An in vitro 96-well plate assay of the mitogen-activated protein kinase cascade

Mitogen-activated protein (MAP) kinases of the extracellular signal-regulat ed kinase (ERK) family are activated in response to many growth and differe ntiation factors as well as some oncogenes. ERK activation follows phosphor ylation by a class of specific upstream MAP kinase/ERK kinase (MEK) exempli fied by MEK-1. Activated ERKs control many short- and long-term changes in cell function through phosphorylating a number of intracellular target subs trates which include stathmin, a phosphoprotein regulating microtubule stab ility. We report here the development of a simple, 96-well plate, quantitat ive in vitro assay measuring purified ERK2 catalytic activation by a consti tutive MEK-1 mutant (S218E S222E). Enzymatic activity was detected by P-33 phosphorylation of purified biotinylated stathmin captured on streptavidin- coated scintillation proximity assay beads which eliminates the need for wa sh steps. The assay was optimized and the K-0.5 value for ATP was found to be 0.9 mu M and the K-m for stathmin was determined to be 16 mu M. The assa y was also used to determine IC50 values for the protein kinase inhibitors PD98059 and staurosporine. This simple assay allows several hundred quantit ative measurements of MEK1-dependent ERK2 activation to be performed in a d ay. (C) 1999 Academic Press.