Development of a Non-Radioactive,384-Well Format Assay to Detect Inhibitors of the Mitogen-Activated Protein Kinase Kinase 4

Mitogen-activated protein kinase (MAPK) kinases (MKKs, also called MAPK/extracellular signal-regulated kinase [ERK] kinase [MEK]) are constituents of numerous signal transduction pathways involved in growth, differentiation, and stress response. One of its members, MKK4, directly phosphorylates and activates the c-Jun terminal kinases (also called stress-activated protein kinase [SAPK]) in response to stress and pro-inflammatory cytokines. Recent evidence suggest that control of MKK4 activity may provide a novel approach for the treatment of cancer or as anti-inflammatory therapy. To screen for novel low-molecular-weight inhibitors of MKK4, we established a quantitative, non-radioactive in vitro kinase assay. Human MKK4 was expressed as fusion protein with glutathione S-transferase (GST) in Escherichia coli. Co-expression of a constitutive active fragment of the MAPK/ERK kinase kinase-1 yielded active GST-MKK4 using GST-SAPKα-kinase-negative (KN) mutant as substrate. We determined the kinetic constants for ATP and GST-SAPKα-KN. The apparentKm value for GST-SAPKα-KN was 3.7 µM, while the apparent Km value for ATP was 0.17 µM. Staurosporine inhibited GST-MKK4 with an IC₅₀ of 70 nM. The kinase assay was adapted to a 384-well non-radioactive format. After the kinase reaction the phosphorylated product was captured onto a streptavidin-coated microtiter plate, and phosphorylation was detected with a europium-labeled antiphosphotyrosine antibody, which allowed time-resolved fluorescence measurement.