title: Synergistic binding of the phosphorylated S233- and S259-binding sites of C-RAF to one 14-3-3ζ dimer.
abstract: C-RAF kinase is a central component of the Ras-RAF-MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase)-ERK (extracellular signal-regulated kinase) pathway, which has been shown to be activated in 30% of human tumors. 14-3-3 proteins inactivate C-RAF by binding to the two N-terminal phosphorylation-dependent binding sites surrounding S233 and S259. 14-3-3 proteins can bind two target sequences located on one polypeptide chain simultaneously, thereby increasing binding affinity compared to single-site binding and possibly allowing regulated 14-3-3 binding through gatekeeper phosphorylation. To date, it was unclear whether 14-3-3 proteins can bind the two N-terminal phosphorylation-dependent binding sites of C-RAF simultaneously. Fluorescence polarization using phosphorylated peptides demonstrated that S233 is the low-affinity and S259 is the high-affinity binding site, while simultaneous engagement of both sites by 14-3-3ζ enhances affinity compared to single-site binding. Determination of a 1:1 stoichiometry for the di-phosphorylated peptide binding to one 14-3-3ζ dimer with isothermal titration calorimetry was supported by the crystal structure of the 14-3-3ζ/C-RAFpS233,pS259 complex. Cellular localization studies validate the significance of these sites for cytoplasmic retention of C-RAF, suggesting an extended mechanism of RAF regulation by 14-3-3 proteins.
authors.affiliation: Chemical Genomics Centre of the Max-Planck-Society, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany.
authors.forename: Manuela
authors.initials: M
authors.lastname: Molzan
authors.forename: Christian
authors.initials: C
authors.lastname: Ottmann
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D048948:14-3-3 Proteins; C482983:YWHAE protein, human; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D016283:Proto-Oncogene Proteins p21(ras)