Analysis of Protein Tyrosine Phosphatases and Substrates

Fatih Mercan1, Anton M. Bennett1

1 Yale University School of Medicine, New Haven, Connecticut
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.16
DOI:  10.1002/0471142727.mb1816s91
Online Posting Date:  July, 2010
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Abstract

Protein tyrosine phosphorylation is a reversible post‐translational modification that is essential for life in eukaryotic cells. The combinatorial action of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs) determines the net level of cellular tyrosine phosphorylation. This unit discusses methods to determine the level of protein tyrosine phosphatase activity and methods for discovering novel substrates for protein tyrosine phosphatases. Curr. Protoc. Mol. Biol. 91:18.16.1‐18.16.17. © 2010 by John Wiley & Sons, Inc.

Keywords: protein tyrosine phosphatase; p‐nitrophenyl phosphate; malachite green; in‐gel phosphatase assay; PTP substrates; substrate‐trapping

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: p‐Nitrophenyl Phosphate (pNPP) Assay to Measure Protein Tyrosine Phosphatase Activity
  • Basic Protocol 2: Malachite Green Assay to Measure Inorganic Phosphate Release
  • Basic Protocol 3: In‐Gel Phosphatase Assay to Determine Relative Protein Tyrosine Phosphatase Activity
  • Basic Protocol 4: PTP Substrate Identification by In Vitro Substrate‐Trapping with PTP Active‐Site Mutants
  • Alternate Protocol 1: Substrate‐Trapping in Cells
  • Basic Protocol 5: Validation of PTP Substrates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: p‐Nitrophenyl Phosphate (pNPP) Assay to Measure Protein Tyrosine Phosphatase Activity

  Materials
  • Sample containing PTP of interest
  • Antibody against PTP of interest for immunoprecipitation
  • p‐Nitrophenyl phosphate (pNPP; mol. wt. = 263.1; Sigma) substrate
  • Lysis buffer I (see recipe)
  • Protease inhibitors
  • 1 mM sodium orthovanadate (NaVO 3)
  • 10 mM sodium fluoride (NaF)
  • 50% (v/v) slurry of protein A or protein G sepharose beads
  • Salt/Tris/EDTA (STE; see recipe)
  • Phosphatase wash buffer (see recipe)
  • 1× and 1.25× pNPP reaction buffers (see reciperecipes), prepared fresh
  • 0.2 N NaOH
  • 1× SDS sample buffer (see recipe)
  • Rotating or nutating platform, 4°C
  • 37°C water bath
  • Plate reader/spectrophotometer (A 405)
  • Additional reagents and equipment for immunoprecipitation (unit 10.16) and SDS‐PAGE (unit 10.2)

Basic Protocol 2: Malachite Green Assay to Measure Inorganic Phosphate Release

  Materials
  • Malachite Green reagent no. 1 (see recipe)
  • Malachite Green reagent no. 2 (see recipe)
  • Tween 20
  • Stock solution of phospho‐tyrosine substrate peptide (custom made)
  • Immunoprecipitated PTP
  • 1× and 10× PTP buffers (see reciperecipes)
  • 1 mM potassium dihydrogen phosphate (KH 2PO 4) in ddH 2O (stable up to 1 year at 4°C)
  • 0.2‐µm filters
  • Microtiter plates (half‐area, tissue culture‐treated, flat‐bottomed 96‐well plates; Sigma)
  • Multichannel pipettor
  • Shaker (shake speed of 120 rpm)
  • Standard ELISA microtiter plate reader (A 650)

Basic Protocol 3: In‐Gel Phosphatase Assay to Determine Relative Protein Tyrosine Phosphatase Activity

  Materials
  • 32P‐labeled substrate (poly (Glu:Tyr) [4:1] or RCML)
  • Affinity purified PTP or total cell lysate
  • Fixation buffer (see recipe)
  • Wash buffer I (see recipe)
  • Denaturation buffer (see recipe)
  • Renaturation buffer with and without DTT (see recipe)
  • Coomassie Blue staining solution (see recipe)
  • Coomassie Blue destaining solution (see recipe)
  • Rocking platform shaker
  • X‐ray film
  • Additional reagents and equipment for SDS gel electrophoresis (unit 10.2)

Basic Protocol 4: PTP Substrate Identification by In Vitro Substrate‐Trapping with PTP Active‐Site Mutants

  Materials
  • 70% confluent cells in 10‐cm dishes
  • Pervanadate (see recipe)
  • Phosphate buffered saline (PBS; appendix 22)
  • Cell lysis buffer II (see recipe)
  • GST‐fusion PTP on gluthathione‐Sepharose beads
  • GST‐PTP wild type
  • GST
  • 10 mM dithiothreitol (DTT; appendix 22)
  • Wash buffer II (see recipe)
  • 1× SDS sample buffer (see recipe)
  • Anti‐phosphotyrosine antibodies (Millipore, cat. no. 4G10)
  • 37°C incubator
  • 1.5‐ml centrifuge tubes
  • Refrigerated centrifuge
  • Platform rocker, 4°C
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2) and immunoblotting (unit 10.8)

Alternate Protocol 1: Substrate‐Trapping in Cells

  • DNA construct of full‐length substrate‐trapping PTP
  • DNA construct of full‐length wild‐type PTP
  • Cells
  • Cell lysis buffer III (see recipe)
  • Additional reagents and equipment for transfections (Chapter 9), immunoprecipitation (unit 10.16), SDS‐PAGE (unit 10.2), and protein detection (unit 10.8)
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Figures

Videos

Literature Cited

Literature Cited
   Agazie, Y.M. and Hayman, M.J. 2003. Development of an efficient “substrate‐trapping” mutant of Src homology phosphotyrosine phosphatase 2 and identification of the epidermal growth factor receptor, Gab1, and three other proteins as target substrates. J. Biol. Chem. 278:13952‐13958.
   Flint, A.J., Tiganis, T., Barford, D., and Tonks, N.K. 1997. Development of “substrate‐trapping” mutants to identify physiological substrates of protein tyrosine phosphatases. Proc. Natl. Acad. Sci. U.S.A. 94:1680‐1685.
   Jia, Z., Barford, D., Flint, A.J., and Tonks, N.K. 1995. Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B. Science 268:1754‐1758.
   Kolli, S., Zito, C.I., Mossink, M.H., Wiemer, E.A., and Bennett, A.M. 2004. The major vault protein is a novel substrate for the tyrosine phosphatase SHP‐2 and scaffold protein in epidermal growth factor signaling. J. Biol. Chem. 279:29374‐29385.
   Kontaridis, M.I., Eminaga, S., Fornaro, M., Zito, C.I., Sordella, R., Settleman, J., and Bennett, A.M. 2004. SHP‐2 positively regulates myogenesis by coupling to the Rho GTPase signaling pathway. Mol. Cell. Biol. 24:5340‐5352.
   Meng, T.C., Hsu, S.F., and Tonks, N.K. 2005. Development of a modified in‐gel assay to identify protein tyrosine phosphatases that are oxidized and inactivated in vivo. Methods 35:28‐36.
   Tiganis, T. and Bennett, A.M. 2007. Protein tyrosine phosphatase function: The substrate perspective. Biochem. J. 402:1‐15.
   Xie, L., Zhang, Y.L., and Zhang, Z.Y. 2002. Design and characterization of an improved protein tyrosine phosphatase substrate‐trapping mutant. Biochemistry 41:4032‐4039.
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