Location and Functional Significance of Retinol-binding Sites on the Serine/Threonine Kinase, c-Raf

Redox activations of serine/threonine kinases represent alternate pathways in which vitamin A plays a crucial co-factor role. Vitamin A binds the zinc finger domain of c-Raf with nanomolar affinity. The retinoid-binding site has been mapped within this structure by scanning mutagenesis. The deduced...

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Bibliographic Details
Published in:The Journal of biological chemistry 2005-02, Vol.280 (8), p.6872-6878
Main Authors: Hoyos, Beatrice, Jiang, Sulin, Hammerling, Ulrich
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Binding Sites - physiology
COS Cells
Humans
Mice
Mutagenesis
Oxidation-Reduction
Protein Kinase C - chemistry
Protein Kinase C - metabolism
Protein Kinase C-alpha
Protein-Serine-Threonine Kinases - chemistry
Proto-Oncogene Proteins c-raf - chemistry
Proto-Oncogene Proteins c-raf - metabolism
Sequence Deletion
Transfection
Vitamin A - metabolism
Zinc Fingers
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Summary:Redox activations of serine/threonine kinases represent alternate pathways in which vitamin A plays a crucial co-factor role. Vitamin A binds the zinc finger domain of c-Raf with nanomolar affinity. The retinoid-binding site has been mapped within this structure by scanning mutagenesis. The deduced contact sites were found anchored on Phe-8, counting from the 1st conserved histidine of the zinc finger. These sites agreed with contact amino acids identified by computational docking. The boundaries of a related binding pocket were identified by mutagenesis and partially confirmed by docking trials in the protein kinase C-α C1A zinc finger. They comprised Phe-7, Phe-8, and Trp-22. This trio was absent from the αC1B domain, explaining why the latter did not bind retinol. Reconfiguring at a minimum the two corresponding amino acids of αC1B, Thr-7 and Tyr-22, to conform to αC1A converted this domain to a binder. Deletion of the predicted retinoid-binding site in the full-length molecule created a mutant c-Raf that was deficient in retinol-dependent redox activation but fully responsive to epidermal growth factor. Our findings indicate that ligation of retinol to a specific site embedded in the regulatory domain is an important feature of c-Raf regulation in the redox pathway.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M412695200