Abstract
A green fluorescent protein (GFP)-Raf-1 fusion protein was used to show that Bcl-2 can target this kinase to mitochondria. Active Raf-1 fused with targeting sequences from an outer mitochondrial membrane protein protected cells from apoptosis and resulted in phosphorylation of BAD, a proapoptotic Bcl-2 homolog. Plasma membrane-targeted Raf-1 did not protect from apoptosis and resulted in phosphorylation of ERK-1 and ERK-2. Untargeted active Raf-1 improved Bcl-2-mediated resistance to apoptosis, whereas a kinase-inactive Raf-1 mutant abrogated apoptosis suppression by Bcl-2. Bcl-2 can therefore target Raf-1 to mitochondrial membranes, allowing this kinase to phosphorylate BAD or possibly other protein substrates involved in apoptosis regulation.
Publication types
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Comparative Study
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Apoptosis / physiology*
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Biological Transport
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism
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Carrier Proteins / metabolism
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Cell Compartmentation*
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Cell Membrane / metabolism
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Cells, Cultured
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Green Fluorescent Proteins
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Mitochondria / metabolism*
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases*
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Phosphorylation
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Protein Binding
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Protein-Tyrosine Kinases / metabolism
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-bcl-2 / metabolism*
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Proto-Oncogene Proteins c-raf
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Recombinant Fusion Proteins / metabolism
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Signal Transduction
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Staurosporine / pharmacology
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Subcellular Fractions
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Transfection
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bcl-Associated Death Protein
Substances
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Carrier Proteins
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Luminescent Proteins
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-bcl-2
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Recombinant Fusion Proteins
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bcl-Associated Death Protein
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Green Fluorescent Proteins
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Protein-Tyrosine Kinases
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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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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases
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Staurosporine