MEK and ERK activation in Ras-disabled RBL-2H3 mast cells and novel roles for geranylgeranylated and farnesylated proteins in Fc epsilon RI-mediated signaling

Cross-linking the high affinity IgE receptor Fc epsilon RI of basophils and mast cells activates receptor-associated protein-tyrosine kinases and stim ulates a signaling cascade leading to secretion, ruffling, spreading, and c ytokine production. Previous evidence that the pan-prenylation inhibitor lo vastatin blocks Ag-stimulated Ca2+ influx, secretion, and membrane/cytoskel etal responses implicated isoprenylated proteins in the Fc epsilon RI-coupl ed signaling cascade but could not distinguish between contributions of C-1 5 (farnesylated) and C-20 (geranylgeranylated) species. Here we establish c oncentrations of lovastatin and the farnesyl-specific inhibitor BZA-5B that inhibit the farnesylation and Ag-induced activation of Ras species in RBL- 2H3 cells (H-Ras, K-RasA, and K-RasB), These inhibitors have little effect on tyrosine kinase activation, which initiates Fc epsilon RI signaling. Alt hough Ras is disabled, only lovastatin substantially blocks Raf-1 activatio n, and neither inhibitor affects mitogen-activated protein kinase kinase/ex tracellular signal regulated kinase kinase (MEK) or ERK1/ERK2 activation. T hus, the pathway to Fc epsilon RI-mediated MEK/ERK and ERK activation can a pparently bypass Ras and Raf-1, Predictably, only lovastatin inhibits Ag-in duced ruffling, spreading, and secretion, previously linked to geranylgeran ylated Rho and Rab family members. Additionally, only lovastatin inhibits p hospholipase Cy-mediated inositol (1,4,5) trisphosphate production, sustain ed Ca2+ influx, and Ca2+-dependent IL-4 production, suggesting novel roles for geranylgeranylated (lovastatin-sensitive, BZA-5B-insensitive) proteins in Fc epsilon RI signal propagation. Remarkably, BZA-5B concentrations too low to inactivate Ras reduce the lag time to Ag-induced Ca2+ stores release and enhance secretion. These results link a non-Pas farnesylated protein(s ) to the negative regulation of Ca2+ release from intracellular stores and secretion. We identified no clear role for Ras in Fc epsilon RI-coupled sig naling but suggest its involvement in mast cell growth regulation based on the inhibition of cell proliferation by both BZA-5B and lovastatin.