Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells

Rony Seger, Natalie G. Ahn, James Posada, Erlynda S. Munar, Amy M. Jensen, Jonathan A. Cooper, Melanie H. Cobb, Edwin G. Krebs

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)14373-14381
Number of pages9
JournalJournal of Biological Chemistry
Volume267
Issue number20
StatePublished - Jan 1 1992

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Seger, R., Ahn, N. G., Posada, J., Munar, E. S., Jensen, A. M., Cooper, J. A., Cobb, M. H., & Krebs, E. G. (1992). Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells. Journal of Biological Chemistry, 267(20), 14373-14381.