A semisynthetic epitope for kinase substrates

Nat Methods. 2007 Jun;4(6):511-6. doi: 10.1038/nmeth1048. Epub 2007 May 7.


The ubiquitous nature of protein phosphorylation makes it challenging to map kinase-substrate relationships, which is a necessary step toward defining signaling network architecture. To trace the activity of individual kinases, we developed a semisynthetic reaction scheme, which results in the affinity tagging of substrates of the kinase in question. First, a kinase, engineered to use a bio-orthogonal ATPgammaS analog, catalyzes thiophosphorylation of its direct substrates. Second, alkylation of thiophosphorylated serine, threonine or tyrosine residues creates an epitope for thiophosphate ester-specific antibodies. We demonstrated the generality of semisynthetic epitope construction with 13 diverse kinases: JNK1, p38alpha MAPK, Erk1, Erk2, Akt1, PKCdelta, PKCepsilon, Cdk1/cyclinB, CK1, Cdc5, GSK3beta, Src and Abl. Application of this approach, in cells isolated from a mouse that expressed endogenous levels of an analog-specific (AS) kinase (Erk2), allowed purification of a direct Erk2 substrate.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives
  • Amino Acid Sequence
  • Animals
  • Epitopes / chemistry*
  • Epitopes / immunology
  • Epitopes / metabolism*
  • Extracellular Signal-Regulated MAP Kinases / genetics
  • Extracellular Signal-Regulated MAP Kinases / metabolism*
  • Gene Duplication
  • Haptens / chemistry*
  • Haptens / immunology
  • Haptens / metabolism*
  • Immunoglobulin G / chemistry
  • Immunoglobulin G / metabolism
  • Isotope Labeling / methods
  • Mice
  • Mice, Knockout
  • Organothiophosphates / chemistry
  • Organothiophosphates / metabolism
  • Substrate Specificity


  • Epitopes
  • Haptens
  • Immunoglobulin G
  • Organothiophosphates
  • adenosine 5'-O-(3-thiotriphosphate)
  • Adenosine Triphosphate
  • Extracellular Signal-Regulated MAP Kinases