Detection of Phosphorylation by Enzymatic Techniques

Shirish Shenolikar1

1 Duke University Medical Center, Durham
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 13.5
DOI:  10.1002/0471140864.ps1305s05
Online Posting Date:  May, 2001
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Abstract

This unit presents protocols for examining the functional effects elicited by nonspecific acid or alkaline phosphatases that dephosphorylate many phosphoproteins in vitro. Additional protocols describe digestion of phosphoproteins with a protein serine/threonine phosphatase and protein tyrosine phosphatase. A support protocol has been included to identify the radiolabel as 32PI based on its ability to form a complex with ammonium molybdate.

     
 
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Table of Contents

  • Basic Protocol 1: Digestion of Phosphoproteins with Nonspecific Acid Phosphatases
  • Alternate Protocol 1: Digestion of Phosphoproteins with Nonspecific Alkaline Phosphatase
  • Basic Protocol 2: Digestion of Phosphoproteins with Protein Serine/Threonine Phosphatases
  • Alternate Protocol 2: Digestion of Phosphoproteins with Protein Tyrosine Phosphatases
  • Support Protocol 1: Measurement and Identification of Released 32P
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Digestion of Phosphoproteins with Nonspecific Acid Phosphatases

  Materials
  • Sample containing 100 to 200 µg total protein
  • 50 mM piperazine‐N,N′‐bis(2‐hydroxypropanesulfonic acid) (PIPES), pH 6.0
  • Sephadex G‐25 column (optional)
  • PIPES/2‐ME or PIPES/DTT buffer, pH 6.0: 50 mM PIPES containing 15 mM 2‐mercaptoethanol or 1 mM dithiothreitol (prepare fresh)
  • Potato acid phosphatase
  • 2× SDS‐PAGE sample buffer: 50 mM Tris⋅Cl/0.4 M glycine (pH 8.3)/0.2% (w/v) SDS
  • 100 mM sodium pyrophosphate or other general phosphatase inhibitor
  • 90°C water bath or heating block
  • Additional reagents and equipment for electrophoresis (unit 10.1)

Alternate Protocol 1: Digestion of Phosphoproteins with Nonspecific Alkaline Phosphatase

  • Tris/MgCl 2, pH 7.5 or HEPES/MgCl 2, pH 7.5 buffer: 50 mM Tris⋅Cl (pH 7.5; appendix 2E)/1 mM MgCl 2 or 50 mM
  • N,‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid (HEPES; pH 7.5)/1 mM MgCl 2
  • Calf intestine alkaline phosphatase (molecular biology grade)

Basic Protocol 2: Digestion of Phosphoproteins with Protein Serine/Threonine Phosphatases

  Materials
  • Sample containing 100 µg total protein
  • Tris/DTT/MnCl 2 buffer, pH 7.5: 50 mM Tris⋅Cl (pH 7.5; appendix 2E)/1 mM dithiothreitol (DTT)/1 mM MnCl 2 (prepare fresh)
  • Microcystin‐LR
  • Protein phosphatase 2A (PP2A), catalytic subunit

Alternate Protocol 2: Digestion of Phosphoproteins with Protein Tyrosine Phosphatases

  Materials
  • Sample containing 10 to 100 µg total protein
  • 50 mM imidazole, pH 7.5
  • Protein tyrosine phosphatase (e.g., PTP‐1B or SH‐PTP)
  • 2× SDS‐PAGE sample buffer (see protocol 1) or 100 mM sodium vanadate

Support Protocol 1: Measurement and Identification of Released 32P

  Materials
  • Trichloroacetic acid
  • Radiolabeled protein/phosphatase reaction mixture (see protocol 1)
  • 1.25 mM potassium phosphate (KH 2PO 4)/1 N H 2SO 4
  • 1:1 (v/v) isobutanol/toluene
  • 5% (w/v) ammonium molybdate
  • Scintillation fluid
  • Liquid scintillation counter
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Figures

Videos

Literature Cited

Literature Cited
   Berger, H.A., Travis, S.M., and Welsh, M.J. 1993. Regulation of the cystic fibrosis transmembrane conductance regulator Cl− channel by specific protein kinases and protein phosphatases. J. Biol.Chem. 268:2037‐2047.
   Charbonneau, H. and Tonks, N.K. 1992. 1002 protein phosphatases? Annu. Rev. Cell Biol. 8:463‐493.
   Shenolikar, S. 1994. Protein serine/threonine phosphatases: New avenues for cell regulation. Annu. Rev. Cell Biol. 10:55‐86.
   Shenolikar, S. and Ingebritsen, T.S. 1984. Protein (serine and threonine) phosphate phosphatases. Methods Enzymol. 107:102‐129.
   Shenolikar, S. and Nairn, A.C. 1991. Protein phosphatases: Recent progress. Adv. Second Messenger Phosphoprotein Res. 23:1‐123.
   Swarup, G., Cohen, S. and Garbers, D.L. 1981. Selective dephosphorylation of proteins containing phosphotyrosine by alkaline phosphatases. J. Biol. Chem. 256:8197‐8201.
   Van Etten, R.L. and Waymack, P.P. 1991. Substrate specificity and pH dependence of homogenous wheat germ acid phosphatase. Arch. Biochem. Biophys. 288:634‐645.
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