In Vitro Enzymatic Assays for Ser/Thr‐Selective Protein Kinases

H. Neal Bramson1

1 GlaxoSmithKline, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.11
DOI:  10.1002/0471141755.ph0311s19
Online Posting Date:  February, 2003
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Abstract

Medium and high throughput methods for measuring the activities of Ser/Thr‐selective protein kinases are described. These methods utilize radiochemical detection, fluorescence polarization, and ultraviolet spectroscopy to monitor transfer of the gamma‐phosphoryl group of ATP to protein or peptide substrates. These assays have utility in characterizing protein kinase inhibitors and in mechanistic studies.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Use of Phosphocellulose Capture to Measure Protein Kinase Activities
  • Alternate Protocol 1: Use of Neutravidin Capture to Measure Protein Kinase Activities
  • Alternate Protocol 2: Coupled Assays to Measure Phosphotransferase Activities
  • Alternate Protocol 3: Scintillation Proximity Assays (SPAs)
  • Alternate Protocol 4: Fluorescence Polarization (FP)
  • Alternate Protocol 5: Fluorescence Intensity Quenching (IQ)
  • Basic Protocol 2: Kinase Cascade Assays
  • Basic Protocol 3: Optimizing Assays for Protein Kinases
  • Basic Protocol 4: Investigations of Protein Kinase Mechanism of Inhibition
  • Basic Protocol 5: Investigation of Protein Kinase Mechanism of Competitive Inhibition
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Use of Phosphocellulose Capture to Measure Protein Kinase Activities

  Materials
  • PKA buffer: assay buffer 1 (see recipe) containing 0.1 mg/ml bovine serum albumin (BSA) and 1 mM dithiothreitol (DTT)
  • [γ‐33P]ATP (0.5 µCi/well)
  • Kemptide (Leu‐Arg‐Arg‐Ala‐Ser‐Leu‐Gly)
  • cAMP‐dependent protein kinase (PKA)
  • 100 mM phosphoric acid or 1% and 0.1% acetic acid
  • Scintillant
  • 96‐well polystyrene plate
  • Phosphocellulose MultiScreen‐PH opaque plates (Millipore)
  • Vacuum manifold (Millipore) and vacuum
  • 96‐well scintillation counter (e.g., Packard Topcount)

Alternate Protocol 1: Use of Neutravidin Capture to Measure Protein Kinase Activities

  • Biotin‐Leu‐Arg‐Arg‐Ala‐Ser‐Leu‐Gly (phosphoacceptor must be biotinylated)
  • 50 mM ethylenediaminetetraacetic acid (EDTA; appendix 2A)
  • Reacti‐Bind NeutrAvidin‐coated polystyrene plates (Pierce)

Alternate Protocol 2: Coupled Assays to Measure Phosphotransferase Activities

  • 4 mM ATP in PKA buffer solution (no label; see protocol 1 for PKA buffer)
  • 4× coupled enzyme mix: 4 mM phosphoenolpyruvate, 0.6 mM NADH in assay buffer 1 (see recipe) containing 60 to 120 U/ml each of pyruvate kinase and lactate dehydrogenase
  • 4× peptide solution (kemptide for PKA) in PKA buffer (see protocol 1 for PKA buffer)
  • 200 mM EDTA in PKA buffer (see protocol 1 for PKA buffer)
  • 4× PKA (or another kinase) in PKA buffer (see protocol 1 for PKA buffer)
  • 1‐ml cuvette or clear‐bottom 96‐well plates that do not absorb significantly at 340 nm
  • UV spectrophotometer or 96‐well plate reader, as appropriate

Alternate Protocol 3: Scintillation Proximity Assays (SPAs)

  • Streptavidin‐coated YSI beads (SPA beads, Amersham Biosciences)
  • Assay buffer 2 (see recipe)
  • Biotin‐Leu‐Arg‐Arg‐Ala‐Ser‐Leu‐Gly (phosphoacceptor must be biotinylated)
  • [γ‐33P]ATP (0.1 µCi/well)
  • 96‐well, white, round‐bottom plates
NOTE: SPA beads are optimized for use with scintillation counters. Amersham Biosciences also markets beads optimized for use with CCD‐camera and very high‐density plate formats.

