Serine/Threonine Protein Phosphatase Assays

Thomas McAvoy1, Angus C. Nairn2

1 The Rockefeller University, New York, New York, 2 Yale University School of Medicine, New Haven, Connecticut
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.18
DOI:  10.1002/0471142727.mb1818s92
Online Posting Date:  October, 2010
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Abstract

Methods for assaying serine/threonine protein phosphatases are discussed. Three commonly used protocols are presented that employ either colorimetric or radiometric assays. These methods can be used to assay a variety of preparations of serine/threonine phosphatases, from crude lysates to purified proteins. Strategies for the application of a particular protocol for a particular purpose are discussed. The assays can be used in either a high‐throughput mode where simple comparison of activities can be done, or in specific assays where kinetic data can be derived. Curr. Protoc. Mol. Biol. 92:18.18.1‐18.18.11. © 2010 by John Wiley & Sons, Inc.

Keywords: protein phosphorylation; protein phosphatase; colorimetric assay; radiometric assay

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Colorimetric Assay of Protein Phosphatases Using p‐Nitrophenyl Phosphate
  • Basic Protocol 2: Colorimetric Assay of Protein Phosphatases Using a Synthetic Phospho‐Peptide Substrate and Malachite Green Reagent
  • Basic Protocol 3: Assay of Protein Phosphatases Using 32P‐Labeled Substrates
  • Support Protocol 1: Preparation of 32P‐Labeled Substrate (Phosphorylase A)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Colorimetric Assay of Protein Phosphatases Using p‐Nitrophenyl Phosphate

  Materials
  • Protein phosphatase sample
  • 1× colorimetric assay buffer (see recipe)
  • p‐Nitrophenyl phosphate (pNPP) solution or tablets (Sigma)
  • 5 N NaOH ( appendix 22)
  • Standard flat‐bottomed 96‐well microtiter plate
  • Microplate reader capable of reading absorbance at 405 nm

Basic Protocol 2: Colorimetric Assay of Protein Phosphatases Using a Synthetic Phospho‐Peptide Substrate and Malachite Green Reagent

  Materials
  • Malachite green phosphate assay reagent or kit (Biomol Green, Enzo Life Sciences; Serine/Threonine Phosphatase Assay System, Promega)
  • 10 mM synthetic peptide stock solution dissolved in water and stored at −20°C
  • 1× colorimetric assay buffer (see recipe)
  • Phosphatase samples
  • 1 mM phosphate standard (KH 2PO 4)
  • 96‐well standard flat‐bottomed microtiter plate
  • Microtiter plate shaker (optional)
  • Microplate reader capable of reading absorbance at 620 nm (or 600 to 630 nm range)

Basic Protocol 3: Assay of Protein Phosphatases Using 32P‐Labeled Substrates

  Materials
  • 32P‐labeled substrate (see protocol 4)
  • 1× labeled substrate assay buffer (see recipe)
  • Phosphatase solution
  • Phosphatase inhibitor or activator (if needed; see Table 18.18.2)
  • 20% aqueous trichloroacetic acid (TCA) solution
  • 1.5‐ml microcentrifuge tubes
  • 30°C water bath
  • 2.0‐ml screw‐cap tubes with caps
  • Scintillation vials
  • Scintillation counter

Support Protocol 1: Preparation of 32P‐Labeled Substrate (Phosphorylase A)

  Materials
  • Phosphorylase b (20 mg protein supplied as lyophilized crystals; Sigma or Calzyme)
  • Phosphorylase kinase (1.4 mg powder; Sigma)
  • 10× phosphorylase kinase reaction buffer (see recipe)
  • 10 mM ATP
  • [γ‐32P]‐ATP (8000 mCi/ml; Perkin Elmer NEG‐502A)
  • 90% saturated ammonium sulfate solution (66.2 g ammonium sulfate in 100 ml aqueous solution)
  • Storage solution (see recipe)
  • Glycerol
  • 15‐ml centrifuge tubes
  • Refrigerated centrifuge
  • 30°C water bath
  • Desalting columns for 100‐µl and 2.5‐ml sample volumes (NICK column and PD‐10 column, respectively, GE Biosciences)
  • 2‐ml screw‐cap tubes
  • Additional reagents and equipment for Bradford assay (unit 10.1)
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Literature Cited

Literature Cited
   Andreeva, A.V. and Kutuzov, M.A. 2001. PPP family of protein Ser/Thr phosphatases: Two distinct branches? Mol. Biol. Evol. 18:448‐452.
   Cohen, P.T. 2002. Protein phosphatase 1–targeted in many directions. J. Cell. Sci. 115:241‐256.
   Collins, E. and Sim, A.T. 1998. Regulation of neuronal PP1 and PP2A during development. Methods Mol. Biol. 93:79‐102.
   Killilea, S.D., Cheng, Q., and Wang, Z.X. 1998. Protein phosphatase type 1 and type 2A assays. Methods Mol. Biol. 93:23‐33.
   Lu, G. and Wang, Y. 2008. Functional diversity of mammalian type 2C protein phosphatase isoforms: New tales from an old family. Clin. Exp. Pharmacol. Physiol. 35:107‐112.
   MacKintosh, C. and Moorhead, G.B. 1999. Assay and purification of protein serine/threonine phosphatases. In Protein Phosphorylation: A Practical Approach (D.G. Hardie, ed.) pp. 153‐181. Oxford University Press, Oxford, United Kingdom.
   Rusnak, F. and Mertz, P. 2000. Calcineurin: Form and function. Physiol. Rev. 80:1483‐1521.
   Shibasaki, F., Hallin, U., and Uchino, H. 2002. Calcineurin as a multifunctional regulator. J. Biochem. 131:1‐15.
   Swingle, M., Ni, L., and Honkanen, R.E. 2007. Small‐molecule inhibitors of ser/thr protein phosphatases: Specificity, use and common forms of abuse. Methods Mol. Biol. 365:23‐38.
   Virshup, D.M. and Shenolikar, S. 2009. From promiscuity to precision: Protein phosphatases get a makeover. Mol. Cell 33:537‐545.
   Wang, B., Zhang, P., and Wei, Q. 2008. Recent progress on the structure of Ser/Thr protein phosphatases. Sci. China C. Life Sci. 51:487‐494.
   Xu, Y., Xing, Y., Chen, Y., Chao, Y., Lin, Z., Fan, E., Yu, J.W., Strack, S., Jeffrey, P.D., and Shi, Y. 2006. Structure of the protein phosphatase 2A holoenzyme. Cell 127:1239‐1251.
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