Assays of Protein Kinases Using Exogenous Substrates

A. Nigel Carter1

1 The 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

  • 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 β‐Casein
  • Alternate Protocol 1: 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
  • recipe5× cyclic nucleotide–dependent kinase reaction buffer (see recipe)
  • 10 mg/ml histone 2B in H 2O
  • recipe[γ‐32P]ATP solution (see recipe)
  • 20× cyclic nucleotide solution: 20 µM cyclic AMP in H 2O/20 µM cyclic GMP in H 2O
  • Enzyme sample containing cyclic nucleotide–dependent kinase activity (see protocol 8), kept on ice until use
  • 30° water bath
  • Additional reagents and equipment for TCA precipitation (see protocol 9), adsorption onto P81 phosphocellulose paper (see protocol 10), or electrophoretic analysis (see protocol 11)

Basic Protocol 2: Assay for Protein Kinase C Isoforms

  Materials
  • recipe5× PKC reaction buffer (see recipe)
  • 10 mg/ml histone H1 in H 2O
  • recipe[γ‐32P]ATP solution (see recipe)
  • Enzyme sample containing PKC activity (see protocol 8)
  • 30°C water bath
  • Additional reagents and equipment for TCA precipitation (see protocol 9), adsorption onto P81 phosphocellulose paper (see protocol 10), or electrophoretic analysis (see protocol 11)

Basic Protocol 3: Assay for Casein Kinases Using β‐Casein

  Materials
  • recipe5× casein kinase reaction buffer (see recipe)
  • 10 mg/ml β‐casein in H 2O
  • recipe[γ‐32P]ATP solution (see recipe)
  • Enzyme sample containing casein kinase activity (see protocol 8), kept on ice
  • 30°C water bath
  • Additional reagents and equipment for TCA precipitation (see protocol 9) or electrophoretic analysis (see protocol 11)

Alternate Protocol 1: Assay for Casein Kinases Using a Peptide Substrate

  • 10 mM recipesynthetic 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 protocol 9), adsorption onto P81 phosphocellulose paper (see protocol 10), or electrophoretic analysis (see protocol 11)

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

  Materials
  • recipe5× CaM kinase reaction buffer (see recipe)
  • recipe10 mM synthetic peptide substrate solution (see recipe): e.g., TyrLeuArgArgArgLeuSerAspSerAsnPhe (for CaM kinase I) or LysLysAlaLeuArgGlnGluThrValAspAlaLeu (autocamtide for CaM kinase II)
  • recipe[γ‐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 protocol 8), kept on ice
  • 30°C water bath
  • Additional reagents and equipment for adsorption onto P81 phosphocellulose paper (see protocol 10)

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
  • recipe5× tyrosine kinase reaction buffer (see recipe)
  • recipe[γ‐32P]ATP solution (see recipe)
  • Enzyme sample containing tyrosine kinase activity (see protocol 8), kept on ice
  • 2× SDS‐PAGE sample buffer (unit 10.2), ice‐cold
  • 30°C and boiling water bath
  • Additional reagents and equipment for immunoprecipitation (unit 10.16) or SDS‐PAGE (see protocol 11 and unit 10.2)

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 protocol 8), kept on ice
  • 20% (v/v) 2‐propanol/ 50 mM Tris⋅Cl (pH 8.0 at room temperature; appendix 22)
  • 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)
  • recipeAppropriate kinase reaction buffer (see recipe)
  • recipe10 mM Mg/ATP solution (see reciperecipes)
  • 10 mCi/ml [γ‐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 10.2) 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 [γ‐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 22), ice‐cold
  • recipeLysis buffer (see recipe)
  • recipeProtease inhibitor stock solutions (see recipe)
  • Microcentrifuge, 4°C

Support Protocol 2: TCA Precipitation to Determine Incorporation of Radioactivity

  Materials
  • Assay samples (see Basic Protocols protocol 11 to protocol 65 and protocol 4)
  • 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 protocol 11 to protocol 65 and protocol 4)
  • 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. )
  • 20‐ml scintillation vial
  • Scintillation counter
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Figures

Videos

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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