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.
Papin
C
C
Unité Mixte de Recherche 146 du CNRS, Institut Curie, Orsay, France.
Denouel
A
A
Calothy
G
G
Eychène
A
A
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D014446:Tyrosine 3-Monooxygenase; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D048948:14-3-3 Proteins; D021382:Protein Sorting Signals; D011506:Proteins; D011518:Proto-Oncogene Proteins; D014446:Tyrosine 3-Monooxygenase; C482128:MAP2K2 protein, human; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins