Protein Tyrosine Kinase Activity Assays

Brian E. Hawes1, Tim van Biesen2

1 Schering‐Plough Research Institute, Kenilworth, New Jersey, 2 Abbott Laboratories, Abbott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.5
DOI:  10.1002/0471141755.ph0305s05
Online Posting Date:  May, 2001
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Abstract

Protein tyrosine kinases (PTKs) are ubiquitous enzymes that are integrally involved in the regulation of transformation mechanisms, normal and pathological growth, cell cycle regulation, immune responses, and a variety of intracellular signaling mechanisms. This rapidly growing family of enzymes is generally divided into two groups: receptor PTKs (with more than twelve distinct families) and nonreceptor PTKs (with more than nine distinct families). PTKs mediate the enzymatic transfer of the gamma phosphate of ATP to the phenolic groups on tyrosine residues to generate phosphate monoesters. In this unit, several assays are provided to measure the ability of PTKs to transphosphorylate protein and peptide substrates, and to autophosphorylate. Phosphorylation of exogenous substrates or autophosphorylation is detected using a 32P‐ or 33P‐phosphorylated protein. Alternatively, antibodies recognizing phosphorylated tyrosine residues can be used to quantify PTK activity. In some cases, antibodies are available for context‐specific phosphotyrosine residues, thereby enabling the detection of PTK‐specific substrate phosphorylation.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Detection of Autophosphorylated PTK by Immunoblotting
  • Basic Protocol 2: Detection of Autophosphorylated PTK by Radiolabeling
  • Basic Protocol 3: Detection of Transphosphorylation of Exogenous Substrates
  • Basic Protocol 4: Determination of Transphosphorylation by ELISA
  • Alternate Protocol 1: Time‐Resolved Fluorometry of Europium Chelates
  • Alternate Protocol 2: Radiolabeling of Solid‐Phase Substrate
  • Basic Protocol 5: Detection of PTK by Fluorescence Polarization Assay
  • Support Protocol 1: Immunoprecipitation of PTK for Subsequent Enzymatic Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Autophosphorylated PTK by Immunoblotting

  Materials
  • Immunoprecipitated PTK preparation (see protocol 8)
  • 2× SDS sample buffer ( appendix 3B)
  • Tris‐glycine gels (Novex): 2% to 20% gradient, 10 wells, 1‐mm thickness
  • Transfer buffer (see recipe)
  • Electrophoresis buffer (see recipe)
  • TTBS (see recipe)
  • Blocking solution: 3% (w/v) BSA in TTBS
  • PY20 anti‐phosphotyrosine antibody (Transduction Labs)
  • Horseradish peroxidase (HRP)–conjugated goat anti–mouse IgG antibody (Transduction Labs)
  • 100°C water bath
  • Gel electrophoresis apparatus (Novex or equivalent)
  • Power supply
  • Transfer apparatus (Novex)
  • Nitrocellulose paper
  • ECL immunoblotting detection kit (Amersham)
  • Autoradiography film
  • Densitometer

Basic Protocol 2: Detection of Autophosphorylated PTK by Radiolabeling

  Materials
  • Cell monolayers (grown in 100‐mm tissue culture plates)
  • [32P]‐ or [33P]orthophosphoric acid (NEN Life Sciences; 8500 to 9120 Ci/mmol)
  • Tissue culture medium
  • 2× SDS sample buffer ( appendix 3B)
  • Tris‐glycine gels (Novex): 2% to 20% gradient, 10 wells, 1‐mm thickness
  • Electrophoresis buffer (see recipe)
  • 95°C water bath
  • Gel electrophoresis apparatus (Novex or equivalent)
  • Power supply
  • Whatman filter paper
  • Gel dryer
  • Autoradiography film
  • Densitometer
  • Additional reagents and equipment for stimulation of cells and isolation of PTK (see protocol 8)

Basic Protocol 3: Detection of Transphosphorylation of Exogenous Substrates

  Materials
  • Immunoprecipitated PTK preparation (see protocol 8)
  • Kinase buffer (see recipe)
  • 100 mM piperazine‐N,N′‐bis (2‐hydroxypropanesulfonic acid) (PIPES)
  • 50 mM angiotensin II peptide (Sigma)
  • ATP mix (see recipe)
  • Stop solution (see recipe)
  • 10% (w/v) trichloroacetic acid (TCA), ice cold
  • 0.425% (v/v) phosphoric acid
  • Acetone
  • Scintillant
  • p81 phosphocellulose paper, 2 cm × 2 cm (Whatman)
  • Beta scintillation counter (Beckman)

