IDENTIFICATION OF A NOVEL INHIBITOR OF MITOGEN-ACTIVATED PROTEIN-KINASE KINASE

The compound U0126 o-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 resul t from direct inhibition of the mitogen-activated protein kinase kinas e family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 a nd -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, MKK6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhi bitor 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, 7686-76 89) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstr ate that the two compounds bind to S218E/S222D MEK in a mutually exclu sive fashion, suggesting that they may share a common or overlapping b inding site(s). Quantitative evaluation of the steady state kinetics o f MEK inhibition by these compounds reveals that U0126 has approximate ly 100-fold higher affinity for Delta N3-S218E/S222D MEK than does PD0 98059. We further tested the effects of these compounds on the activit y of wild type MEK isolated after activation from stimulated cells. Su rprisingly, we observe a significant diminution in affinity of both co mpounds for wild type MEK as compared with the Delta N3-S218E/S222D mu tant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two acti vated MEK forms. The MEK affinity of U0126, its selectivity for MEK ov er other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigation s of mitogen-activated protein kinase-mediated signal transduction.