Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro

D. J. Robbins, E. Zhen, H. Owaki, C. A. Vanderbilt, D. Ebert, T. D. Geppert, M. H. Cobb

Research output: Contribution to journalArticle

419 Scopus citations

Abstract

Extracellular signal-regulated protein kinases (ERK) 1 and 2 and mutants of each were expressed in bacteria with a hexahistidine tag and purified using nickel-chelate chromatography. Basal activity of wild type ERK2 was ∼2 nmol/min/mg. Self-catalyzed phosphorylation occurred in vitro on the major physiological site of tyrosine phosphorylation in an intramolecular reaction. Rabbit muscle ERK activator activated ERK2 500-1000-fold up to a specific activity (∼2 μmol/min/mg) approximating that of ERK1 purified from stimulated cells (Boulton, T. G., Gregory, J. S., and Cobb, M. H. (1991) Biochemistry 30, 278-286). ERK1 could also be activated by the ERK activator to the same extent. Mutants lacking the major site of tyrosine phosphorylation were autophosphorylated at a greatly reduced rate and were no longer highly activated by the ERK kinase. Mutants lacking the major site of threonine phosphorylation were autophosphorylated at the same or an enhanced rate, but the kinase activity of these mutants depended on the residue used to replace the threonine. Replacement by glutamate rendered the kinase capable of being activated by ERK activator, while replacement by alanine did not. Thus, the carboxyl group of glutamate can provide at least some of the features introduced by phosphothreonine in activated ERKs.

Original languageEnglish (US)
Pages (from-to)5097-5106
Number of pages10
JournalJournal of Biological Chemistry
Volume268
Issue number7
StatePublished - Jan 1 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Robbins, D. J., Zhen, E., Owaki, H., Vanderbilt, C. A., Ebert, D., Geppert, T. D., & Cobb, M. H. (1993). Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro. Journal of Biological Chemistry, 268(7), 5097-5106.