Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells.
Two peaks of mitogen-activated protein (MAP) kinase activator activity are resolved upon ion exchange chromatography of cytosolic extracts from epidermal growth factor-stimulated A431 cells. Two forms of the activator (1 and 2) have been purified from these peaks, using chromatography on Q-Sepharose, heparin-agarose, hydroxylapatite, ATP-agarose, Sephacryl S-300, Mono S, and Mono Q. The two preparations each contained one major protein band with an apparent molecular mass of 46 or 45 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Evidence identifying the MAP kinase activators as the 46- and 45-kDa proteins is presented. Using inactive mutants of MAP kinase as potential substrates, it was found that each preparation of MAP kinase activator catalyzes phosphorylation of the regulatory residues, threonine 188 and tyrosine 190, of Xenopus MAP kinase. These results support the concept that the MAP kinase activators are protein kinases. These MAP kinase kinases demonstrate an apparent high degree of specificity toward the native conformation of MAP kinase, although slow autophosphorylation on serine, threonine, and tyrosine residues and phosphorylation of myelin basic protein on serine and threonine residues is detected as well.
Seger
R
R
Department of Pharmacology, University of Washington, Seattle 98195.
Ahn
N G
NG
Posada
J
J
Munar
E S
ES
Jensen
A M
AM
Cooper
J A
JA
Cobb
M H
MH
Krebs
E G
EG
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D017886:Durapatite; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D006882:Hydroxyapatites; D020929:Mitogen-Activated Protein Kinase Kinases; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D014981:Xenopus
D006882:Hydroxyapatites; D011506:Proteins; D004815:Epidermal Growth Factor; D017886:Durapatite; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases