Activated transcription factor AP-1 is composed of c-Jun homodimers or c-Jun/c-Fos heterodimers and mediates expression of several gene products that have been implicated in disease pathogenesis. Activation of AP-1 is dependent on phosphorylation of c-Jun by Jun N-terminal kinase (JNK). Therefore, identification of inhibitors of JNK-mediated phosphorylation of c-Jun may lead to a novel class of therapeutics. A nonradioactive, high-through-put, time-resolved fluorescence assay was developed to measure and identify inhibitors of JNK activity. This assay utilized a lanthanide (europium)-labeled antibody that was specific for N-terminally phosphorylated c-Jun. The optimized europium-based assay was approximately 15-fold more sensitive compared to a similar 32P-based JNK assay. Compounds that were identified as inhibitors of JNK using the europium-based assay also inhibited JNK activity in the 32P-based assay with similar IC50 values. The europium-based JNK assay eliminates the contamination problems associated with the use of radioactivity. The sensitivity and safety of the europium-based assay make it amenable to robotics that will significantly increase screening throughput.
References
1.
Karin, M., Hunter, T. (1995). Transcriptional control by protein phosphorylation: Signal transmission from the cell surface to the nucleus. Curr. Biol.5:747-757.
2.
Stein, B., Anderson, D. (1996). The MAP kinase family: New "MAPs" for signal transduction pathways and novel targets for drug discovery. In: Annual Reports in Medicinal Chemistry. J.A. Bristol (ed), 289-298. Academic Press, San Diego, CA.
3.
Hibi, M., Lin, A., Smeal, T., Minden, A., Karin, M. (1993). Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev.7:2135-2148.
4.
Derijard, B., Hibi, M., Wu, I.H., Barrett, T., Su, B., Deng, T., Karin, M., Davis, R.J. (1994). A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell76:1025-1037.
5.
Adler, J., Schaffer, A., Kim, J., Dolan, L., Ronai, Z. (1995). UV irradiation and heat shock mediate JNK activation via alternate pathways. J. Biol. Chem.270:26071-26077.
6.
Hambleton, J., Weinstein, S.L., Lem, L., DeFranco, A.L. (1996). Activation of c-Jun N-terminal kinase in bacterial lipopolysac-charide-stimulated macrophages. Proc. Natl. Acad. Sci. USA93:2774-2778.
7.
Kallunki, T., Su, B., Tsigelny, I., Sluss, H.K., Derijard, B., Moore, G., Davis, R., Karin, M. (1994). JNK2 contains a specificity-determining region responsible for efficient c-Jun binding and phosphorylation. Genes Dev.8:2996-3007.
8.
Gupta, S., Barrett, T., Whitmarsh, A.J., Cavanagh, J., Sluss, H.K., Derijard, B., Davis, R.J. (1996). Selective interaction of JNK protein kinase isoforms with transcription factors. EMBO J.15:2760-2770.
9.
Lange-Carter, C.A., Pleiman, C.M., Gardner, A.M., Blumer, K.J., Johnson, G.L. (1993). A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science260:315-319.
10.
Braunwalder, A.F., Yarwood, D.R., Sills, M.A., Lipson, K.E. (1996). Measurement of the protein tyrosine kinase activity of c-src using time-resolved fluorometry of europium chelates. Anal. Biochem.238:159-164.
11.
Goldman, M.E., Walton, T.D., Ransone, L.J., Suto, C.M. (1995). An integrated laboratory automation system for quantitative high throughput screening (QHTS) with cell-based assays. ISLAR '95 Proceedings125-137.
12.
Smith, S.B., Johnson, K.S. (1988). Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene67:31-40.
13.
Vaughn, J.M., Rivier, J., Corrigan, A.Z., McClintock, R., Campen, C.A., Jolley, D., Voglmayer, J.K., Bardin, C.W., Rivier, C., Vale, W. (1989). Detection and purification of inhibin using antisera generated against synthetic peptide fragments. In: Methods in Enzymology, P.M. Conn (ed.) 168:588-617. Academic Press, Orlando, FL.
14.
Ginty, D.D., Kornhauser, J.M., Thompson, M.A., Bading, H., Mayo, K.E., Takahashi, J.S., Greenberg, M.E. (1993). Regulation of CREB phosphorylation in the superchiasmatic nuclus by light and circadian clock. Science260:238-241.
15.
Miranti, C.K., Ginty, D.D., Huang, G., Chatila, T., Greenberg, M.E. (1995). Calcium activates serum response factor-dependent transcription by a ras- and Elk-l-independent mechanism that involves a Ca2+/calmodulin-dependent kinase. Mol. Cell. Biol.15:3672-3684.
16.
Levitzki, A., Gazit, A. (1995). Tyrosine kinase inhibition: An approach to drug development. Science267:1782-1788.
17.
Kovalinko, M., Ronnstrand, L., Heldin, C.H., Loubtchenkov, M., Gazit, A., Levitzki, A., Bohmer, F.D. (1997). Phosphorylation site-specific inhibition of platelet-derived growth factor-receptor autophosphorylation by the receptor blocking tyrphostin AG1296. Biochem.36:6260-6269.
18.
Hemmila, I.A. (1991). Fluorescence energy-transfer immunoassays. In: Application of Fluorescence in Immunoassays. J.D. Wineforder, I.M. Kolthoff (eds.), 117:199-201. Wiley, Inc., New York.
19.
Mathis, G. (1995). Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer. Clin. Chem.41:1391-1397.
