Detection of Phosphorylation by Enzymatic Techniques

Shirish Shenolikar1

1 Duke University Medical Center, Durham, North Carolina
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.5
DOI:  10.1002/0471142727.mb1805s33
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Reversible protein phosphorylation is an important mechanism for regulating physiological processes in both plant and animal cells. There are a number of techniques to demonstrate the presence of covalently bound phosphate in proteins. The general strategy of the protocols in this unit is to first examine the functional effects elicited by nonspecific acid or alkaline phosphatases that dephosphorylate many phosphoproteins in vitro. Protein phosphatases that selectively hydrolyze phosphoserine and phosphothreonine or phosphotyrosine residues can then be used to identify a functionally important covalent modification. 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.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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 (pH 7.5; appendix 22)/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 10.2)

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 22)/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 22)/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
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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.
   Li, M., Guo, H., and Damuni, Z. 1995. Purification and characterization of two potent heat‐stable inhibitors of protein phosphatase 2A from bovine kidney. Biochemistry 34:1988‐1996.
   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. 256:1447‐1452.
   Van Etten, R.L. and Waymack, P.P. 1991. Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase. Arch. Biochem. Biophys. 288:634‐645.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library