Protein Tyrosine Phosphatase Assays

Ulrike Lorenz1

1 The Beirne Carter Center for Immunology Research and Department of Microbiology, University of Virginia, Charlottesville, Virginia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 11.7
DOI:  10.1002/0471142735.im1107s93
Online Posting Date:  April, 2011
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Abstract

Tyrosine phosphorylation and dephosphorylation of proteins play a critical role in many processes of the immune system, from early development to fully differentiated effector function. Since the opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) determine the steady‐state level of tyrosine phosphorylation on a given protein, it is often important for mechanistic studies to determine the specific activities of PTKs and PTPs. PTPs are defined by their enzymatic activity that catalyzes the dephosphorylation of phosphotyrosine residues. This unit focuses on methods to determine the enzymatic activity of PTPs. While there are many varieties of PTP assays, the focus in this unit is on immune complex PTP assays, which do not require elaborate biochemical purifications and are commonly used to test the activities of specific PTPs in the immune system. Curr. Protoc. Immunol. 93:11.7.1‐11.7.12. © 2011 by John Wiley & Sons, Inc.

Keywords: protein tyrosine phosphatase; protein tyrosine phosphatase assay; immunoprecipitation; pNPP; phosphoprotein; phosphopeptide; malachite green assay; radioactively labeled; phosphoproteins

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Immune Complex Assays for Protein Tyrosine Phosphatases
  • Basic Protocol 2: Phosphatase Assay Using pNPP as Substrate
  • Alternate Protocol 1: Phosphatase Assay Using Phosphoprotein as Substrate Followed by the Malachite Green Assay
  • Alternate Protocol 2: Phosphatase Assay Using Radioactively Labeled Phosphoprotein as Substrate
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Immune Complex Assays for Protein Tyrosine Phosphatases

  Materials
  • Cells of desired type
  • Phosphate‐buffered saline (PBS, appendix 2A), ice cold
  • 1× NP‐40 lysis buffer (see recipe for 5× buffer), freshly supplemented with 1× protease inhibitors (see recipe), ice‐cold
  • Antibodies against protein of interest (polyclonal or monoclonal)
  • Protein A– or protein G–Sepharose (GE Healthcare)
  • 1× phosphatase buffer (see recipe for 2× buffer)
  • Refrigerated centrifuge and microcentrifuge
  • Nutator mixer (BD Diagnostics)

Basic Protocol 2: Phosphatase Assay Using pNPP as Substrate

  Materials
  • 250 mM p‐nitrophenyl phosphate (pNPP) in 1× phosphatase buffer
  • Immunoprecipitated protein (resuspended beads from protocol 1, step 16)
  • 1 N NaOH
  • 30°C water bath or heat block
  • Spectrophotometer capable of measuring absorbance at 405 nm

Alternate Protocol 1: Phosphatase Assay Using Phosphoprotein as Substrate Followed by the Malachite Green Assay

  Materials
  • Phosphate standard: 100 µM KH 2PO 4
  • 1× phosphatase buffer (see recipe for 2× buffer)
  • Immunoprecipitated protein (resuspended beads from protocol 1, step 16)
  • 10 to 100 µM phosphopeptides: can be chemically synthesized to order or, alternatively, some more commonly used peptides are commercially available, e.g., those peptides provided as part of the Tyrosine Phosphatase Assay System from Promega
  • Solution A: molybdate (see recipe)
  • Solution B: malachite green (see recipe)
  • Solution C: 7.8% (v/v) H 2SO 4
  • Disposable cuvettes
  • 30°C water bath or heat block
  • Spectrophotometer capable of measuring absorbance in the 600‐ to 630‐nm range

Alternate Protocol 2: Phosphatase Assay Using Radioactively Labeled Phosphoprotein as Substrate

  Materials
  • 2× Abl kinase buffer (see recipe)
  • 5 mg/ml myelin basic protein (MBP; e.g., Invitrogen, Sigma, Active Motif, http://www.activemotif.com)
  • 100 mM adenosine triphosphate (ATP)
  • 100 mM MnCl 2
  • 100 mM Na 3VO 4
  • 100 mg/ml bovine serum albumin (BSA)
  • [γ‐32P]ATP (sp. act., 3000 Ci/mmol)
  • Trichloroacetic acid (TCA), solid
  • 15% and 20% (v/v) TCA in H 2O
  • Acetone, ice cold
  • 1× phosphatase buffer (see recipe for 2× buffer)
  • Immunoprecipitated protein (resuspended beads from protocol 1, step 16)
  • Scintillation cocktail and scintillation counter
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Figures

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Literature Cited

Literature Cited
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   Carter, S.G. and Karl, D.W. 1982. Inorganic phosphate assay with malachite green: An improvement and evaluation. J. Biochem. Biophys. Methods 7:7‐13.
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   Gallagher, S. 2001. Immunoblot detection. Curr. Protoc. Protein Sci. 4:10.10.1‐10.10.12.
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   Huntington, N.D. and Tarlinton, D.M. 2004. CD45: Direct and indirect government of immune regulation. Immunol. Lett. 94:167‐174.
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   Markova, B., Gulati, P., Herrlich, P.A., and Bohmer, F.D. 2005. Investigation of protein‐tyrosine phosphatases by in‐gel assays. Methods 35:22‐27.
   Montalibet, J., Skorey, K.I., and Kennedy, B.P. 2005. Protein tyrosine phosphatase: Enzymatic assays. Methods 35:2‐8.
   Pao, L.I., Badour, K., Siminovitch, K.A., and Neel, B.G. 2007. Nonreceptor protein‐tyrosine phosphatases in immune cell signaling. Annu. Rev. Immunol. 25:473‐523.
   Segel, I.H. 1976. Biochemical Calculations: How to Solve Mathematical Problems in General Biochemistry. John Wiley & Sons, New York.
   Sozio, M.S., Mathis, M.A., Young, J.A., Walchli, S., Pitcher, L.A., Wrage, P.C., Bartok, B., Campbell, A., Watts, J.D., Aebersold, R., Hooft van Huijsduijnen, R., and van Oers, N.S. 2004. PTPH1 is a predominant protein‐tyrosine phosphatase capable of interacting with and dephosphorylating the T cell receptor zeta subunit. J. Biol. Chem. 279:7760‐7769.
   Tiganis, T. and Bennett, A.M. 2007. Protein tyrosine phosphatase function: The substrate perspective. Biochem. J. 402:1‐15.
   Van Veldhoven, P.P. and Mannaerts, G.P. 1987. Inorganic and organic phosphate measurements in the nanomolar range. Anal. Biochem. 161:45‐48.
   Zhang, Z.Y. 1998. Protein‐tyrosine phosphatases: Biological function, structural characteristics, and mechanism of catalysis. Crit. Rev. Biochem. Mol. Biol. 33:1‐52.
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