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Analysis of Protein Tyrosine Phosphatases and Substrates

Fatih Mercan1,  Anton M. Bennett1

1Yale 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: 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 (NaVO3)
  • 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 recipes), 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 (A405)
  • Additional reagents and equipment for immunoprecipitation (unit 10.16) and SDS-PAGE (unit 10.2A)

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 recipes)
  • 1 mM potassium dihydrogen phosphate (KH2PO4) in ddH2O (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 (A650)

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 10.2A)

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 2)
  • Cell lysis buffer II (see recipe)
  • GST-fusion PTP on gluthathione-Sepharose beads
  • GST-PTP wild type
  • GST
  • 10 mM dithiothreitol (DTT; appendix 2)
  • 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: Substrate-Trapping in Cells

 Additional Materials (also see Basic Protocol 4)
  • 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 10.2A), and protein detection (unit 10.8)
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Figures

  • Figure 18.16.1
    pNPP chemistry.

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  • Figure 18.16.2
    An example 96-well plate layout for the Malachite Green assay.

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  • Figure 18.16.3
    An example standard curve for the Malachite Green assay.

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  • Figure 18.16.4
    A hypothetical data set for the Malachite Green assay.

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  • Figure 18.16.5
    Schematic representation of an in-gel phosphatase assay. Cell lysates are prepared and resolved as described in the text. Lane 1: unstimulated, and lane 2: stimulated cells. Immunoprecipitated phosphatase complex from, lane 3: unstimulated, and lane 4: stimulated cells. The gel is processed as described in the text and the “white” areas represent regions in which there is phosphatase activity towards the in-gel substrate. The arrow indicates the position of the immunoprecipitated phosphatase at 69 kD, which exhibits increased phosphatase activity following stimulation. In addition, upon stimulation there is an associated phosphatase activity (~150 kDa).

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  • Figure 18.16.6
    Flow chart of a substrate-trapping experiment.

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  • Figure 18.16.7
    Serum-deprived WI38 cells were pervanadate-treated and lysates were subjected to affinity precipitation with the indicated PTP wild type and substrate-trapping GST fusion proteins. Affinity purified protein complexes were detected by immunoblotting with anti-phosphotyrosine (pTyr) antibodies. The arrow at right indicates the substrate-trapped tyrosyl phosphorylated proteins p100 and p80. Modified from Kolli et al. (2004).

    View Image

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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