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Phospholipase D Promotes Lipid Microdomain-Associated Signaling Events in Mast Cells¹

Felipe A. Lisboa,^* Ze Peng,^* Christian A. Combs, and Michael A. Beaven^2*

^*Laboratory of Molecular Immunology and Light Microscopy Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892

Initial IgE-dependent signaling events are associated with detergent-resistant membrane microdomains. Following Ag stimulation, the IgE-receptor (FcRI) accumulates within these domains. This facilitates the phosphorylation of FcRI 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 FcRI 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 FcRI and Lyn in these fractions as well as the phosphorylation of Src kinases, FcRI, 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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the intramural program of the National Heart, Lung, and Blood Institute at the National Institutes of Health.

F.A.L. performed all experiments, created the figures, and prepared a draft of the manuscript in consultation with other authors; Z.P. prepared and evaluated PLD siRNA constructs which were used in the initial studies of this project; C.A.C. advised and assisted in the microscopic studies; and M.A.B. was responsible for the direction and scope of research and for the final version of the manuscript.

² Address correspondence and reprint requests to Michael A. Beaven, Room 8N109/Building 10, National Institutes of Health, Bethesda, MD 20892-1760. E-mail address: beavenm{at}nhlbi.nih.gov

³ Abbreviations used in this paper: LAT, linker for activation of T cells; PL, phospholipase; PA, phosphatidic acid; CTx, cholera toxin; GM1, ganglioside1; MβCD, methyl beta cyclodextrin; PIP2, phosphatidylinositol bisphosphate; siRNA, small inhibitory RNA.