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Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP

Abstract

Raf-1 phosphorylates and activates MEK-1, a kinase that activates the extracellular signal regulated kinases (ERK). This kinase cascade controls the proliferation and differentiation of different cell types1,2. Here we describe a Raf-1-interacting protein, isolated using a yeast two-hybrid screen. This protein inhibits the phosphorylation and activation of MEK by Raf-1 and is designated RKIP (Raf kinase inhibitor protein). In vitro, RKIP binds to Raf-1, MEK and ERK, but not to Ras. RKIP co-immunoprecipitates with Raf-1 and MEK from cell lysates and colocalizes with Raf-1 when examined by confocal microscopy. RKIP is not a substrate for Raf-1 or MEK, but competitively disrupts the interaction between these kinases. RKIP overexpression interferes with the activation of MEK and ERK, induction of AP-1-dependent reporter genes and transformation elicited by an oncogenically activated Raf-1 kinase. Downregulation of endogenous RKIP by expression of antisense RNA or antibody microinjection induces the activation of MEK-, ERK- and AP-1-dependent transcription. RKIP represents a new class of protein-kinase-inhibitor protein that regulates the activity of the Raf/MEK/ERK module.

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Figure 1: In vitro interaction of RKIP with components of the ERK pathway.
Figure 2: Inhibition of endogenous RKIP activates AP-1-dependent transcription.
Figure 3: RKIP inhibits Raf-induced AP-1 activation and transformation.
Figure 4: RKIP specifically blocks MEK phosphorylation by Raf-1.
Figure 5: RKIP regulates MEK and ERK activation in vivo.
Figure 6: During mitogenic stimulation RKIP binding to Raf-1 decreases.

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Acknowledgements

We thank M. Karin, M. Weber, F. Hofer, P. Shaw, M. Baccarini and B. Schaffhausen for various expression plasmids; D. K. Grandy for the full-length rat cDNA homologue of RKIP; and L. Black for help with microinjections. We appreciate critical reading and helpful comments by members of the Beatson laboratories. This work was in part supported by grants of the AICR, DFG and the Sanders Stiftung to W.K. and H.M. and by an NIH grant to J.M.S.

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Correspondence to John M. Sedivy or Walter Kolch.

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Yeung, K., Seitz, T., Li, S. et al. Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature 401, 173–177 (1999). https://doi.org/10.1038/43686

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