反应。在此，我们描述了一种体外方法，表明PPM1H是一种针对Smad1/5/8的真正磷酸酶。PPM1H作为GST融合蛋白在大肠杆菌中产生，并通过谷胱甘肽琼脂糖珠进行纯化。细菌纯化的重组PPM1H对人工底物对硝基苯磷酸酯（pNPP）具有磷酸酶活性。重组PPM1H还能在试管中使免疫纯化的磷酸化Smad1去磷酸化。这些直接的体外磷酸酶测定提供了令人信服的证据，证明了PPM1H作为P-Smad1特异性磷酸酶的作用。
