Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase).
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.
Payne
D M
DM
Department of Internal Medicine and Pharmacology, University of Virginia, Charlottesville 22908.
Rossomando
A J
AJ
Martino
P
P
Erickson
A K
AK
Her
J H
JH
Shabanowitz
J
J
Hunt
D F
DF
Weber
M J
MJ
Sturgill
T W
TW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin
D010446:Peptide Fragments; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin