JNK3 AlphaScreen Assay
The c-jun N-terminal kinase (JNK) family are serine/threonine protein kinases that phosphorylate c-jun, a component of the transcription factor protein-1 (AP-1). JNK kinases are members of the mitogen-activated protein kinase family including the extracellular regulated kinases and p38 kinases. Three JNK genes (JNK 1, 2, and 3) have been identified in humans so far. JNK1 and JNK2 have a broad distribution and JNK3 is primarily found in neuronal tissues and cardiac myocytes. ..more
BioActive Compounds: 34
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
NCGC Assay Overview:
The c-jun N-terminal kinase (JNK) family are serine/threonine protein kinases that phosphorylate c-jun, a component of the transcription factor protein-1 (AP-1). JNK kinases are members of the mitogen-activated protein kinase family including the extracellular regulated kinases and p38 kinases. Three JNK genes (JNK 1, 2, and 3) have been identified in humans so far. JNK1 and JNK2 have a broad distribution and JNK3 is primarily found in neuronal tissues and cardiac myocytes.
JNK3 has been under consideration as a target for the treatment of neurodegenerative disorders. Inhibition of JNK3 has displayed neuroprotective actions on transient brain ischemia and reperfusion-induced neuronal death in the rat hippocampal CA1 region (Yang et al., 1997; Herdegen et al., 1998). JNK pathway is also involved in pathological process in a mouse model of Parkinson's disease (Xia et al., 2001).
Given these potential tissue distributions and links to neuroprotection, JNK3 inhibitors should provide useful investigational tools to study the role of this kinase in several diseases including, arthritis, stroke, myocardial infarction, ischemic injury and Parkinson's disease.
A homogeneous AlphaScreen is developed to measure the activity of JNK3. AlphaScreen is a two bead-based proximity-dependent chemical energy transfer luminescent assay platform. The phosphorylation of a biotinylated c-jun substrate by JNK3 can be detected with an antibody raised against phosphorylated c-jun. This process brings streptavidin donor beads and Protein-A acceptor beads to within a proximity range of <200 nm, and subsequently lead a chemical luminescent reaction upon exciting donor beads with 680 nm laser excitation light. Inhibition of JNK3 activity by small molecules and subsequent decrease in the antibody binding to biotinylated c-jun could attenuate or abolish this chemical luminescent reaction.
Yang DD, Kuan CY, Whitmarsh AJ, Rincon M, Zheng TS, Davis RJ, Rakic P and Flavell RA (1997) Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature 389:865-870. [PMID: 9349820]
Herdegen T, Claret FX, Kallunki T, Martin-Villalba A, Winter C, Hunter T, and Karin M (1998) Lasting N-terminal phosphorylation of c-Jun and activation of c-Jun N-terminal kinases after neuronal injury. J.Neurosci. 18:5124-5135. [PMID: 9651196]
Xia XG, Harding T, Weller M, Bieneman A, Uney JB and Schulz JB (2001) Gene transfer of the JNK interacting protein-1 protects dopaminergic neurons in the MPTP model of Parkinson's disease. Proc.Natl.Acad.Sci.U.S.A. 98:10433-10438. [PMID: 11504916]
NCGC Assay Protocol Summary:
GST-cJun (1-79) (Stratagene, La Jolla CA, #205145) was biotnylated by PerkinElmer (PerkinElmer-BioSignalInc. Montreal, Canada) and was detectable by the streptavidin Donor beads. Anti-(phospho)cJun (Ser73) antibody was from UpState (Charlottesvilles, VA, #06-659). JNK3-containing lysates were generated from HepG2 cells and pre-activated by interleukin1-beta and tumor necrosis factor alpha. All reagents were stored at -20 C before using. Phosphorylated biotinylated-GST-cJun was recognized by Anti-(phospho)cJun (Ser73) antibody and this process brings acceptor and donor beads together. Therefore JNK3 phosphorylation of cJun can be measured by Alpha reading by EnVision (PerkinElmer, Boston, MA).
The composition of kinase buffer was (in mM) 20 tris- HCl (pH 7.6) 10 MgCl2, 1 DTT, and 0.1 Na3VO4 and 0.01% Tween-20. Beads were diluted in detection buffer containing (in mM) 20 tris-HCl (pH 7.7), 20 NaCl and 80 EDTA and 0.3% BSA. The assay was performed at room temperature. All compounds were screened as titrations from 0.6 nM to 46 uM final concentration. The assay was performed in 1536-well format (white solid bottom and untreated) as follows:
1,Reagent,3 uL,Biotin-GST-cJun, Anti-(phospho)cJun and pre-activated JNK3-containing lysates
2,Compound,23 nL,Final concentration was 0.6nM to 46uM
3,Time,15 minutes,Room Temperature
4,Reagent,1 uL,ATP final concentration 10uM
5,Time,2 hour,Room temp
6,Reagent,0.5 uL,2ng/well bead mixture
7,Time,3 hour,Room temp
8,Detector,EX 680 nm / singlet O2 / EM 520-620 nm,Envision (PerkinElmer, detected through aperture 1536A1)
Keywords: cJun, JNK3, AlphaScreen, MLSMR, MLSCN, NIH Roadmap, qHTS and NCGC
1. Compounds are first classified as being probable inhibitors (compounds interfering with the alpha screen), activators (compounds causing an increase in alpha screen readout), inactive, or inconclusive.
2. Within the inhibitors, compounds were ranked by efficacy and potency.
Data Table (Concise)