Identification of signalling proteins interacting with B-Raf in the yeast two-hybrid system.

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.
Mesh Terms:
14-3-3 Proteins, Amino Acid Sequence, Animals, Base Sequence, Calcium-Calmodulin-Dependent Protein Kinases, Cricetinae, Humans, MAP Kinase Kinase 1, MAP Kinase Kinase 2, Mice, Mitogen-Activated Protein Kinase Kinases, Molecular Sequence Data, Protein Sorting Signals, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, Proteins, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-raf, Saccharomyces cerevisiae, Sequence Homology, Amino Acid, Signal Transduction, Tyrosine 3-Monooxygenase, ras Proteins
Oncogene
Date: May. 16, 1996
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