RKIP (Raf kinase inhibitor protein) is a novel protein that inhibits Raf-1 kinase activity and the MAP kinase signalling pathway. It was identified through a yeast two-hybrid screen using the Raf-1 kinase domain as bait. RKIP binds to Raf-1, MEK, and ERK but not to Ras. It co-immunoprecipitates with Raf-1 and MEK from cell lysates and colocalizes with Raf-1 in cells. RKIP is not a substrate for Raf-1 or MEK but competitively disrupts the interaction between these kinases. Overexpression of RKIP interferes with MEK and ERK activation, AP-1-dependent reporter gene induction, and transformation by oncogenically activated Raf-1. Downregulation of endogenous RKIP by antisense RNA or antibody microinjection activates 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. In metazoans, the Ras/Raf-1/MEK/ERK module is a ubiquitously expressed signalling pathway that conveys mitogenic and differentiation signals from the cell membrane to the nucleus. This kinase cascade is spatially organized in a signalling complex nucleated by Ras proteins. The regulation of this module is complex and may involve associations with scaffolding and regulatory proteins. To isolate such proteins, the Raf-1 kinase domain, BXB, was used in a yeast two-hybrid screen. Screening 500,000 clones of a human T-cell library yielded nine clones that specifically interacted with BXB. Five clones corresponded to 14-3-3 proteins. One clone, RKIP, bound to both kinase-active and kinase-negative BXB, but not to control baits. RKIP binds to BXB, full-length Raf-1, MEK-1, and ERK-2, but not to Ras. RKIP binding is independent of Raf-1 kinase activity and not affected by phosphatidylethanolamine. RKIP interactions were also shown between endogenous mammalian proteins. An RKIP antiserum co-immunoprecipitated Raf-1, MEK, and ERK from Rat-1 cells. These interactions were also observed in reciprocal immunoprecipitations with antisera to Raf-1, MEK, or ERK. Confocal microscopy revealed extensive colocalization between Raf-1 and RKIP in both quiescent and Ras-transformed cells. To investigate the relevance of the interaction between RKIP and the kinases of the Raf/MEK/ERK module in mammalian cells, endogenous RKIP was inhibited by antibody microinjection or expression of antisense RNA. RKIP significantly reduced the transformation efficiency of BXB in three distinct assays: morphological transformationRKIP (Raf kinase inhibitor protein) is a novel protein that inhibits Raf-1 kinase activity and the MAP kinase signalling pathway. It was identified through a yeast two-hybrid screen using the Raf-1 kinase domain as bait. RKIP binds to Raf-1, MEK, and ERK but not to Ras. It co-immunoprecipitates with Raf-1 and MEK from cell lysates and colocalizes with Raf-1 in cells. RKIP is not a substrate for Raf-1 or MEK but competitively disrupts the interaction between these kinases. Overexpression of RKIP interferes with MEK and ERK activation, AP-1-dependent reporter gene induction, and transformation by oncogenically activated Raf-1. Downregulation of endogenous RKIP by antisense RNA or antibody microinjection activates 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. In metazoans, the Ras/Raf-1/MEK/ERK module is a ubiquitously expressed signalling pathway that conveys mitogenic and differentiation signals from the cell membrane to the nucleus. This kinase cascade is spatially organized in a signalling complex nucleated by Ras proteins. The regulation of this module is complex and may involve associations with scaffolding and regulatory proteins. To isolate such proteins, the Raf-1 kinase domain, BXB, was used in a yeast two-hybrid screen. Screening 500,000 clones of a human T-cell library yielded nine clones that specifically interacted with BXB. Five clones corresponded to 14-3-3 proteins. One clone, RKIP, bound to both kinase-active and kinase-negative BXB, but not to control baits. RKIP binds to BXB, full-length Raf-1, MEK-1, and ERK-2, but not to Ras. RKIP binding is independent of Raf-1 kinase activity and not affected by phosphatidylethanolamine. RKIP interactions were also shown between endogenous mammalian proteins. An RKIP antiserum co-immunoprecipitated Raf-1, MEK, and ERK from Rat-1 cells. These interactions were also observed in reciprocal immunoprecipitations with antisera to Raf-1, MEK, or ERK. Confocal microscopy revealed extensive colocalization between Raf-1 and RKIP in both quiescent and Ras-transformed cells. To investigate the relevance of the interaction between RKIP and the kinases of the Raf/MEK/ERK module in mammalian cells, endogenous RKIP was inhibited by antibody microinjection or expression of antisense RNA. RKIP significantly reduced the transformation efficiency of BXB in three distinct assays: morphological transformation