title: Analysis of Smad Phosphatase Activity In Vitro.
abstract: 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.
authors.affiliation: 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.
authors.forename: Tao
authors.initials: T
authors.lastname: Shen
authors.affiliation: Michael E. DeBakey Department of Surgery, Baylor College of Medicine, One Baylor Plaza, R711, Houston, TX, 77030, USA.
authors.forename: Lan
authors.initials: L
authors.lastname: Qin
authors.affiliation: Michael E. DeBakey Department of Surgery, Baylor College of Medicine, One Baylor Plaza, R711, Houston, TX, 77030, USA. xialin@bcm.edu.
authors.forename: Xia
authors.initials: X
authors.lastname: Lin
mesh_terms: D018929:Cell Culture Techniques; D004789:Enzyme Activation; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051785:Smad Proteins / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D051785:Smad Proteins; C521058:PPM1H protein, human; D010749:Phosphoprotein Phosphatases; D010744:Phosphoric Monoester Hydrolases
keywords: Immunoprecipitation; In vitro; Phosphatase; Western blotting; pNPP