Abstract
Recent studies suggested the existence of Ras/B-Raf/ MEK-1 complexes and a critical role for B-Raf in regulating the MAP kinase/ERKs signalling pathway. We report, here, that both Ras and MEK-1 proteins interact physically with B-Raf proteins in the yeast two-hybrid system. In addition, by screening a mouse brain cDNA library, we isolated additional B-Raf interacting proteins. These include three members of the 14-3-3 proteins family (eta, theta and zeta) and the MEK-2 protein. We also show that c-Raf-1, previously reported to interact with beta and zeta 14-3-3 proteins, also interacts with eta and theta 14-3-3 proteins in the two-hybrid system. By using different portions of the B-Raf protein, we mapped the regions of the protein involved in these interactions. Specifically, we have characterized B-Raf specific sequences required for an efficient interaction with MEK proteins. We show that, consequently, B-Raf interacts with MEK-1 and MEK-2 with a better affinity than does c-Raf-1, thus strengthening the notion that B-Raf is a stronger MEK activator than c-Raf-l. Our results also suggest that a MEK specific sequence, not present in MAP kinase kinases which are not activated by members of the Raf family, is required for the interaction with Raf proteins.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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14-3-3 Proteins
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Amino Acid Sequence
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Animals
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Base Sequence
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism
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Cricetinae
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Humans
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MAP Kinase Kinase 1
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MAP Kinase Kinase 2
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Mice
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Mitogen-Activated Protein Kinase Kinases*
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Molecular Sequence Data
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Protein Sorting Signals / metabolism*
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Protein-Tyrosine Kinases / genetics
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Protein-Tyrosine Kinases / metabolism
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Proteins / genetics
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Proteins / 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-raf
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Saccharomyces cerevisiae / enzymology
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Saccharomyces cerevisiae / metabolism*
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Sequence Homology, Amino Acid
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Signal Transduction / physiology*
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Tyrosine 3-Monooxygenase*
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ras Proteins / genetics
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ras Proteins / metabolism
Substances
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14-3-3 Proteins
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Protein Sorting Signals
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Proteins
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Proto-Oncogene Proteins
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Tyrosine 3-Monooxygenase
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MAP2K2 protein, human
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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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MAP Kinase Kinase 1
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MAP Kinase Kinase 2
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MAP2K1 protein, human
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Map2k1 protein, mouse
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Mitogen-Activated Protein Kinase Kinases
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ras Proteins