Phospholipase d promotes lipid microdomain-associated signaling events in mast cells.
Initial IgE-dependent signaling events are associated with detergent-resistant membrane microdomains. Following Ag stimulation, the IgE-receptor (Fc(epsilon)RI ) accumulates within these domains. This facilitates the phosphorylation of Fc(epsilon)RI subunits by the Src kinase, Lyn, and the interaction with adaptor proteins, such as the linker for activation of T cells. Among the phospholipases (PL) subsequently activated, PLD is of interest because of its presence in lipid microdomains and the possibility that its product, phosphatidic acid, may regulate signal transduction and membrane trafficking. We find that in Ag-stimulated RBL-2H3 mast cells, the association of Fc(epsilon)RI with detergent-resistant membrane fractions is inhibited by 1-butanol, which subverts production of phosphatidic acid to the biologically inert phosphatidylbutanol. Furthermore, the knockdown of PLD2, and to a lesser extent PLD1 with small inhibitory RNAs, also suppressed the accumulation of Fc(epsilon)RI and Lyn in these fractions as well as the phosphorylation of Src kinases, Fc(epsilon)RI , linker for activation of T cells, and degranulation. These effects were accompanied by changes in distribution of the lipid microdomain component, ganglioside 1, in the plasma membrane as determined by binding of fluorescent-tagged cholera toxin B subunit and confocal microscopy in live cells. Collectively, these findings suggest that PLD activity plays an important role in promoting IgE-dependent signaling events within lipid microdomains in mast cells.
Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1760, USA.
Felipe A
FA
Lisboa
Ze
Z
Peng
Christian A
CA
Combs
Michael A
MA
Beaven
D020001:1-Butanol / Q000494:pharmacology; D048868:Adaptor Proteins, Signal Transducing / Q000276:immunology / Q000378:metabolism; D000818:Animals; D015550:Cell Degranulation / Q000276:immunology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004140:Dinitrophenols / Q000276:immunology; D055785:Gene Knockdown Techniques; D020404:Glycerophospholipids / Q000276:immunology / Q000378:metabolism; D008407:Mast Cells / Q000201:enzymology* / Q000276:immunology; D021962:Membrane Microdomains / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D010712:Phosphatidic Acids / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation / Q000276:immunology; D034741:RNA, Small Interfering / Q000276:immunology / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism; D017455:Receptors, IgE / Q000187:drug effects / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000276:immunology; D015398:Signal Transduction / Q000276:immunology; D018800:Thy-1 Antigens / Q000276:immunology / Q000378:metabolism; D014162:Transfection; D047392:beta-Cyclodextrins / Q000494:pharmacology; D019061:src-Family Kinases / Q000276:immunology / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D004140:Dinitrophenols; D020404:Glycerophospholipids; C491733:Lat protein, rat; D008565:Membrane Proteins; D010712:Phosphatidic Acids; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; D017455:Receptors, IgE; D018800:Thy-1 Antigens; D047392:beta-Cyclodextrins; C034638:dinitrophenyl-bovine serum albumin; C034993:ganglioside receptor; C108732:methyl-beta-cyclodextrin; C068564:phosphatidylbutanol; D012710:Serum Albumin, Bovine; D020001:1-Butanol; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1