Detection of Protein Phosphorylation in Tissues and Cells

Katherine W. Roche1, Richard L. Huganir2

1 National Institute on Deafness and other Communication Disorders, NIH, Bethesda, Maryland, 2 The Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.14
DOI:  10.1002/0471142301.ns0514s11
Online Posting Date:  May, 2001
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Abstract

Phosphorylation is one of the principal regulatory mechanisms in the nervous system. Several different procedures used to characterize the phosphorylation state of neuronal proteins are described in this unit, including analysis of phosphorylation in situ, phosphoamino acid analysis, and phosphopeptide map analysis. In addition, there is a protocol describing in vitro phosphorylation of fusion proteins. These methods are often combined to provide a comprehensive evaluation of the phosphorylation state of a particular protein.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Labeling of Phosphoproteins In Situ
  • Basic Protocol 2: Phosphopeptide Map Analysis
  • Basic Protocol 3: Phosphoamino Acid Analysis
  • Support Protocol 1: Phosphorylation of Fusions Protein In Vitro
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Labeling of Phosphoproteins In Situ

  Materials
  • Cultured cells or tissues in 10‐cm dishes
  • Phosphate‐free MEM (Life Technologies), 37°C
  • Neurotransmitters, protein kinases, or phosphatase activators/inhibitors of interest
  • [32P]orthophosphate (9000 mCi/mol; NEN Life Sciences)
  • Phosphate‐buffered saline (PBS; appendix 2A), 4°C
  • Lysis buffer (see recipe) with or without detergents
  • Antibody against protein of interest (units 5.4 & 5.5), attached to protein A Sepharose beads according to manufacturer's recommendations (Amersham Pharmacia Biotech)
  • Lysis buffer (see recipe) containing 2% (v/v) Triton X‐100 (4°C)
  • Lysis buffer (see recipe) containing 500 to 750 mM NaCl (4°C)
  • SDS sample buffer ( appendix 2A)
  • Super Stain (see recipe)
  • Destaining solution (see recipe)
  • Cellophane sheets (BioRad)
  • 10–cm culture dishes
  • Disposable cell scrapers
  • Tabletop ultracentrifuge (e.g., Optima TL with TLA 100.2 rotor; Beckman)
  • Probe sonicator
  • Platform rocker
  • Phosphor imager (optional)
  • Additional reagents and equipment for SDS‐PAGE (CPMB UNIT and appendix 1A in this manual) and autoradiography (CPMB APPENDIX and appendix 1A in this manual)
CAUTION: Safety equipment is important when using [32P]orthophosphate.Utmost care must be exercised to avoid radioactive contamination and exposure. See Strategic Planning for more information.

Basic Protocol 2: Phosphopeptide Map Analysis

  Materials
  • Dried acrylamide gel containing the radiolabeled protein of interest (see protocol 1)
  • 50% (v/v) methanol
  • 0.3 mg/ml L‐tosylamide‐2‐phenylethyl chloromethyl ketone (TPCK)–treated trypsin or thermolysin (both compounds available from Sigma) in 0.4% (w/v) NH 4HCO 3
  • 1 mg/ml basic fuschin (Sigma)
  • 1 mg/ml phenol red
  • Peptide map electrophoresis buffer, pH 3.5 (see recipe)
  • Ascending chromatography buffer (see recipe)
  • Glass vials
  • 65°C water bath
  • 20‐cm cellulose TLC plates (Analtech)
  • Hair dryer
  • Whatman 3MM filter paper
  • Paint thinner (Varsol)
  • Electrophoresis tank designed to accommodate TLC plates (Fisher) or Hunter thin‐layer peptide mapping electrophoresis system (CBS Scientific, dist. by VWR)
  • 500‐V power supply
  • Ascending chromatography tank
  • Phosphor imager (optional)
  • Additional reagents and materials for isolating the radiolabeled peptide of interest (see protocol 1) and autoradiography (CPMB APPENDIX and appendix 1A in this manual)
CAUTION: Safety equipment is important when using [32P]orthophosphate.Utmost care must be exercised to avoid radioactive contamination and exposure. See Strategic Planning for more information.

