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 types. 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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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3T3 Cells
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Androgen-Binding Protein*
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Animals
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COS Cells
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Calcium-Calmodulin-Dependent Protein Kinases / antagonists & inhibitors*
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism
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Carrier Proteins / isolation & purification
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Carrier Proteins / metabolism*
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Cell Transformation, Neoplastic
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Cloning, Molecular
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Enzyme Activation
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Enzyme Inhibitors / metabolism
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Gene Expression Regulation
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Green Fluorescent Proteins
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Humans
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Luminescent Proteins / genetics
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MAP Kinase Kinase Kinase 1*
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Mice
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Phosphatidylethanolamine Binding Protein
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Phospholipid Transfer Proteins
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Prostatein
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Protein Serine-Threonine Kinases / metabolism
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Proto-Oncogene Proteins c-raf / antagonists & inhibitors*
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Proto-Oncogene Proteins c-raf / metabolism
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Rats
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Secretoglobins
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Signal Transduction / drug effects*
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Transcription Factor AP-1 / metabolism
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Uteroglobin
Substances
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Androgen-Binding Protein
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Carrier Proteins
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Enzyme Inhibitors
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Luminescent Proteins
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PEBP1 protein, human
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Phosphatidylethanolamine Binding Protein
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Phospholipid Transfer Proteins
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Prostatein
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Recombinant Proteins
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Scgb1d2 protein, rat
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Scgb1d4 protein, rat
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Scgb2a2 protein, rat
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Secretoglobins
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Transcription Factor AP-1
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Green Fluorescent Proteins
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Uteroglobin
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-raf
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Calcium-Calmodulin-Dependent Protein Kinases
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MAP Kinase Kinase Kinase 1
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MAP3K1 protein, human
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Map3k1 protein, mouse