Use of Protein Phosphatase Inhibitors

Douglas C. Weiser1, Shirish Shenolikar1

1 Duke University Medical Center, Durham
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 13.10
DOI:  10.1002/0471140864.ps1310s31
Online Posting Date:  May, 2003
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Abstract

Reversible protein phosphorylation is recognized as a major mechanism regulating the physiology of plant and animal cells. Virtually every biochemical process within eukaryotic cells is controlled by the covalent modification of key regulatory proteins. This in turn dictates the cellular response to a variety of physiological and environmental stimuli; errors in signals transduced by phosphoproteins contribute to many human diseases. Thus, defining protein phosphorylation events, and specifically, the phosphoproteins involved, is crucial for obtaining a better understanding of the physiological events that distinguish normal and diseased states. Protein phosphatase inhibitors are useful when deciphering physiological events regulated by reversible protein phosphorylation but the hormonal stimuli or signaling pathways involved are not known. They are also useful in analyzing the impact of hormones and other physiological stimuli on the function of a specific phosphoprotein. This unit describes protocols for inhibiting the cellular PP1/PP2A activity with okadaic acid, microcystin‐LR, and PP2B/calcineurin and a widely utilized strategy for inhibiting protein tyrosine phosphatases.

     
 
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Table of Contents

  • Basic Protocol 1: Inhibition of Cellular PP1/PP2A Activity with Okadaic Acid
  • Basic Protocol 2: Inhibition of PP1/PP2A Activity In Vitro with Microcystin‐LR
  • Basic Protocol 3: Inhibition of Cellular PP2B/Calcineurin Activity with Cyclosporin A
  • Basic Protocol 4: In Vitro Inhibition of PP2B/Calcineurin Activity with Cypermethrin
  • Basic Protocol 5: Inhibition of Protein Tyrosine Phosphatases with Sodium Orthovanadate
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Inhibition of Cellular PP1/PP2A Activity with Okadaic Acid

  Materials
  • Cultured mammalian cells grown to <50% confluence ( appendix 3C)
  • 0.1 or 1.0 mM okadaic acid sodium salt (Alexis Biochemicals or Sigma), in DMSO (store at −20°C in a light‐proof container under N 2)
  • 0.1 or 1.0 mM 1‐norokadaone (Calbiochem or Sigma) in DMSO (store at −20°C in a light‐proof container under N 2)
NOTE: Okadaic acid (OA), either as sodium, potassium, or ammonium salt, shows increased solubility in aqueous solutions compared to the free acid. The solutions of the OA salts are also more stable during storage. However, for reasons that are not entirely clear, aqueous solutions of OA lose their activity as phosphatase inhibitors over time. By comparison, the stock solutions of OA in DMSO (or ethanol or methanol) can be stored in a dark glass container under nitrogen at −20°C for many months without significant loss of activity.

Basic Protocol 2: Inhibition of PP1/PP2A Activity In Vitro with Microcystin‐LR

  Materials
  • Tissue or cells
  • 0.1 or 1.0 mM microcystin‐LR (Alexis Biochemicals or Sigma) in DMSO (store at −20°C)

Basic Protocol 3: Inhibition of Cellular PP2B/Calcineurin Activity with Cyclosporin A

  Materials
  • 0.1 mM cyclosporin A (Alexis Biochemical or Sigma) in DMSO
  • Cultured cells

Basic Protocol 4: In Vitro Inhibition of PP2B/Calcineurin Activity with Cypermethrin

  Materials
  • 1.0 mM cypermethrin (Alexis Biochemicals) in DMSO and store in dark glass containers pretreated with 1% polyethylene glycol (PEG)
  • Sample cell extracts or subcellular fractions
NOTE: Cypermethrin and other type II pyrethroids are sold in both solid and liquid form. The liquid form represents cypermethrin dissolved in organic solvents, such as DMSO, acetone, or ethanol. Hydrophobicity of these compounds means that long‐term storage in plastic or polyethylene containers should be avoided because these compounds adhere to such surfaces and may be slowly extracted from solution. Many pyrethroids are also light‐sensitive. If stored in dark glass containers pretreated with 1% (w/v) PEG to prevent surface adherence, stock solutions of cypermethrin are stable for many months. For cellular studies using cypermethrin, the compound should be added directly to the culture medium as its PP2B inhibitory activity is reduced when made up in fresh medium. While the underlying basis for a time‐dependent loss of cypermethrin activity is not clear, this provides a strong argument for using cypermethrin and the pyrethroid inhibitors solely for in vitro studies of PP2B function.

Basic Protocol 5: Inhibition of Protein Tyrosine Phosphatases with Sodium Orthovanadate

  Materials
  • Sodium orthovanadate (Sigma)
  • 0.1 M NaOH
  • Sample cells
  • Boiling water bath or heating block
  • Additional reagents and equipment for metabolic labeling (unit 13.2) or immunoblotting with an anti‐phosphotyrosine antibody (unit 13.6)
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Literature Cited

Literature Cited
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Key References
   Current Medicinal Chemistry. November, 2002, vol. 9.
  A special “hot topics” issue, which is devoted to the synthesis of serine/threonine phosphatase inhibitors.
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