Basic Protocol 3: Phosphoamino Acid Analysis

  Materials
  • 6 N HCl
  • N 2 gas
  • 10 mg/nm phosphorylated amino acid standards (see recipe)
  • Peptide map electrophoresis buffer, pH 1.9 (see recipe)
  • 1% (w/v) ninhydrin (Sigma) in acetone
  • 16 × 75–mm Kimax screw‐top tube with Teflon top
  • 105°C oven
  • 20‐cm cellulose TLC plates (Analtech)
  • Electrophoresis tanks (2) designed to accommodate TLC plates (Fisher) or Hunter thin‐layer peptide mapping electrophoresis system (CBS Scientific, dist. by VWR)
  • Additional reagents and equipment for separating and extracting phosphopeptides on a gel (see protocol 1Basic Protocols 1 and protocol 22)
CAUTION: Safety equipment is important when using [32P]orthophosphate. Utmost care must be exercised to avoid radioactive contamination and exposure. See Strategic Planning for more information.

Support Protocol 1: Phosphorylation of Fusions Protein In Vitro

  Materials
  • Purified fusion protein (unit 5.7)
  • Protein kinase, e.g.:
  •  cAMP‐dependent protein kinase (PKA)
  •  Protein kinase C (PKC)
  •  Calcium/calmodulin‐dependent protein kinase II (CAM‐KII)
  • Appropriate kinase buffer, e.g.:
  •  PKA buffer (see recipe)
  •  PKC buffer (see recipe)
  •  CAM‐KII buffer (see recipe)
  • 10 Ci/mmol [γ‐32P]ATP
  • 3× SDS sample buffer ( appendix 2A)
  • Super Stain (see recipe)
  • Destaining solution (see recipe)
  • Cellophane sheets (BioRad)
  • Phosphor imager (optional)
  • Additional reagents and materials for SDS‐PAGE (CPMB UNIT and appendix 1A in this manual) and autoradiography (CPMB APPENDIX and appendix 1A in this manual)
CAUTION: Safety equipment is important when using [γ‐32P]ATP. Utmost care must be exercised to avoid radioactive contamination and exposure. See Strategic Planning for more information.
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Figures

Videos

Literature Cited

Literature Cited
   Boyle, W.J., van der Geer, P., and Hunter, T. 1991. Phosphopeptide mapping and phosphoamino acid analysis by two‐dimensional separation on thin‐layer cellulose plates. Methods Enzymol. 201:110‐149.
   Czernik, A.J., Girault, J.A., Nairn, A.C., Chen, J., Snyder, G., Kebabian, J., and Greengard, P. 1991. Production of phosphorylation state–specific antibodies. Methods Enzymol. 201:264‐283.
   Ginty, D.D., Kornhauser, J.M., Thompson, M.A., Bading, H., Mayo, K.E., Takahashi, J.S., and Greenberg, M.E. 1993. Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science 260:238‐241.
   Greengard, P., Valtorta, F., Czernik, A.J., and Benfenati, F. 1993. Synaptic vesicle phosphoproteins and regulation of synaptic function. Science 259:780‐785.
   Huganir, R.L. and Greengard, P. 1990. Regulation of neurotransmitter receptor desensitization by protein phosphorylation. Neuron 5:555‐567.
   Kaplan, D.R. 1998. Studying signal transduction in neuronal cells: The Trk/NGF system. Prog. Brain Res. 117:35‐46.
   Levitan, I.B. 1994. Modulation of ion channels by protein phosphorylation and dephosphorylation. Annu. Rev. Physiol. 56:193‐212.
   Mammen, A.L., Kameyama, K., Roche, K.W., and Huganir, R.L. 1997. Phosphorylation of the alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptor GluR1 subunit by calcium/calmodulin‐dependent kinase II. J. Biol. Chem. 272:32528‐32533.
   Patton, B.L., Molloy, S.S., and Kennedy, M.B. 1993. Autophosphorylation of type II CaM kinase in hippocampal neurons: Localization of phospho‐ and dephosphokinase with complementary phosphorylation site‐specific antibodies. Mol. Biol. Cell 4:159‐172.
   Pearson, R.B. and Kemp, B.E. 1991. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 200:62‐81.
   Roche, K.W., Tingley, W.G., and Huganir, R.L. 1994. Glutamate receptor phosphorylation and synaptic plasticity. Curr. Opin. Neurobiol. 4:383‐388.
   Roche, K.W., O'Brien, R.J., Mammen, A.L., Bernhardt, J., and Huganir, R.L. 1996. Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit. Neuron 16:1179‐1188.
   Tingley, W.G., Ehlers, M.D., Kameyama, K., Doherty, C., Ptak, J.B., Riley, C.T., and Huganir, R.L. 1997. Characterization of protein kinase A and protein kinase C phosphorylation of the N‐methyl‐D‐aspartate receptor NR1 subunit using phosphorylation site‐specific antibodies. J. Biol. Chem. 272:5157‐5166.
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