Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox‐Based Sensors

Jon R. Beck1, Laura B. Peterson2, Barbara Imperiali2, Cliff I. Stains1

1 Department of Chemistry, University of Nebraska–Lincoln, Lincoln, Nebraska, 2 Department of Biology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140106
Online Posting Date:  September, 2014
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Abstract

Defining perturbations in protein kinase activity within biological samples can provide insight into disease mechanisms as well as potential targets for drug development. In this article, we present a method that utilizes a phosphorylation‐sensitive amino acid, termed CSox, to afford kinase‐selective biosensors capable of reporting on enzymatic activity directly in biological samples. These sensors produce an increase in fluorescence in response to phosphorylation of an amino acid residue adjacent to CSox. Probes can be designed for either serine/threonine or tyrosine kinases, and analysis can be performed using standard fluorescence equipment. The procedures provided herein represent our optimized protocols for the design, validation, and application of CSox‐based protein kinase activity sensors. Curr. Protoc. Chem. Biol. 6:135‐156 © 2014 by John Wiley & Sons, Inc.

Keywords: cell signaling; kinase activity assay; kinase activity profiling; phosphorylation; fluorescence‐based biosensor

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: General Cell Lysate Assay Format
  • Support Protocol 1: Design and Evaluation of Initial Sensor Constructs
  • Support Protocol 2: Validation of Sensors in Cell Lysates
  • Support Protocol 3: Sox‐Br Synthesis and On‐Resin Alkylation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: General Cell Lysate Assay Format

  Materials
  • Appropriate adherent mammalian cell line (e.g., ATCC)
  • Cell culture medium appropriate for cell line used
  • Stimulation method of choice for the KOI
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no.10010‐023), ice cold
  • Nondenaturing lysis buffer (see recipe)
  • Liquid nitrogen in Dewar flask
  • 10× Mg2+ assay buffer (see recipe, the concentration of Mg2+ is optimized in Support Protocol protocol 2)
  • 10× CSox peptide sensor solution (see recipe, the optimal concentration of CSox‐based sensor is determined in Support Protocol protocol 2)
  • 10× ATP solution (10 mM, see recipe)
  • Corning 150‐mm culture dishes (Sigma, cat. no. CLS430599)
  • Cell scrapers (Fisher Scientific, cat. no. 08‐100‐242)
  • Temperature‐controlled microcentrifuge
  • Fluorescence plate reader (SynergyH2 or similar)
  • Corning 96‐well half‐area flat‐bottom white microplate (Sigma, cat. no. CLS3693)
  • Multichannel pipettor
  • Computer running spreadsheet program, e.g., Microsoft Excel

Support Protocol 1: Design and Evaluation of Initial Sensor Constructs

  Materials
  • Potential kinase peptide substrates (synthesize as described in Support Protocol protocol 4 or purchase from Life Technologies)
  • 0.1 M NaOH containing 1 mM disodium EDTA
  • 500 mM (10×) Tris·Cl (pH = 7.5 at 22°C)
  • 1.5 M (10×) NaCl
  • MgCl 2 (Alfa Aesar, cat. no. 10797)
  • 10× Mg2+ assay buffer (see recipe, the concentration of Mg2+ is optimized in step 11 of this protocol)
  • 10× ATP solution (10 mM, see recipe)
  • Enzyme dilution buffer (see recipe)
  • Recombinant KOI (e.g., Life Technologies, EMD Millipore)
  • Recombinant panel of kinases (Life Technologies, EMD Millipore, etc.)
  • Nonlinear curve‐fitting program (e.g., KaleidaGraph)
  • Spectrophotometer
  • Fluorescence plate reader (SynergyH2 or similar)
  • Corning 96‐well half‐area flat‐bottom white microplates (Sigma, cat. no. CLS3693)
  • Multichannel pipettor
  • Computer running spreadsheet program, e.g., Microsoft Excel
  • Additional reagents and equipment for synthesis of peptide substrates (Support Protocol protocol 4)

Support Protocol 2: Validation of Sensors in Cell Lysates

  Materials
  • Cell lysates (Basic Protocol protocol 1)
  • BioRad protein assay dye reagent concentrate (BioRad, cat. no. 500‐0006)
  • Nondenaturing lysis buffer (see recipe)
  • 10× Mg+2 assay buffer (see recipe, the concentration of Mg2+ is optimized in Support Protocol protocol 2)
  • CSox peptide substrate (optimal sequence identified from Support Protocol protocol 2)
  • 10× ATP solution (10 mM, see recipe)
  • Anti‐KOI antibody (appropriate for immunoprecipitation)
  • Naïve IgG antibody (isotype matched to anti‐KOI antibody)
  • Protein A– or G–Sepharose (e.g., GE Life Sciences)
  • 100× stock(s) of small‐molecule inhibitor(s) of the KOI
  • Cell culture‐grade DMSO (e.g., Sigma)
  • Fluorescence plate reader (SynergyH2 or similar)
  • Corning 96‐well half‐area flat‐bottom white microplates (Sigma, cat. no. CLS3693)
  • Multichannel pipettor
  • Orbital rocker/shaker
  • Nonlinear regression analysis software (e.g., KaleidaGraph)
  • Additional reagents and equipment for kinase activity measurements (Basic Protocol protocol 1)

Support Protocol 3: Sox‐Br Synthesis and On‐Resin Alkylation

  Materials
  • 8‐hydroxy‐2‐methyl‐quinoline (Sigma, cat. no. H57602; structure 1 in Fig. )
  • Chlorosulfonic acid (ClSO 3H)
  • Brine (saturated aqueous NaCl)
  • Dichloromethane (CH 2Cl 2)
  • Potassium carbonate (K 2CO 3)
  • Tetrahydrofuran (THF)
  • Dimethylamine (NHMe 2)
  • Nitrogen source
  • Ethyl acetate (EtOAc)
  • Hexanes
  • Dimethylformamide (DMF)
  • Imidazole
  • Tert‐butyldiphenylsilyl chloride (TBDPSiCl; Sigma‐Aldrich, cat. no. 195537)
  • Saturated aqueous ammonium chloride
  • Dioxane, dry
  • Molecular sieves (4 Å)
  • Selenium dioxide (SeO 2)
  • Celite (diatomaceous earth)
  • Saturated aqueous potassium carbonate
  • Magnesium sulfate (MgSO 4)
  • Sodium borohydride (NaBH 4)
  • Ethanol (EtOH), absolute
  • Diethyl ether (Et 2O)
  • Phosphorus pentoxide (P 2O 5)
  • N‐bromosuccinimide (NBS)
  • Triphenylphosphine (PPh 3)
  • Florisil (100 to 200 mesh, Sigma‐Aldrich, cat. no. 220736)
  • Fmoc‐Cys(Mmt)‐OH (EMD Millipore, cat. no. 852031)
  • Trifluoroacetic acid (TFA)
  • Triisopropylsilane (TIS)
  • Tetramethylguanidine (TMG)
  • Appropriately sized separatory funnels
  • Oil bath (used for all heating steps)
  • Rotary evaporator
  • Appropriately sized chromatography column
  • Appropriately sized round‐bottom flasks
  • 3‐ml syringe with frit (Fisher Scientific, cat. no. NC9214213)
  • ½‐dram vials
  • Orbital shaker or vortex mixer
  • Additional reagents and equipment for solid‐phase peptide synthesis (SPPS: Stawikowski and Fields, )
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Figures

Videos

Literature Cited

Literature Cited
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