Enzyme-linked immunosorbent assay for measurement of JNK, ERK, and p38 kinase activities.
A rapid enzyme-linked immunosorbent assay for the enzyme activity measurement of three well-known mitogen-activated protein (MAP) kinases, JNK2, ERK2, and p38 is described. The assay involves immobilization of the respective kinase substrates c-Jun, Elk1, or ATF2 on microtiter plates, addition of the kinase reaction mixture, and measurement of substrate phosphorylation using phospho-epitope-specific antibodies. This novel procedure represents a marked improvement to conventional radioactive MAP kinase assays in terms of quantification, precision, performance at physiological ATP concentration, high throughput, time consumption and amenability to automation. In addition to the standard solid phase assay using plastic-bound protein substrates, we developed an alternative solution phase protocol using soluble protein substrates. By comparing the results of the two assays, we found that MAP kinases retained much of their substrate specificity in the phosphorylation of immobilized protein substrates. Interestingly, we observed a strong preference of JNK2 and p38 for the phosphorylation of dimeric over monomeric substrates. We further characterized the kinase inhibitory activity of olomoucine, staurosporine, and SB 203580 for JNK2, ERK2, and p38. Taken together, this assay could assist in the biochemical characterization of MAP kinases and in identifying potent and specific inhibitors of these enzymes.
Forrer
P
P
Institute of Medical Radiobiology of the Paul Scherrer Institute and the University of Zürich, Villigen PSI, Switzerland.
Tamaskovic
R
R
Jaussi
R
R
D016475:3T3 Cells; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D007701:Kinetin; D051379:Mice; D010766:Phosphorylation; D011687:Purines / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D000939:Epitopes; D007093:Imidazoles; D011687:Purines; D011725:Pyridines; D011994:Recombinant Proteins; C090046:olomoucine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019311:Staurosporine; C093642:SB 203580; D007701:Kinetin