METHODS OF THE IDENTIFICATION OF PHARMACEUTICALLY ACTIVE COMPOUNDS

CSBP/p38 is a MAP kinase that is activated in response to stress, endotoxin, interleukin 1 and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, a kinase has been cloned which shares ~70% amino acid identity to MAPKAP kinase...

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Hauptverfasser:	YOUNG, PETER, RONALD, KUMAR, SANJAY, LIVI, GEORGE, PETRO, MCLAUGHLIN, MEGAN, MCHALE
Format:	Patent
Sprache:	eng
Schlagworte:	BEER BIOCHEMISTRY CHEMISTRY COMPOSITIONS OR TEST PAPERS THEREFOR COMPOSITIONS THEREOF CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES CULTURE MEDIA ENZYMOLOGY HUMAN NECESSITIES HYGIENE INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIRCHEMICAL OR PHYSICAL PROPERTIES MEASURING MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS MEDICAL OR VETERINARY SCIENCE METALLURGY MICROBIOLOGY MICROORGANISMS OR ENZYMES MUTATION OR GENETIC ENGINEERING PHYSICS PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES PROCESSES OF PREPARING SUCH COMPOSITIONS PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS ORMEDICINAL PREPARATIONS SPIRITS TESTING VINEGAR WINE
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Beschreibung
Zusammenfassung:	CSBP/p38 is a MAP kinase that is activated in response to stress, endotoxin, interleukin 1 and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, a kinase has been cloned which shares ~70% amino acid identity to MAPKAP kinase-2, and thus was designated MAPKAP kinase-3. The binding of CSBP to MAPKAP kinase 3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2 and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially-expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co-expressed in HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2, and was then able to phosphorylate HSP27 in vitro. Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preincubation of cells with 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-4-pyridyl)-1H-imidazole, a specific inhibitor of CSBP kinase activity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo. The use of MAPKAP kinase-3 in screens for the identification of pharmaceutically active compounds is disclosed.