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J Mol Biol. 2012 Nov 2;423(4):486-95. doi: 10.1016/j.jmb.2012.08.009. Epub 2012 Aug 24.

Synergistic binding of the phosphorylated S233- and S259-binding sites of C-RAF to one 14-3-3ζ dimer.

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1
Chemical Genomics Centre of the Max-Planck-Society, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany.

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.

PMID:
22922483
DOI:
10.1016/j.jmb.2012.08.009
[Indexed for MEDLINE]

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