title: The antitumor ether lipid 1-Q-octadecyl-2-O-methyl-rac-glycerophosphocholine (ET-18-OCH3) inhibits the association between Ras and Raf-1.
abstract: BACKGROUND
  Previous studies have shown that the antitumor ether lipid, 1-O-Octadecyl-2-O-methyl-rac-glycerophosphocholine (ET-18-OCH3), inhibits the activation of the MAPK pathway in EGF- and serum-stimulated MCF-7 cells. The activation of the MAPK pathway subsequent to growth factor stimulation requires the recruitment of Raf-1 from the cytosol to the membrane. ET-18-OCH3 decreased the level of membrane-associated Raf-1 relative to untreated control cells. Since ET-18-OCH3 did not inhibit the activities of the kinases in the cascade, the reduced Raf-1 levels appeared to be the cause for the reduction in the magnitude and duration of MAPK activity. In this study we have investigated whether the reduced Raf-1 levels arise from a perturbation of the interaction of Raf-1 with activated Ras, which is the event that mediates the membrane recruitment.
  
  
  METHODS
  The interaction of Raf-1 with Ras was examined by investigating the association of cytosolic Raf-1, from ET-18-OCH3-treated and untreated cells with purified GST-Ras-GTP-gamma-S bound to agarose beads. The level of associating Raf was determined by Western blot analysis. The effect of naturally occurring phospholipids on the Raf-1-Ras interaction was also examined to assess the specificity of the results.
  
  
  RESULTS
  In cells preincubated with ET-18-OCH3, the interaction of GST-Ras-GTP-gamma-S with cytosolic Raf was reduced. The addition of ET-18-OCH3 to the cytosolic fraction isolated from untreated cells also reduced the binding of Raf to activated GST-Ras-GTP-gamma-S. Cytosolic Raf-1 from cells incubated with natural lysophospholipids was similar to that of controls.
  
  
  CONCLUSIONS
  Our findings suggest that ET-18-OCH3 associates specifically with Raf-1 in the cytosol and interferes in the interaction of Raf-1 with activated Ras, thereby reducing the levels that are translocated to the membrane for activation. Thus Raf-1 appears to be a molecular target of ET-18-OCH3.
authors.affiliation: Department of Biochemistry and Medical Genetics, University of Manitaoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0W3.
authors.forename: Pranati
authors.initials: P
authors.lastname: Samadder
authors.forename: Christina
authors.initials: C
authors.lastname: Richards
authors.forename: Robert
authors.initials: R
authors.lastname: Bittman
authors.forename: Rajinder Pal
authors.initials: RP
authors.lastname: Bhullar
authors.forename: Gilbert
authors.initials: G
authors.lastname: Arthur
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008246:Lysophospholipids / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010742:Phospholipid Ethers / Q000494:pharmacology*; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D007328:Insulin; D008246:Lysophospholipids; D010742:Phospholipid Ethers; C026659:edelfosine; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins