In Vitro Enzymatic Assays of Protein Tyrosine Phosphatase 1B

Thomas Lubben1, Jill Clampit1, Michael Stashko1, James Trevillyan1, Michael R. Jirousek1

1 Abbott Laboratories, Abbott Park, IL
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.8
DOI:  10.1002/0471141755.ph0308s13
Online Posting Date:  August, 2001
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Abstract

Many hormone or growth factor receptors signal via the activation of protein‐tyrosine kinases and phosphatases. Alteration of the phosphorylation state of tyrosine residues in certain proteins can directly regulate enzyme activity or cause formation of protein complexes necessary for transducing intracellular signals. Genetic and biochemical evidence also implicates protein‐tyrosine phosphatases in several disease processes, including negative regulation of insulin receptor signaling at the level of the insulin receptor and perhaps in signaling at the IRS‐1 level. The expression of protein tyrosine phosphatase‐1B (PTP1B) is elevated in muscle and adipose tissue in insulin‐resistant states both in man and rodents suggesting that PTP1B may play a role in the insulin‐resistant state associated with diabetes and obesity. As described in this unit, PTP1B activity can be determined with the small molecule substrate, p‐nitrophenyl phosphate (pNPP), in which the cleavage of the phosphate results in production of p‐nitrophenol (pNP) and an increase in absorbance at 405 nm. Alternatively, PTP1B activity can be measured as described using model phosphotyrosyl‐containing peptide substrates in which the release of free phosphate from the peptide is determined using a malachite green colorimetric assay.

     
 
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Table of Contents

  • Basic Protocol 1: Continuously Monitored Phosphatase Assay Using p‐Nitrophenylphosphate (pNPP) as Substrate
  • Basic Protocol 2: Time‐point PTP1B Assay Using a Synthetic Phosphopeptide as Substrate and Malachite Green Reagent to Detect Released Phosphate
  • Support Protocol 1: Purification of Human PTP1B from E. COLI
  • Support Protocol 2: Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Continuously Monitored Phosphatase Assay Using p‐Nitrophenylphosphate (pNPP) as Substrate

  Materials
  • p‐nitrophenyl phosphate (pNPP; MW = 263.1; Sigma) substrate
  • PTP1B assay buffer (see recipe)
  • Inhibitors/test compounds
  • 10% (v/v) DMSO
  • ∼1 mg/ml PTP1B (see protocol 3) in PTP1B enzyme buffer (see recipe) (Human PTP1B residues 1 to 321)
  • PTP1B enzyme buffer (see recipe)
  • BSA (Sigma or other of similar or better purity; optional)
  • 2 mM (0.278 mg/ml) p‐nitrophenol (pNP; Sigma) in PTP1B assay buffer recipe (freeze in aliquots for up to 6 months at −20°C)
  • SpectraMax plate reader (Molecular Devices) or any other 96‐well plate reader capable of measuring absorbance at 405 nm and creating a file of A 405 values at the various time points
  • Any 96‐well clear‐bottom plates for reading absorbance
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 2: Time‐point PTP1B Assay Using a Synthetic Phosphopeptide as Substrate and Malachite Green Reagent to Detect Released Phosphate

  Materials
  • 1% (v/v) Tween 20
  • Malachite green reagent (Upstate Biotechnology)
  • Appropriate phosphopeptide substrate
  • PTP1B assay buffer (see recipe)
  • Inhibitors/test compounds
  • 10% (v/v) DMSO
  • ∼1 mg/ml PTP1B (see protocol 3) in PTP1B enzyme buffer (see recipe)
  • 10 mM Na 2HPO 4 (phosphate standard)
  • Any 96‐well clear‐bottom plates
  • SpectraMax plate reader (Molecular Devices) or any other 96‐well plate reader capable of measuring at 620 nm and creating a file of A 620 values
NOTE: All volumes in this protocol are based on a reaction of 225 µl for four time points (50 µl per time point plus 25 µl extra), but the reaction volume can be scaled to accommodate a different number of time points.

Support Protocol 1: Purification of Human PTP1B from E. COLI

  Materials
  • Human PTP‐1B (1‐321)–expressed E. coli BL21(DE3)
  • Lysis buffer (see recipe)
  • 5 × 21–cm S‐Sepharose FF column (Amersham Pharmacia Biotech)
  • S‐Sepharose buffer A (see recipe)
  • Liquid nitrogen
  • Rannie homogenizer or French Press
  • Centrifuge and rotor (e.g., Beckman JA‐14 rotor)
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Figures

Videos

Literature Cited

Literature Cited
   Ahmad, F., Azevedo, J.L., Cortwright, R., Dohm, G.L., and Goldstein, B.J. 1997. Alterations in skeletal muscle protein‐tyrosine phosphatase activity and expression in insulin‐resistant human obesity and diabetes. J. Clin. Invest. 100:449‐458.
   Cornish‐Bowden, A. 1995. Fundamentals of Enzyme Kinetics. Revised edition. Portland Press, London.
   Elchebly, M., Payette, P., Michaliszyn, E., Cromlish, W., Collins, S., Loy, A.L., Normandin, D., Cheng, A., Himms‐Hagen, J., Chan, C.‐C., Ramachandran, C., Gresser, M.J., Tremblay, M.L., and Kennedy, B.P. 1999. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase‐1B gene. Science 283:1544‐1548.
   Geladopoulos, T.P., Sotiroudis, T.G., and Evangelopoulos, A.E. 1991. A malachite green colorimetric assay for protein phosphatase activity. Anal. Biochem. 192:112‐116.
   Goldstein, B.J., Bittner‐Kowalczyk, A., White, M.F., and Harbeck, M. 2000. Tyrosine dephosphorylation and deactivation of insulin receptor substrate‐1 by protein‐tyrosine phosphatase 1B. Possible facilitation by the formation of a ternary complex with the GRB2 adaptor protein. J. Biol. Chem. 275:4283‐4289.
   Iversen, L.F. and Andersen, H.S., Branner, S., Mortensen, S.B., Peters, G.H., Norris, K., Olsen, O.H., Jeppesen, C.B., Lundt, B.F., Ripka, W., Mϕller, K.B., and Mϕsller, N.P.H. 2000. Structure‐based design of a low molecular weight, nonphosphorus, nonpeptide and highly selective inhibitor of protein‐tyrosine phosphatase 1 B. J. Biol. Chem. 275:10300‐10307.
   Kenner, K.A., Anyanwu, E., Olefsky, J.M., and Kusari, J. 1996. Protein‐tyrosine phosphatase 1B is a negative regulator of insulin‐ and insulin‐like growth factor 1‐stimulated signaling. J. Biol. Chem. 271:19810‐19816.
   Salmeen, A. and Andersen, J.N., Myers, M.P., Tonks, N.K., and Barford, D. 2000. Molecular basis for the dephosphorylation of the activation segment of the insulin receptor by protein tyrosine phosphatase 1B. Mol. Cell. 6:1401‐1412.
   Sarmiento, M., Zhao, Y., Gordon, S.J., and Zhang, Z.‐Y. 1998. Molecular basis for substrate specificity of protein‐tyrosine phosphatase‐1B. J. Biol. Chem. 273:26368‐26374.
   Skorey, K., Kelly, J., Hammond, M., Ramachandran, C., Huang, Z., Gresser, M.J., and Wang, Q. 1997. How does alendronate inhibit protein‐tyrosine phosphatases? J. Biol. Chem. 272:22472‐22480.
   Vetter, S.W., Keng, Y.‐F., Lawrence, D.S., and Zhang, Z.‐Y. 2000. Assessment of protein‐tyrosine phosphatase 1B substrate specificity using “inverse alanine scanning”. J. Biol. Chem. 275:2265‐2268.
   Zhang, Z.‐Y. 1998. Protein‐tyrosine phosphatases: Biological function, structural characteristics, and mechanism of catalysis. CRC Crit. Rev. Biochem. Mol. Biol. 33:1‐52.
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