title: Development of a non-radioactive, 384-well format assay to detect inhibitors of the mitogen-activated protein kinase kinase 4.
abstract: 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-SAPK alpha-kinase-negative (KN) mutant as substrate. We determined the kinetic constants for ATP and GST-SAPK alpha-KN. The apparent Km value for GST-SAPKalpha-KN was 3.7 microM, while the apparent Km value for ATP was 0.17 microM. 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 anti-phosphotyrosine antibody, which allowed time-resolved fluorescence measurement.
authors.affiliation: Bayer Yakuhin, Ltd., Research Center, Kyoto, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Togame
authors.forename: K
authors.initials: K
authors.lastname: Fuchikami
authors.forename: A
authors.initials: A
authors.lastname: Sagara
authors.forename: H
authors.initials: H
authors.lastname: Inbe
authors.forename: K
authors.initials: K
authors.lastname: Ziegelbauer
mesh_terms: D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D016231:Fluorescence Polarization Immunoassay / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012371:Robotics / Q000295:instrumentation / Q000379:methods*
chemical_list: D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D048670:MAP Kinase Kinase 4