title: MEK and ERK activation in ras-disabled RBL-2H3 mast cells and novel roles for geranylgeranylated and farnesylated proteins in Fc epsilonRI-mediated signaling.
abstract: Cross-linking the high affinity IgE receptor Fc epsilonRI of basophils and mast cells activates receptor-associated protein-tyrosine kinases and stimulates a signaling cascade leading to secretion, ruffling, spreading, and cytokine production. Previous evidence that the pan-prenylation inhibitor lovastatin blocks Ag-stimulated Ca2+ influx, secretion, and membrane/cytoskeletal responses implicated isoprenylated proteins in the Fc epsilonRI-coupled signaling cascade but could not distinguish between contributions of C15 (farnesylated) and C20 (geranylgeranylated) species. Here we establish concentrations 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 epsilonRI signaling. Although Ras is disabled, only lovastatin substantially blocks Raf-1 activation, and neither inhibitor affects mitogen-activated protein kinase kinase/extracellular signal regulated kinase kinase (MEK) or ERK1/ERK2 activation. Thus, the pathway to Fc epsilonRI-mediated MEK/ERK and ERK activation can apparently bypass Ras and Raf-1. Predictably, only lovastatin inhibits Ag-induced ruffling, spreading, and secretion, previously linked to geranylgeranylated Rho and Rab family members. Additionally, only lovastatin inhibits phospholipase Cgamma-mediated inositol (1,4,5) trisphosphate production, sustained Ca2+ influx, and Ca2+-dependent IL-4 production, suggesting novel roles for geranylgeranylated (lovastatin-sensitive, BZA-5B-insensitive) proteins in Fc epsilonRI 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-Ras farnesylated protein(s) to the negative regulation of Ca2+ release from intracellular stores and secretion. We identified no clear role for Ras in Fc epsilonRI-coupled signaling but suggest its involvement in mast cell growth regulation based on the inhibition of cell proliferation by both BZA-5B and lovastatin.
authors.affiliation: Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque 87131, USA.
authors.forename: T E
authors.initials: TE
authors.lastname: Graham
authors.forename: J R
authors.initials: JR
authors.lastname: Pfeiffer
authors.forename: R J
authors.initials: RJ
authors.lastname: Lee
authors.forename: D F
authors.initials: DF
authors.lastname: Kusewitt
authors.forename: A M
authors.initials: AM
authors.lastname: Martinez
authors.forename: T
authors.initials: T
authors.lastname: Foutz
authors.forename: B S
authors.initials: BS
authors.lastname: Wilson
authors.forename: J M
authors.initials: JM
authors.lastname: Oliver
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors; D000818:Animals; D001569:Benzodiazepines / Q000494:pharmacology; D020013:Calcium Signaling / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002455:Cell Division; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051231:Farnesyltranstransferase; D007073:Immunoglobulin E / Q000276:immunology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D015471:Leukemia, Basophilic, Acute / Q000473:pathology; D008148:Lovastatin / Q000494:pharmacology; D008407:Mast Cells / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009363:Neoplasm Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D051966:Phospholipase C gamma; D020033:Protein Isoforms / Q000172:deficiency / Q000378:metabolism*; D017368:Protein Prenylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000276:immunology*; D015398:Signal Transduction / Q000502:physiology*; D014407:Tumor Cells, Cultured / Q000187:drug effects; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D018631:ras Proteins / Q000172:deficiency / Q000378:metabolism*
chemical_list: C090156:BZA 5B; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009363:Neoplasm Proteins; D009842:Oligopeptides; D020033:Protein Isoforms; D017455:Receptors, IgE; D001569:Benzodiazepines; D007073:Immunoglobulin E; D015544:Inositol 1,4,5-Trisphosphate; D008148:Lovastatin; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D018631:ras Proteins