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

• Published in: The Journal of biological chemistry 1993-03, Vol.268 (7), p.5097-5106
• Main Authors: ROBBINS, D. J, ZHEN, E, OWAKI, H, VANDERBILT, C. A, EBERT, D, GEPPERT, T. D, COBB, M. H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; DNA; enzymatic activity; Enzyme Activation; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Histidine - metabolism; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molecular Sequence Data; mutants; Mutation; Phosphorylation; properties; protein kinase 1; protein kinase 2; Protein Kinases - genetics; Protein Kinases - isolation & purification; Protein Kinases - metabolism; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - isolation & purification; Protein-Serine-Threonine Kinases - metabolism; Rabbits; Rats; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; regulation; Transferases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)53507-9

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 approximately 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 (approximately 2 mumol/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.