Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase)

EMBO J. 1991 Apr;10(4):885-92.

Abstract

Mitogen-activated protein kinase (MAP kinase) is a 42 kd serine/threonine protein kinase whose enzymatic activity requires phosphorylation of both tyrosyl and threonyl residues. As a step in elucidating the mechanism(s) for activation of this enzyme, we have determined the sites of regulatory phosphorylation. Following proteolytic digestion of 32P-labeled pp42/MAP kinase with trypsin, only a single phosphopeptide was detected by two-dimensional peptide mapping, and this peptide contained both phosphotyrosine and phosphothreonine. The amino acid sequence of the peptide, including the phosphorylation sites, was determined using a combination of Fourier transform mass spectrometry and collision-activated dissociation tandem mass spectrometry with electrospray ionization. The sequence for the pp42/MAP kinase tryptic phosphopeptide is similar (but not identical) to a sequence present in the ERK1- and KSS1-encoded kinases. The two phosphorylation sites are separated by only a single residue. The regulation of activity by dual phosphorylations at closely spaced threonyl and tyrosyl residues has a functional correlate in p34cdc2, and may be characteristic of a family of protein kinases regulating cell cycle transitions.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Cell Line
  • Electrophoresis, Polyacrylamide Gel
  • Mass Spectrometry
  • Molecular Sequence Data
  • Peptide Fragments / isolation & purification
  • Phosphorylation
  • Protein Kinases / genetics*
  • Protein Kinases / isolation & purification
  • Protein Kinases / metabolism
  • Sequence Homology, Nucleic Acid
  • Trypsin

Substances

  • Peptide Fragments
  • Protein Kinases
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Trypsin