Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the Raf kinase inhibitor protein

We have recently identified the Raf kinase inhibitor protein (RKIP) as a ph ysiological endogenous inhibitor of the Raf-1/MEK/extracellular signal-regu lated kinase (ERK) pathway. RKIP interfered with MEK phosphorylation and ac tivation by Raf-1, resulting in the suppression of both Raf-1-induced trans formation and AP-1-dependent transcription. Here we report the molecular me chanism of RKIP's inhibitory function. RKIP can form ternary complexes with Raf-1, MEK, and ERK. However, whereas MEK and ERK can simultaneously assoc iate with RKIP, Raf-1 binding to RKIP and that of MEK are mutually exclusiv e. RKIP is able to dissociate a Raf-1-MEK complex and behaves as a competit ive inhibitor of MEK phosphorylation. Mapping of the binding domains showed that MEK and Raf-1 bind to overlapping sites in RKIP, whereas MEK and RKIP associate with different domains in Raf-1, and Raf-1 and RKIP bind to diff erent sites in MEK. Both the Raf-1 and the MEK binding sites in RKIP need t o be destroyed in order to relieve RKIP-mediated suppression of the Raf-1/M EK/ERK pathway, indicating that binding of either Raf-1 or MEK is sufficien t for inhibition. The properties of RKIP reveal the specific sequestration of interacting components as a novel motif in the cell's repertoire for the regulation of signaling pathways.