Alternate Protocol 4: Fluorescence Polarization (FP)

  Materials
  • Dithiothreitol (DTT)
  • Assay buffer 3 (see recipe)
  • IMAP SGK assay kit (Molecular Devices) containing:
  •  Kinase reaction buffer
  •  Fluorescein‐labeled crosstide substrate,
  •  (5‐carboxyfluorescein)‐GlyArgProArgThrSerSerPheAlaGluGly
  •  400× IMAP binding reagent
  •  IMAP binding buffer
  •  SGK, active
  • 10 µM ATP
  • 384‐well black plates
  • Analyst or Acquest (Molecular Devices) or another FP‐plate reader

Alternate Protocol 5: Fluorescence Intensity Quenching (IQ)

  Materials
  • IQ PKA assay kit (Pierce Chemical) containing:
  •  Reaction buffer concentrate (or assay buffer 1, see recipe)
  •  ATP stock solution (not radiolabeled)
  •  96‐well, white round‐bottom plates (or a higher density plate, if desired)
  •  Dye‐labeled peptide substrate (kemptide for PKA)
  •  IQ reagents A and B
  • PKA
  • Plate‐compatible fluorescence spectrometer

Basic Protocol 2: Kinase Cascade Assays

  • Inhibitors or other test substance
  • DMSO
  • 500 mM EDTA
  • Assay buffer 6 (see recipe) containing 30 µM [γ‐33P]ATP
  • Biotin‐AlaAlaAlaThrGlyProLeuSerProGlyProPheAla
  • Enzyme mixture containing 30 nM cRaf1, 300 nM MEK1, and 900 nM ERK2
  • Assay buffer 7 (see recipe)
  • Packard Topcount scintillation counter (Packard Instrument) or other appropriate reader

Basic Protocol 3: Optimizing Assays for Protein Kinases

  • Potent competitive inhibitor
  • Fully optimized buffer
  • 96‐well deep titer plates (2 ml/well)
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Figures

Videos

Literature Cited

Literature Cited
   Barker, S.C., Kassel, D.B., Weigl, D., Huang, X., Luther, M.A., and Knight, W. 1995. Characterization of pp60c‐src tyrosine kinase activities using a continuous assay: Autoactivation of the enzyme is an intermolecular autophosphorylation process. Biochemistry 34:14843‐14851.
   Chen, G., Porter, M.D., Bristol, J.R., Fitzgibbon, M.J., and Pazhanisamy, S. 2000. Kinetic mechanism of the p38‐MAP kinase: Phosphoryl transfer to synthetic peptides. Biochemistry 39:2079‐2087.
   Clare, P.M., Poorman, R.A., Kelley, L.C., Watenpaugh, K.D., Bannow, C.A., and Leach, K.L. 2001. The cyclin‐dependent kinases cdk2 and cdk5 act by a random, anticooperative kinetic mechanism. J. Biol. Chem. 276:48292‐48299.
   Copeland, R.A. 2000. Enzymes, 2nd ed. John Wiley & Sons, New York.
   Hanks, S. and Hunter, T. 1995. The eukaryotic protein kinase superfamily: Kinase (catalytic) domain structure and classification. FASEB J. 9:576‐596.
   McDonald, O.B., Chen, W.‐J., Ellis, B., Hoffman, C., Overton, L., Rink, M., Smith, A., Marshall, C.J., and Wood, E.R. 1999. A scintillation proximity assay for the Raf/MEK/ERK kinase cascade: High‐throughput screening and identification of selective enzyme inhibitors. Anal. Biochem. 268:318‐329.
   Morrison, J.F. and Walsh, C.T. 1988. The behavior and significance of slow‐binding enzyme inhibitors. Adv. Enzymol. Relat. Areas Mol. Biol. 61:201‐301.
   Newton, A. 2001. Protein kinase C: Structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem. Rev. 101:2353‐2364.
   Obata, T., Yaffe, M.B., Leparc, G.G., Piro, E.T., Maegawa, H., Kashiwagi, A., Kikkawa, R., and Cantley, L.C. 2000. Peptide and protein library screening defines optimal substrate motifs for AKT/PKB. J. Biol. Chem. 275:36108‐36115.
   Segel, I.H. 1993. Enzyme kinetics: Behavior and analysis of rapid equilibrium and steady‐state enzyme systems. John Wiley & Sons, New York.
   Sonyang, Z. and Cantley, L.C. 1998. The use of peptide library for the determination of kinase peptide substrates. Methods in Molecular Biology 87:87‐98.
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