ERK-MAP Kinase signaling in the cytoplasm. Academic Article uri icon

Overview

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.

publication date

  • January 1, 2010

Research

keywords

  • Cytoplasm
  • Enzyme Assays
  • Extracellular Signal-Regulated MAP Kinases
  • MAP Kinase Signaling System
  • Mitogen-Activated Protein Kinases

Identity

Scopus Document Identifier

  • 79952114793

Digital Object Identifier (DOI)

  • 10.1007/978-1-60761-795-2_11

PubMed ID

  • 20811984

Additional Document Info

volume

  • 661