Basic Protocol 4: Determination of Transphosphorylation by ELISA

  Materials
  • Coating solution (see recipe)
  • Random polymer poly(Glu,Tyr) 4:1 (Sigma)
  • TTBS (see recipe), 4°C
  • Immunoprecipitated PTK preparation (see protocol 8)
  • Assay buffer 1 (see recipe)
  • 7.5 µM ATP in assay buffer 1
  • Blocking solution: 3.0% (w/v) BSA in TTBS
  • Anti‐phosphotyrosine PY20 antibody (Transduction Labs)
  • Horseradish peroxidase (HRP)–conjugated goat anti–mouse IgG antibody (Transduction Labs)
  • HRP substrate (Bio‐Rad)
  • Maxisorp microtiter plates (Nunc)
  • 96‐well plate washer
  • Spectrophotometric plate reader (Titertek, multiscan; MTX Lab Systems)

Alternate Protocol 1: Time‐Resolved Fluorometry of Europium Chelates

  • PY20 anti‐phosphotyrosine antibody (Transduction Labs)
  • DELFIA europium labeling kit (Wallac)
  • Enhancement solution (Wallac)
  • DELFIA Research Fluorometer (Wallac)
  • Additional reagents and equipment for europium labeling of antibodies (see manufacturer's instructions)

Alternate Protocol 2: Radiolabeling of Solid‐Phase Substrate

  • Scintistrip microtiter plates (Wallac)
  • [γ‐33P]ATP (NEN Life Sciences; 2000 to 4000 Ci/mmol)
  • 96‐well scintillation counter

Basic Protocol 5: Detection of PTK by Fluorescence Polarization Assay

  Materials
  • Immunoprecipitated PTK preparation (see protocol 8)
  • Assay buffer 2 (see recipe)
  • 40 µM fluorescein‐labeled substrate peptide
  • ATP
  • Anti‐phosphotyrosine antibody PY54 (Transduction Labs)
  • 20 mM EDTA in FP buffer (see recipe)
  • 12 × 70–mm borosilicate glass tubes
  • FPM‐1 fluorescence polarization analyzer (Jolley Consulting and Research)

Support Protocol 1: Immunoprecipitation of PTK for Subsequent Enzymatic Assay

  Materials
  • Cell lines expressing PTK of interest, grown in 100‐mm cell culture plates
  • Chemical stimulant (optional): e.g., growth factor, hormone, neurotransmitter
  • PBS (Life Technologies), 4°C
  • Lysis buffer (see recipe), 4°C
  • Monoclonal or polyclonal anti‐PTK primary antibody
  • Protein G plus/protein A–agarose beads (Oncogene Research Products, Calbiochem)
  • Kinase buffer (see recipe)
  • Cell lifters
  • Incubator, 37°C humidified, equipped with 5% to 10% CO 2 in air
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Figures

Videos

Literature Cited

Literature Cited
   Braunwalder, A.F., Yarwood, D.R., Hall, T., Missbach, M., Lipson, K.E., and Sills, M.A. 1996a. A solid‐phase assay for the determination of protein tyrosine kinase activity of c‐src using scintillation microtiter plates. Anal. Biochem. 234:23‐26.
   Braunwalder, A.F., Yarwood, D.R., Sills, M.A., and Lipson, K.E. 1996b. Measurement of the protein tyrosine kinase activity of c‐src using time‐resolved fluorometry of europium chelates. Anal. Biochem. 238:159‐164.
   Farley, K., Mett, H., McGlynn, E., Murray, B., and Lydon, N.B. 1992. Development of solid‐phase enzyme‐linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60c‐src tyrosine protein kinase activity. Anal. Biochem. 203:151‐157.
   Lazaro, I., Gonzalez, M., Roy, G., Villar, L.M., and Gonzalez‐Porque, P. 1991. Description of an Enzyme‐Linked Immunosorbant Assay for the detection of protein tyrosine kinase. Anal. Biochem. 192:257‐261.
   Luttrell, L.M., Daaka, Y., Della Rocca, G.J., and Lefkowitz, R.J. 1997. G protein coupled receptors mediate two functionally distinct pathways of tyrosine phosphorylation in Rat 1a fibroblasts. J. Biol. Chem. 272:31648‐31656.
   Seethala, R. and Menzel, R. 1997. A homogeneous, fluorescence polarization assay for Src‐family tyrosine kinases. Anal. Biochem. 253:210‐218.
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