Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells.

摘要: Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., and Bell, R. M.(1994) Biol. Chem. 269, 10000-10007). Further analysis showed full-length bound to both phosphatidic acid (PA). Specifically, a carboxyl-terminal (RafC; residues 295-648 human Raf-1) interacted strongly acid. The binding RafC PA displayed positive cooperativity Hill numbers between 3.3 6.2; apparent Kd ranged from 4.9 ± 0.6 7.8 0.9 mol % PA. interaction pH dependence distinct Also, RafC-PA was unaffected at high ionic strength. Of all lipids tested, only cardiolipin exhibited affinity binding; other acidic were either ineffective or weakly effective. By deletion mutagenesis, site within narrowed down 35-amino segment 389 423. did not bind phosphatidyl alcohols; also, inhibition formation in Madin-Darby canine kidney cells by treatment 1% ethanol significantly reduced translocation cytosol membrane following stimulation 12-O-tetradecanoylphorbol-13-acetate. These results suggest potential role lipid second messenger, PA, regulation subsequent activation vivo.