title: Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay.
abstract: To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca(2+)/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.
authors: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan. Electronic address: sugiyama@ag.kagawa-u.ac.jp.
authors: Yasunori
authors: Y
authors: Sugiyama
authors: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors: Sho
authors: S
authors: Yamashita
authors: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors: Yuuki
authors: Y
authors: Uezato
authors: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan; Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki 305-8566, Japan.
authors: Yukako
authors: Y
authors: Senga
authors: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors: Syouichi
authors: S
authors: Katayama
authors: Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan.
authors: Naoki
authors: N
authors: Goshima
authors: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Osaka 563-8577, Japan.
authors: Yasushi
authors: Y
authors: Shigeri
authors: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors: Noriyuki
authors: N
authors: Sueyoshi
authors: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors: Isamu
authors: I
authors: Kameshita
mesh_terms: D000818:Animals; D006801:Humans; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases
keywords: Malachite green assay; Myelin basic protein; Phosphatase assay; Protein phosphatase; Protein substrate