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

Previous studies demonstrated that the cysteine-rich amino-terminal do main of Raf-1 kinase interacts selectively with phosphatidylserine (Gh osh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., an d Bell, R. M. (1994) J. Biol. Chem. 269, 10000-10007). Further analysi s showed that full-length Raf-1 bound to both phosphatidylserine and p hosphatidic acid (PA). Specifically, a carboxyl-terminal domain of Raf -1 kinase (RafC; residues 295-648 of human Raf-1) interacted strongly with phosphatidic acid. The binding of RafC to PA displayed positive c ooperativity with Hill numbers between 3.3 and 6.2; the apparent K-d r anged from 4.9 +/- 0.6 to 7.8 +/- 0.9 mol % PA. The interaction of Raf C with PA displayed a pH dependence distinct from the interaction betw een the cysteine-rich domain of Raf-1 and PA Also, the RafC-PA interac tion was unaffected at high ionic strength. Of all the lipids tested, only PA and cardiolipin exhibited high affinity binding; other acidic lipids were either ineffective or weakly effective. By deletion mutage nesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphat idyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the tr anslocation of Raf-1 from the cytosol to the membrane following stimul ation with 12-O-tetradecanoylphorbol-13-acetate. These results suggest a potential role of the lipid second messenger, PA, in the regulation of translocation and subsequent activation of Raf-1 in vivo.