ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR MEASUREMENT OF JNK, ERK, AND P38 KINASE-ACTIVITIES

A rapid enzyme-linked immunosorbent assay for the enzyme activity meas urement of three well-known mitogen-activated protein (MAP) kinases, J NK2, ERK2, and p38 is described, The assay involves immobilization of the respective kinase substrates c-Jun, Elk1, or ATF2 on microtiter pl ates, addition of the kinase reaction mixture, and measurement of subs trate phosphorylation using phospho-epitope-specific antibodies. This novel procedure represents a marked improvement to conventional radioa ctive MAP kinase assays in terms of quantification, precision, perform ance at physiological ATP concentration, high throughput, time consump tion and amenability to automation. In addition to the standard solid phase assay using plastic-bound protein substrates, we developed an al ternative solution phase protocol using soluble protein substrates. By comparing the results of the two assays, we found that MAP kinases re tained much of their substrate specificity in the phosphorylation of i mmobilized protein substrates, Interestingly, we observed a strong pre ference of JNK2 and p38 for the phosphorylation of dimeric over monome ric substrates. We further characterized the kinase inhibitory activit y of olomoucine, staurosporine, and SE 203580 for JNK2, ERK2, and p38, Taken together, this assay could assist in the biochemical characteri zation of MAP kinases and in identifying potent and specific inhibitor s of these enzymes.