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 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-alpha C1A zinc finger. They comprised Phe-7, Phe-8, and Trp-22. This trio was absent from the alphaC1B domain, explaining why the latter did not bind retinol. Reconfiguring at a minimum the two corresponding amino acids of alphaC1B, Thr-7 and Tyr-22, to conform to alphaC1A 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.
Hoyos
Beatrice
B
Immunology Program, Memorial Sloan Kettering Cancer, New York, New York, 10021, USA. b-hoyos@ski.mskcc.org
Jiang
Sulin
S
Hammerling
Ulrich
U
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D019556:COS Cells; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D010084:Oxidation-Reduction; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism; D051571:Protein Kinase C-alpha; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D014801:Vitamin A / Q000378:metabolism*; D016335:Zinc Fingers
D014801:Vitamin A; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; C496628:PRKCA protein, human; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha