Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 423, Issue 4, Pages 486-495Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2012.08.009
Keywords
gatekeeper phosphorylation; protein protein interactions; X-ray; fluorescence polarization; MAPK pathway
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Funding
- PhD scholarship from the International Max Planck Research School
- AstraZeneca
- Bayer CropScience
- Bayer Healthcare
- Boehringer Ingelheim
- Merck KGaA
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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 14-3-3 zeta enhances affinity compared to single-site binding. Determination of a 1:1 stoichiometry for the di-phosphorylated peptide binding to one 14-3-3 zeta dimer with isothermal titration calorimetry was supported by the crystal structure of the 14-3-3 zeta 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. (C) 2012 Elsevier Ltd. All rights reserved.
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