Permeabilization Strategies to Study Protein Phosphorylation

A. Nigel Carter1

1 The Salk Institute for Biological Studies, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.8
DOI:  10.1002/0471142727.mb1808s40
Online Posting Date:  May, 2001
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Abstract

This unit deals with the use of nucleotide triphosphates to label proteins in vitro in permeabilized cells and isolated cellular fractions. These experiments generally utilize [g‐32P]ATP as an exogenously added phosphate donor, although [g‐32P]GTP can be used in specific cases. Procedures are outlined for performing a protein phosphorylation experiment using permeabilized cells and isolated intracellular organelles. Both of these procedures result in lysates from which the protein of interest may be easily immunoprecipitated; however alternative techniques are described for preparing the final lysate for electrophoretic analysis. A related procedure that does not involve permeabilization is outlined for direct analysis of cytosolic or membrane‐bound kinases. Two different methods for determining the specific radioactivity of 32P ‐containing compounds are also included.

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of Protein Phosphorylation in Permeabilized Cells
  • Alternate Protocol 1: Intact Cell Sample Preparation for SDS‐PAGE
  • Alternate Protocol 2: Intact Cell Sample Preparation for Isoelectric Focusing
  • Basic Protocol 2: Analysis of Protein Phosphorylation Using Isolated Subcellular Fractions
  • Alternate Protocol 3: Organelle Sample Preparation for SDS‐PAGE
  • Alternate Protocol 4: Organelle Sample Preparation for Isoelectric Focusing
  • Alternate Protocol 5: Direct Analysis of Cytosolic or Membrane‐Bound Kinases
  • Support Protocol 1: Determination of Specific Radioactivity
  • Support Protocol 2: Determination of the Specific Activity of the γ‐Phosphate of [32P]ATP
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Protein Phosphorylation in Permeabilized Cells

  Materials
  • Cell culture to be labeled
  • Cell culture medium appropriate for cells being studied, buffered with 20 mM HEPES, pH 7.2, prewarmed to 37°C
  • Cells under study (normally cultured cell lines or isolated primary cells in culture)
  • recipeExtracellular buffer (see recipe), 37°C
  • recipePermeabilization buffer (see recipe), 37°C
  • recipe60 U/ml streptolysin O working solution (see recipe), freshly prepared
  • recipe10 mM cold ATP stock solution (see recipe)
  • 10 mCi/ml [γ‐32P]ATP (3000 Ci/mmol; e.g., DuPont NEN)
  • Pharmacological agents, peptides, or Fab′ fragments to be studied
  • Appropriate receptor agonists
  • recipe2× lysis buffer for immunoprecipitation (see recipe), ice‐cold
  • Protease inhibitor stock solutions (in absolute ethanol; store up to 6 months at −20°C):
  •  100 mM phenylmethylsulfonyl fluoride (PMSF)
  •  100 mM benzamidine
  •  1 mg/ml pepstatin A
  •  1 mg/ml leupeptin
  •  1 mg/ml antipain
  • 100 mM DTT
  • Dry ice/ethanol bath
  • Noncirculating 37°C water bath containing a flat shelf (preferably made of wire mesh) with space for the number of cell plates in the experiment
  • 50‐ml centrifuge tubes
  • Tabletop centrifuge
  • Cell scrapers
  • Screw‐cap microcentrifuge tubes

Alternate Protocol 1: Intact Cell Sample Preparation for SDS‐PAGE

  • recipe2× SDS‐PAGE sample buffer (see recipe), 4°C
  • Bath sonicator
  • Boiling water bath

Alternate Protocol 2: Intact Cell Sample Preparation for Isoelectric Focusing

  • recipeTwo‐dimensional‐PAGE lysis buffer (see recipe), 4°C
  • recipeTwo‐dimensional‐PAGE sample buffer (see recipe)
  • Bath sonicator
  • Dry ice/ethanol bath
  • Lyophilizer

Basic Protocol 2: Analysis of Protein Phosphorylation Using Isolated Subcellular Fractions

  Materials
  • Suspension of isolated intracellular organelles
  • recipeIntracellular buffer (see recipe), 4°C
  • recipePermeabilization buffer (see recipe), 37°C
  • recipe60 U/ml streptolysin O working solution (see recipe)
  • recipe10 mM cold ATP stock solution (see recipe)
  • 10 mCi/ml [γ‐32P]ATP (3000 Ci/mmol; e.g., DuPont NEN)
  • Reagents under study: e.g., kinase/phosphatase inhibitors, protein kinases, or phosphatases
  • recipe2× lysis buffer for immunoprecipitation (see recipe), 4°C
  • Screw‐cap microcentrifuge tubes
  • Benchtop ultracentrifuge (e.g., Beckman TL‐100 or Airfuge)
  • 37°C circulating water bath

Alternate Protocol 3: Organelle Sample Preparation for SDS‐PAGE

  • recipe2× SDS‐PAGE sample buffer (see recipe)
  • Boiling water bath

Alternate Protocol 4: Organelle Sample Preparation for Isoelectric Focusing

  • recipeTwo‐dimensional‐PAGE lysis buffer (see recipe)
  • Lyophilizer

Alternate Protocol 5: Direct Analysis of Cytosolic or Membrane‐Bound Kinases

  Materials
  • Appropriate kinase assay buffer (unit 18.7), 4°C
  • [γ‐32P]ATP solution (see recipe in unit 18.7)
  • Suspension of isolated intracellular organelles, 4°C
  • Purified protein kinase of interest (keep on ice)
  • recipe2× SDS‐PAGE sample buffer (see recipe), ice‐cold
  • Screw‐cap microcentrifuge tubes
  • 30°C and boiling water baths

Support Protocol 1: Determination of Specific Radioactivity

  Materials
  • 32P‐labeled cells or cell lysates
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Standard ATP samples of known concentration in 4% perchloric acid
  • 4% or 8% (v/v) perchloric acid, ice‐cold
  • Tri‐n‐octylamine (Sigma)
  • 1,1,2‐trichlorotrifluoroethane (Freon; e.g., Aldrich, Sigma)
  • Linear gradient consisting of:
  •  Solution A: Milli‐Q water filtered through 0.2‐mm filter
  •  Solution B: 1.25 M NH 4H 2PO 4, pH 3.8 (adjust pH with H 3PO 4; filter through 0.2‐mm filter)
  • Nucleotide standard mix: e.g., 1 mM each of AMP, ADP, ATP, GMP, GDP, and GTP
  • Cell scrapers
  • Polypropylene screw‐cap microcentrifuge tubes or 5‐ml polypropylene tubes with very tight‐fitting caps
  • Tabletop centrifuge
  • HPLC system consisting of:
  •  Whatman Partisphere SAX HPLC column and guard column
  •  Apparatus for producing linear gradient
  •  UV detector set at 254 nm
  •  Fraction collector
  • Whatman low‐dead‐volume 0.2‐mm syringe filters
  • Additional reagents and equipment for HPLC (units 10.12 10.14)

Support Protocol 2: Determination of the Specific Activity of the γ‐Phosphate of [32P]ATP

  Materials
  • Cell lysates containing unknown [32P]ATP concentrations
  • cA‐PrK (purified subunit from bovine heart; Calbiochem) and recipe10× cA‐PrK assay buffer (see recipe)
  • 0.5 mM cyclic AMP (cAMP; Calbiochem)
  • 10 mg/ml kemptide (Sigma)
  • 1 M DTT (store at −20°C)
  • 0.5 M MgCl 2
  • 75 mM orthophosphoric acid
  • Acetone
  • Screw‐cap microcentrifuge tubes
  • P 81 phosphocellulose paper, precut and labeled ( protocol 10 in unit 18.7)
  • 30°C circulating water bath
  • Additional reagents and equipment for precipitating 32P‐labeled peptide on P 81 paper ( protocol 10 in unit 18.7)
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Figures

Videos

Literature Cited

Literature Cited
   Alexander, D.R., Brown, M.H., Tutt, A.L., Crumpton, M.J., and Shivnan, E. 1992. CD3 and CD2 antigen–mediated CD3 γ‐chain phosphorylation in permeabilized human T cells: Regulation by cytosolic phosphatases. Biochem. J. 288:69‐77.
   Castle, D.J. 1995. Purification of organelles from mammalian cells. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, H.L. Ploegh, D.W. Speicher, and P.T. Wingfields, eds.) pp. 4.2.1‐4.2.55. John Wiley & Sons, New York.
   Cunningham, E., Thomas, G.M., Ball, A., Hiles, I., and Cockcroft, S. 1995. Phosphatidylinositol transfer protein dictates the rate of inositol triphosphate production by promoting the synthesis of PIP2. Curr. Biol. 5:775‐783.
   Hawkins, P.T., Michell, R.H., and Kirk, C.J. 1983. A simple assay method for determination of the specific radioactivity of the γ‐phosphate group of 32P‐labeled ATP. Biochem. J. 210:717‐720.
   Martys, J.L., Shevell, T., and McGraw, T.E. 1995. Studies of transferrin recycling reconstituted in streptolysin O–permeabilized Chinese hamster ovary cells. J. Biol. Chem. 270(43):25976‐25984.
   Slowiejko, D.M., Levey, A.I., and Fisher, S.K. 1994. Sequestration of muscarinic cholinergic receptors in permeabilized neuroblastoma cells. J. Neurochem. 62:1795‐1803.
   Stephens, L.R. and Downes, C.P. 1990. Product‐precursor relationships amongst inositol polyphosphates: Incorporation of [32P]Pi into myo‐inositol 1,3,4,6‐tetrakisphosphate, myo‐inositol 1,3,4,5‐tetrakisphosphate, myo‐inositol 3,4,5,6‐tetrakisphosphate and myo‐inositol 1,3,4,5,6‐pentakisphosphate in intact avian erythrocytes. Biochem. J. 265:435‐452.
   Stephens, L.R., Jackson, T., and Hawkins, P.T. 1994. Synthesis of phosphatidylinositol‐3,4,5‐trisphosphate in permeabilized neutrophils regulated by receptors and G‐proteins. J. Biol. Chem. 268:17162‐17172.
   Stutchfield, J. and Cockcroft, S. 1993. Correlation between secretion and phospholipase D activation in differentiated HL60 cells. Biochem. J. 293:649‐655.
   Taylor, J.A., Karas, J.L., Ram, M.K., Green, O.M., and Seidel‐Dugan, C. 1995. Activation of the high‐affinity immunoglobulin E receptor Fc ɛ RI in RBL‐2H3 cells is inhibited by Syk SH2 domains. Mol. Cell. Biol. 15:4149‐4157.
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