Design and synthesis of fluorescent activity probes for protein phosphatases

Methods Enzymol. 2019:622:29-53. doi: 10.1016/bs.mie.2019.02.002. Epub 2019 Mar 8.

Abstract

Protein phosphatases act in concert with protein kinases to regulate and maintain the phosphoproteome. However, the catalog of chemical tools to directly monitor the enzymatic activity of phosphatases has lagged behind their kinase counterparts. In this chapter, we provide protocols for repurposing the phosphorylation-sensitive sulfonamido-oxine fluorophore known as Sox to afford direct activity probes for phosphatases. With validated activity probes in-hand, inhibitor screens can be conducted with recombinant enzyme and the role of phosphatases in cell signaling can be investigated in unfractionated cell lysates.

Keywords: Biosensor; Cell signaling; Fluorescent probes; Phosphatase; Sensor.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Biosensing Techniques / methods
  • Chemistry Techniques, Synthetic / methods
  • Enzyme Assays / methods
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / metabolism
  • Humans
  • Oxyquinoline / analogs & derivatives*
  • Oxyquinoline / chemical synthesis
  • Oxyquinoline / metabolism
  • Phosphoprotein Phosphatases / analysis
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphorylation
  • Signal Transduction
  • Spectrometry, Fluorescence / methods
  • Sulfonamides / chemical synthesis
  • Sulfonamides / chemistry*
  • Sulfonamides / metabolism

Substances

  • Fluorescent Dyes
  • Sulfonamides
  • Oxyquinoline
  • Phosphoprotein Phosphatases