Identification of a Novel Inhibitor of Mitogen-activated Protein Kinase Kinase

doi:10.1074/jbc.273.29.18623

Identification of a Novel Inhibitor of Mitogen-activated Protein Kinase Kinase*

From the ^‡Inflammatory Diseases Research,^¶Chemical Enzymology and ^§Chemical and Physical Sciences, The DuPont Merck Research Laboratories, Wilmington, Delaware 19880-0400

Abstract

The compound U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686–7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to ΔN3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for ΔN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the ΔN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ To whom correspondence should be addressed: Inflammatory Diseases Research, The DuPont Merck Research Laboratories, P. O. Box 80400, Wilmington, DE 19880-0400. Tel.: 302-695-7110; Fax: 302-695-9401; E-mail: James.M.Trzaskos{at}dupontmerck.com.
↵1 The abbreviations used are: GR, glucocorticoid receptors; GRE, glucocorticoid response element(s); MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAP kinase kinase; MEKK, MEK kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; DMEM, Dulbecco’s modified Eagle’s medium; PMA, phorbol 12-myristate 13-acetate; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DTT, dithiothreitol; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; TRE, TPA response element; GST, glutathione S-transferase; FCS, fetal calf serum; TPA, 12-O-tetradecanoylphorbol-13-acetate; PCR, polymerase chain reaction; PHA, phytohemaglutinin; IL, interleukin; ATF, activating transcription factor; BSA, bovine serum albumin, CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
↵2 B. Schnyder, B. Car, and J. M. Trzaskos, unpublished observations.
↵3 G. Fisher and J. M. Trzaskos, unpublished observations.
- Received January 23, 1998.
- Revision received April 16, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.