The phorbol ester-dependent activator of the mitogen-activated protein kinase p42mapk is a kinase with specificity for the threonine and tyrosine regulatory sites.
Mitogen-activated protein kinases (MAP kinases) are activated by dual tyrosine and threonine phosphorylations in response to various stimuli, including phorbol esters. To define the mechanism of activation, recombinant wild-type 42-kDa MAP kinase (p42mapk) and a kinase-defective mutant of p42mapk (K52R) were used to assay both activator activity for p42mapk and kinase activity toward K52R in stimulated EL4.I12 mouse thymoma cells. Phorbol 12,13-dibutyrate (10 min, 650 nM) stimulated a single peak of MAP kinase activator that was coeluted from Mono Q at pH 7.5 and 8.9 with K52R kinase activity. Both activities were inactivated by the serine/threonine-specific phosphatase 2A but not by the tyrosine-specific phosphatase CD45. Phosphorylation of K52R occurred specifically on Thr-183 and Tyr-185, as determined by tryptic phosphopeptide mapping in comparison with synthetic marker phosphopeptides. These findings indicate that phorbol ester-stimulated MAP kinase kinase can activate p42mapk by threonine and tyrosine phosphorylations, and that p42mapk thus does not require an autophosphorylation reaction.
Rossomando
A
A
Department of Internal Medicine, University of Virginia, Charlottesville 22908.
Wu
J
J
Weber
M J
MJ
Sturgill
T W
TW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004789:Enzyme Activation; D007700:Kinetics; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010748:Phosphopeptides / Q000302:isolation & purification; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011994:Recombinant Proteins / Q000378:metabolism; D013945:Thymoma; D013953:Thymus Neoplasms
D010748:Phosphopeptides; D011994:Recombinant Proteins; D015240:Phorbol 12,13-Dibutyrate; D010770:Phosphotransferases; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2