Dual Enzyme-Linked Immunosorbent Assay System for Detection of Endogenous Kinase Activities of Mitogen- and Stress-Activated Protein Kinase-1/2
Autor: | Hiroko Toyoda, Takato Noumi, Keizo Yamashita, Hiroshi Sootome, Mikiya Suda, Tsuyoshi Ishii |
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Rok vydání: | 2007 |
Předmět: |
Immunoblotting
Enzyme-Linked Immunosorbent Assay Biology Mitogen-activated protein kinase kinase Transfection p38 Mitogen-Activated Protein Kinases MAP2K7 Mitogen-Activated Protein Kinase 11 Drug Discovery Humans Immunoprecipitation Mitogen-Activated Protein Kinase 8 c-Raf Phosphorylation Cyclic AMP Response Element-Binding Protein Heat-Shock Proteins Mitogen-Activated Protein Kinase 1 Protein Synthesis Inhibitors Epidermal Growth Factor MAP kinase kinase kinase Cyclin-dependent kinase 4 Cyclin-dependent kinase 2 Protein kinase R Biochemistry biology.protein Molecular Medicine Cyclin-dependent kinase 9 Anisomycin HeLa Cells Plasmids |
Zdroj: | ASSAY and Drug Development Technologies. 5:523-534 |
ISSN: | 1557-8127 1540-658X |
DOI: | 10.1089/adt.2007.063 |
Popis: | The kinase signaling cascades related to mitogen- and stress-activated protein kinase-1 and -2 (MSK1 and MSK2, respectively) are attractive targets for pharmaceutical intervention, especially for neural injury. Therefore, we have developed a high throughput and cost-effective detection platform for measuring selective activity of MSK1/MSK2 in cells. Through the serial monitoring of both the p38 mitogen-activated protein kinase (stress-activated protein kinase 2B)-MSK1/MSK2- cyclic AMP response element binding protein (CREB)/activating transcription factor 1 (ATF1) pathway and the p38-mammalian heat shock protein 27 (Hsp27) pathway in HeLa cells treated with anisomycin, two selective MSK1 inhibitors showed inhibition of CREB (Ser-133) and ATF1 (Ser-63) phosphorylation and no interference with Hsp-27 phosphorylation (Ser-82). On the other hand, the p38 inhibitor SB-220025 showed equipotent inhibition of CREB/ATF1 and Hsp27 phosphorylation. This study demonstrated that the specific inhibition of a target kinase could be subsequently monitored by a secondary assay that measures the intervention arising from the modulation of off-target kinases. Our established system is applicable to inhibitor screening and drug discovery related to MSK1/MSK2. |
Databáze: | OpenAIRE |
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