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 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) J. Biol. Chem. 269, 10000-10007). Further analysis showed that full-length Raf-1 bound to both phosphatidylserine and phosphatidic 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 cooperativity with Hill numbers between 3.3 and 6.2; the apparent Kd ranged from 4.9 +/- 0.6 to 7.8 +/- 0.9 mol % PA. The interaction of RafC with PA displayed a pH dependence distinct from the interaction between the cysteine-rich domain of Raf-1 and PA. Also, the RafC-PA interaction 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 mutagenesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphatidyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the translocation of Raf-1 from the cytosol to the membrane following stimulation 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.
Ghosh
S
S
Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Strum
J C
JC
Sciorra
V A
VA
Daniel
L
L
Bell
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002451:Cell Compartmentation / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004285:Dogs; D020404:Glycerophospholipids; D008969:Molecular Sequence Data; D010712:Phosphatidic Acids / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D011993:Recombinant Fusion Proteins; D012995:Solubility; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017931:DNA Primers; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D010718:Phosphatidylserines; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C051521:phosphatidylethanol; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate