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Assays of Protein Kinases Using Exogenous Substrates

A. Nigel Carter1

1The Salk Institute for Biological Studies, La Jolla, California

Publication Name: 
Current Protocols in Molecular Biology
Unit Number: 
Unit 18.7
DOI: 
10.1002/0471142727.mb1807s40
Online Posting Date: 
May, 2001
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Abstract

In studies of the regulation of specific biochemical events by reversible phosphorylation, assaying the protein kinases themselves can often lead to significant progress in understanding the mechanistic details of a system under study. This unit describes assays for a variety of protein kinases that require different conditions to detect and measure their activities: cyclic nucleotide-dependent kinases, protein kinase C and isoforms, casein kinases, Ca2+/calmodulin-dependent kinases, and tyrosine kinase. A protocol for in-gel assays for specific kinase activity is also provided. The unit is not meant to be a catalog of individual protein kinase assays; however, the general principles of these assays should apply to most if not all known protein kinases.

     
 
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Table of Contents

  • Unit Introduction
  • Strategic Planning
  • Basic Protocol 1: Assay for Cyclic Nucleotide–Dependent Protein Kinases
  • Basic Protocol 2: Assay for Protein Kinase C Isoforms
  • Basic Protocol 3: Assay for Casein Kinases Using -Casein
  • Alternate Protocol: Assay for Casein Kinases Using a Peptide Substrate
  • Basic Protocol 4: Assay for Ca2+/Calmodulin–Dependent Kinases
  • Basic Protocol 5: Assay for Tyrosine Kinases
  • Basic Protocol 6: In-Gel Protein Kinase Assays
  • Support Protocol 1: Preparing a Cell Lysate for Kinase Assays
  • Support Protocol 2: TCA Precipitation to Determine Incorporation of Radioactivity
  • Support Protocol 3: Adsorption onto P81 Phosphocellulose Paper
  • Support Protocol 4: Electrophoretic Analysis of Phosphorylation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assay for Cyclic Nucleotide–Dependent Protein Kinases

 Materials
  • 5× cyclic nucleotide–dependent kinase reaction buffer (see recipe)
  • 10 mg/ml histone 2B in H2O
  • [-32P]ATP solution (see recipe)
  • 20× cyclic nucleotide solution: 20 µM cyclic AMP in H2O/20 µM cyclic GMP in H2O
  • Enzyme sample containing cyclic nucleotide–dependent kinase activity (see Support Protocol 1), kept on ice until use
  • 30° water bath
  • Additional reagents and equipment for TCA precipitation (see Support Protocol 2), adsorption onto P81 phosphocellulose paper (see Support Protocol 3), or electrophoretic analysis (see Support Protocol 4)

Basic Protocol 2: Assay for Protein Kinase C Isoforms

 Materials
  • 5× PKC reaction buffer (see recipe)
  • 10 mg/ml histone H1 in H2O
  • [-32P]ATP solution (see recipe)
  • Enzyme sample containing PKC activity (see Support Protocol 1)
  • 30°C water bath
  • Additional reagents and equipment for TCA precipitation (see Support Protocol 2), adsorption onto P81 phosphocellulose paper (see Support Protocol 3), or electrophoretic analysis (see Support Protocol 4)

Basic Protocol 3: Assay for Casein Kinases Using -Casein

 Materials
  • 5× casein kinase reaction buffer (see recipe)
  • 10 mg/ml -casein in H2O
  • [-32P]ATP solution (see recipe)
  • Enzyme sample containing casein kinase activity (see Support Protocol 1), kept on ice
  • 30°C water bath
  • Additional reagents and equipment for TCA precipitation (see Support Protocol 2) or electrophoretic analysis (see Support Protocol 4)

Alternate Protocol: Assay for Casein Kinases Using a Peptide Substrate

 Additional Materials (also see Basic Protocol 3)
  • 10 mM synthetic peptide substrate solution (see recipe) for casein kinase: e.g., AspAspAspGluGluSerIleThrArgArg (for casein kinase I) or ArgArgArgGluGluGluThrGluGluGlu (for casein kinase II)
  • Additional reagents and equipment for TCA precipitation (see Support Protocol 2), adsorption onto P81 phosphocellulose paper (see Support Protocol 3), or electrophoretic analysis (see Support Protocol 4)

Basic Protocol 4: Assay for Ca2+/Calmodulin–Dependent Kinases

 Materials
  • 5× CaM kinase reaction buffer (see recipe)
  • 10 mM synthetic peptide substrate solution (see recipe): e.g., TyrLeuArgArgArgLeuSerAspSerAsnPhe (for CaM kinase I) or LysLysAlaLeuArgGlnGluThrValAspAlaLeu (autocamtide for CaM kinase II)
  • [-32P]ATP solution (see recipe)
  • 1 mg/ml calmodulin in Milli-Q-purified water (store small aliquots at –70°C)
  • Enzyme sample containing CaM kinase activity (see Support Protocol 1), kept on ice
  • 30°C water bath
  • Additional reagents and equipment for adsorption onto P81 phosphocellulose paper (see Support Protocol 3)

Basic Protocol 5: Assay for Tyrosine Kinases

 Materials
  • Rabbit muscle enolase (enolase EC 4.2.1.11; ammonium sulfate precipitate or suspension purified from rabbit muscle)
  • 1 mM DTT/50 mM HEPES, pH 7.0
  • Glycerol
  • 100 mM acetic acid
  • 5× tyrosine kinase reaction buffer (see recipe)
  • [-32P]ATP solution (see recipe)
  • Enzyme sample containing tyrosine kinase activity (see Support Protocol 1), kept on ice
  • 2× SDS-PAGE sample buffer (UNIT 10.2A), ice-cold
  • 30°C and boiling water bath
  • Additional reagents and equipment for immunoprecipitation (UNIT 10.16) or SDS-PAGE (see Support Protocol 4 and UNIT 10.2A)

Basic Protocol 6: In-Gel Protein Kinase Assays

 Materials
  • 10 mg/ml kinase substrate: e.g., myelin basic protein
  • Enzyme sample containing kinase activity (see Support Protocol 1), kept on ice
  • 20% (v/v) 2-propanol/50 mM Tris×Cl (pH 8.0 at room temperature; APPENDIX 2)
  • 1 mM DTT/50 mM Tris×Cl (pH 8.0 at room temperature)
  • 6 M guanidine×HCl/1 mM DTT/50 mM Tris×Cl (pH 8.0 at room temperature) or 8 M urea/1 mM DTT/50 mM Tris×Cl (pH 8.0 at room temperature)
  • 1 mM DTT/0.05% (v/v) Tween 20/50 mM Tris×Cl (pH 8.0 at 4°C)
  • Appropriate kinase reaction buffer (see recipe)
  • 10 mM Mg/ATP solution (see recipes)
  • 10 mCi/ml [-32P]ATP (3000 Ci/mmol; Amersham, DuPont NEN, or ICN Biomedicals)
  • 5% (w/v) trichloroacetic acid (TCA)
  • 1% (w/v) sodium pyrophosphate/5% (w/v) TCA
  • Container for radioactive incubation: e.g., small tray with tight cover, or heat-sealable polyethylene bag (Seal-a-Meal)
  • Seal-a-Meal apparatus (optional)
  • Additional reagents and equipment for SDS-PAGE (UNIT 10.2A) and autoradiography (APPENDIX 3A)

CAUTION: Because the samples are radioactive, great care should be exercised in sample preparation and loading. Perform the reactions and subsequent manipulations in screw-cap microcentrifuge tubes to minimize the risk of 32P contamination. Run gels until the dye front passes out of the gel, as all residual [-32P]ATP will migrate with the dye front. This means that the SDS-PAGE running buffer will be contaminated with 32P, and it should be disposed of as radioactive waste according to safety regulations.See APPENDIX 1F for proper handling and disposal.

Support Protocol 1: Preparing a Cell Lysate for Kinase Assays

 Materials
  • Cultured cells: adherent cells at ~70% confluence in 100-mm tissue culture dishes or suspension cells at 106 cells/ml
  • PBS (APPENDIX 2), ice-cold
  • Lysis buffer (see recipe)
  • Protease inhibitor stock solutions (see recipe)
  • Microcentrifuge, 4°C

Support Protocol 2: TCA Precipitation to Determine Incorporation of Radioactivity

 Materials
  • Assay samples (see Basic Protocols 1 to 5 and Alternate Protocol)
  • 5% and 10% (w/v) trichloroacetic acid (TCA), ice-cold
  • 95% ethanol
  • Diethyl ether
  • Whatman GF-C glass-fiber filters
  • Vacuum manifold (e.g., Fisher)
  • 20-ml scintillation vials
  • Scintillation counter

Support Protocol 3: Adsorption onto P81 Phosphocellulose Paper

 Materials
  • Assay samples (see Basic Protocols 1 to 5 and Alternate Protocol)
  • 75 mM orthophosphoric acid (stored up to 6 months at room temperature)
  • Acetone
  • 2 × 2–cm squares of P81 phosphocellulose paper (Whatman)
  • 500-ml plastic beaker with the bottom replaced with solvent-resistant plastic or wire mesh (see Fig. 18.7.2)
  • 20-ml scintillation vial
  • Scintillation counter
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Figures

  • Figure 18.7.1
    Addition of sample to P81 phosphocellulose paper. The 2 × 2–cm squares of P81 paper are labeled using a pencil (ink is dissolved by the acetone wash), and 50% to 75% of the reaction product is applied to the paper.

    View Image
  • Figure 18.7.2
    Setup for washing P81 phosphocellulose paper with reaction samples. The paper squares are washed five times for 5 min each in 75 mM orthophosphoric acid and once in acetone. They are then air dried and counted in a scintillation counter.

    View Image

Literature Cited

Literature Cited
    Hanson, P.I., Kapiloff, M.S., Lou, L.L., Rosenfeld, M.G., and Shulman, H. 1989. Expression of a multifunctional Ca2+/calmodulin–dependent protein kinase and mutational analysis of its autoregulation. Neuron 3:59-70.
    Kameshita, I. and Fujisawa, H. 1989. A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate polyacrylamide gel. Anal. Biochem. 183:139-143.
    Pearson, R.B. and Kemp, B.E. 1991. Protein kinase phosphorylation site sequences and consensus specificity motifs. Methods Enzymol. 200:62-81.
    Songyang, Z., Carraway, K.L. III, Eck, M.J., Harrison, S.C., Feldman, R.A., Mohammadi, M., Schlessinger, J., Hubbard, S.R., Smith, D.P., Eng, C., Lorenzo, M.J., Ponder, B.A.J., Mayer, B.J., and Cantley, L.C. 1995. Catalytic specificity of protein tyrosine kinases is critical for selective signalling. Nature 373:536-539.
    Takai, Y., Kishimoto, A., Kikkawa, U., Mori, T., and Nishizuka, Y. 1979. Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system. Biochem. Biophys. Res. Commun. 91:1218-1224.
     
 
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