The synthetic peptide RPRAATF allows specific assay of Akt activity in cell lysates

Anal Biochem. 2002 Jun 1;305(1):32-9. doi: 10.1006/abio.2002.5659.

Abstract

The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immunoprecipitation assays.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Animals
  • COS Cells
  • Cattle
  • Cells, Cultured
  • Humans
  • Immunoblotting
  • Mice
  • Mice, Inbred BALB C
  • Myelin Basic Protein / chemistry
  • Myelin Basic Protein / metabolism
  • Oligopeptides / chemical synthesis
  • Oligopeptides / metabolism*
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphotransferases / analysis
  • Phosphotransferases / chemistry
  • Platelet-Derived Growth Factor / pharmacology
  • Protein-Serine-Threonine Kinases / analysis
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proto-Oncogene Proteins / analysis
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-akt
  • Receptors, Platelet-Derived Growth Factor / analysis
  • Receptors, Platelet-Derived Growth Factor / metabolism
  • Substrate Specificity

Substances

  • Myelin Basic Protein
  • Oligopeptides
  • Platelet-Derived Growth Factor
  • Proto-Oncogene Proteins
  • Phosphotransferases
  • Phosphatidylinositol 3-Kinases
  • Receptors, Platelet-Derived Growth Factor
  • AKT1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt