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

doi:10.1016/s0021-9258(18)53507-9

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 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.

Highlights

Purified recombinant ERKZ autophosphorylates in a manner that generates a small amount of the tyrosine- and threoninephosphorylated peptide whose appearance is associated with enzyme activation [29]
A second mechanism of activation has been suggested by the identification of an enzyme, known variously as MAP kinase/Extracellular signal-regulated protein kinases (ERK) activator or MAP kinase/ERK kinase [28, 31, 32] which appears to catalyze the phosphorylation of both the tyrosine and the threonine required for maximum activity of these enzymes
Nondenatured ERK2 is the only substrate identified far that is phosphorylated at a rapid rate

Summary

RESULTS

Itol (DTT), EGTA, EDTA, and benzamidine to give final concentra- Purification of Recombinant ERKs-To examine the proptions of 1, 1, 1, and 10 mM, respectively. Fractions were pooledbased on protein staining, dialyzed overnight against ERK purification buffer (20 mM Tris, pH 7.5, 1 mM DTT, 1 mM EGTA, 10 mM benzamidine, and 10%glycerol),and stored at -80 "C.Approximately 10 mg of purified ERKZ and 2-5 mg of purified ERKl were recovered erties and activation of ERKs in vitro, the wild type proteins and mutants were expressed with Hiss tags to allow their rapid purification on nickel-chelate resin. ERKZ began to elute from the resin as the imidazole gradient progressed as shown by the appearance of a 42-kDa protein by both Coomassie Blue staining (Fig. lA)and immunoblotting with antiserum 691 was collected by sedimentation for 2 min in a microfuge, and the (Fig. 1B).

C Silver

Findings

DISCUSSION