Metabolic labeling of mitogen-activated protein kinase kinase in A431 cells demonstrates phosphorylation on serine and threonine residues.
Mitogen-activated protein (MAP) kinase kinase is an enzyme that activates the growth factor-regulated MAP kinase in vitro by a mechanism that involves direct phosphorylation of MAP kinase on tyrosine and threonine residues. MAP kinase kinase is stimulated by growth factor treatment of cells and has been shown to be inactivated with protein phosphatases, suggesting that it is regulated by protein phosphorylation. Analysis of two epidermal growth factor-stimulated forms of MAP kinase kinase, purified from 32P-labeled A431 cells, shows that the kinase is phosphorylated on serine and threonine residues and that treatment with protein phosphatases leads to serine dephosphorylation. Under conditions that lead to complete inactivation, only partial dephosphorylation of MAP kinase kinase is observed. Consistent with this finding, inactive forms of MAP kinase kinase, which separate from active forms during the course of purification, are also observed to be phosphorylated in intact cells.
Department of Biochemistry, University of Washington, Seattle 98195.
N G
NG
Ahn
J S
JS
Campbell
R
R
Seger
A L
AL
Jensen
L M
LM
Graves
E G
EG
Krebs
D002460:Cell Line; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D006801:Humans; D006882:Hydroxyapatites; D007700:Kinetics; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis / Q000378:metabolism*; D010769:Phosphothreonine / Q000032:analysis / Q000378:metabolism*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D006882:Hydroxyapatites; D010769:Phosphothreonine; D010768:Phosphoserine; D017886:Durapatite; D011494:Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases