ERK-MAP Kinase signaling in the cytoplasm

Methods Mol Biol. 2010;661:185-203. doi: 10.1007/978-1-60761-795-2_11.

Abstract

ERK-MAPK is activated by dual phosphorylation of its activation loop TEY motif by the MEK-MAPKK. ERK cytoplasmic activity should be measured by assaying both the level of dually phosphorylated ERK and the level of phosphorylated substrate. We describe two complementary methods for quantitatively measuring ERK activity toward the cytoplasmic p90 ribosomal S6 kinase (RSK). The first method is a straightforward immunoblot of endogenous ERK and RSK phosphoepitopes using phospho-specific antibodies. Infrared fluorescent secondary antibodies provide a linear readout that is quantitated using an Odyssey scanner (LI-COR). The second method is an immunoprecipitation of ERK followed by an in vitro immune complex kinase assay with purified GST-RSK as substrate. The level of ERK phosphotransferase activity, or (32)P-labeled phosphate transfer, is quantitated using a PhosphorImager.

MeSH terms

  • Animals
  • Biocatalysis
  • Cytoplasm / enzymology*
  • Cytoplasm / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Assays / methods*
  • Extracellular Signal-Regulated MAP Kinases / metabolism*
  • HEK293 Cells
  • Humans
  • Immunoblotting
  • Immunoprecipitation
  • MAP Kinase Signaling System*
  • Mitogen-Activated Protein Kinases / metabolism*
  • Phosphorylation
  • Phosphotransferases / metabolism
  • Protein Structure, Tertiary
  • Ribosomal Protein S6 Kinases, 90-kDa / chemistry
  • Ribosomal Protein S6 Kinases, 90-kDa / metabolism

Substances

  • Phosphotransferases
  • Ribosomal Protein S6 Kinases, 90-kDa
  • Extracellular Signal-Regulated MAP Kinases
  • Mitogen-Activated Protein Kinases