Dual enzyme-linked immunosorbent assay system for detection of endogenous kinase activities of mitogen- and stress-activated protein kinase-1/2.
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.
Ishii
Tsuyoshi
T
GlaxoSmithKline K.K., Tsukuba-shi, Ibaraki, Japan. tsu_ishii01@yahoo.co.jp
Sootome
Hiroshi
H
Toyoda
Hiroko
H
Suda
Mikiya
M
Noumi
Takato
T
Yamashita
Keizo
K
D000841:Anisomycin / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048290:Mitogen-Activated Protein Kinase 11 / Q000378:metabolism*; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D017362:Cyclic AMP Response Element-Binding Protein; D006360:Heat-Shock Proteins; D011500:Protein Synthesis Inhibitors; D004815:Epidermal Growth Factor; D000841:Anisomycin; D019950:Mitogen-Activated Protein Kinase 1; D048290:Mitogen-Activated Protein Kinase 11; D048055:Mitogen-Activated Protein Kinase 8; D048051:p38 Mitogen-Activated Protein Kinases