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WIP regulates signaling via the high affinity receptor for immunoglobulin E in mast cells.

Kettner A, Kumar L, Antón IM, Sasahara Y, de la Fuente M, Pivniouk VI, Falet H, Hartwig JH, Geha RS - J. Exp. Med. (2004)

Bottom Line: Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs.However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs.These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Wiskott-Aldrich syndrome protein-interacting protein (WIP) stabilizes actin filaments and is important for immunoreceptor-mediated signal transduction leading to actin cytoskeleton rearrangement in T and B cells. Here we report a role for WIP in signaling pathways downstream of the high affinity receptor for immunoglobulin (Ig)E (FcepsilonRI) in mast cells. WIP-deficient bone marrow-derived mast cells (BMMCs) were impaired in their capacity to degranulate and secrete interleukin 6 after FcepsilonRI ligation. Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs. WIP was found to associate with Syk after FcepsilonRI ligation and to inhibit Syk degradation as evidenced by markedly diminished Syk levels in WIP-deficient BMMCs. WIP-deficient BMMCs exhibited no apparent defect in their subcortical actin network and were normal in their ability to form protrusions when exposed to an IgE-coated surface. However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs. These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.

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Calcium mobilization and tyrosine phosphorylation of PLC-γ2 in response to FcεRI ligation. (A) Change of fluorescence of the calcium-sensitive dye indo 1-AM was monitored for the indicated time. IgE-sensitized cells were stimulated with F(ab′)2 anti–rat Ig at the indicated time points. Values were plotted as ratio of fluorescence at 405 and 485 nm. (B) Tyrosine phosphorylation of PLC-γ2 in WIP−/− BMMCs. IgE-sensitized BMMCs were activated by FcεRI cross-linking with F(ab′)2 anti–rat Ig for the indicated time at 37°C. Cell lysates were immunoprecipitated with anti–PLC-γ2. PLC-γ2 tyrosine phosphorylation was analyzed by immunoblotting with antiphosphotyrosine antibody. The membrane was reprobed with anti–PLC-γ2 to control for loading. (C) Tyrosine phosphorylation of SLP-76. Cells were stimulated as for PLC-γ2 tyrosine phosphorylation and cell lysates were immunoprecipitated with anti–SLP-76 antibody. The membrane was reprobed with anti–SLP-76 to control for loading.
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fig4: Calcium mobilization and tyrosine phosphorylation of PLC-γ2 in response to FcεRI ligation. (A) Change of fluorescence of the calcium-sensitive dye indo 1-AM was monitored for the indicated time. IgE-sensitized cells were stimulated with F(ab′)2 anti–rat Ig at the indicated time points. Values were plotted as ratio of fluorescence at 405 and 485 nm. (B) Tyrosine phosphorylation of PLC-γ2 in WIP−/− BMMCs. IgE-sensitized BMMCs were activated by FcεRI cross-linking with F(ab′)2 anti–rat Ig for the indicated time at 37°C. Cell lysates were immunoprecipitated with anti–PLC-γ2. PLC-γ2 tyrosine phosphorylation was analyzed by immunoblotting with antiphosphotyrosine antibody. The membrane was reprobed with anti–PLC-γ2 to control for loading. (C) Tyrosine phosphorylation of SLP-76. Cells were stimulated as for PLC-γ2 tyrosine phosphorylation and cell lysates were immunoprecipitated with anti–SLP-76 antibody. The membrane was reprobed with anti–SLP-76 to control for loading.

Mentions: Upon FcεRI cross-linking, PLC-γ becomes rapidly phosphorylated and activated to hydrolyze membrane phosphoinositides to generate IP3, which initiates an increase in intracellular calcium. A rapid sustained increase in intracellular calcium levels is important for FcεRI-mediated degranulation and cytokine release in mast cells (33, 34). After FcεRI ligation, intracellular calcium levels in WIP−/− BMMCs rapidly increased but remained significantly below those observed in WT BMMCs throughout the 6-min observation period (Fig. 4 A). Similarly decreased calcium fluxes were still observed under conditions of higher FcεRI cross-linking (10 mg/ml rat IgE and 50 mg F(ab′)2 anti–rat Ig).


WIP regulates signaling via the high affinity receptor for immunoglobulin E in mast cells.

Kettner A, Kumar L, Antón IM, Sasahara Y, de la Fuente M, Pivniouk VI, Falet H, Hartwig JH, Geha RS - J. Exp. Med. (2004)

Calcium mobilization and tyrosine phosphorylation of PLC-γ2 in response to FcεRI ligation. (A) Change of fluorescence of the calcium-sensitive dye indo 1-AM was monitored for the indicated time. IgE-sensitized cells were stimulated with F(ab′)2 anti–rat Ig at the indicated time points. Values were plotted as ratio of fluorescence at 405 and 485 nm. (B) Tyrosine phosphorylation of PLC-γ2 in WIP−/− BMMCs. IgE-sensitized BMMCs were activated by FcεRI cross-linking with F(ab′)2 anti–rat Ig for the indicated time at 37°C. Cell lysates were immunoprecipitated with anti–PLC-γ2. PLC-γ2 tyrosine phosphorylation was analyzed by immunoblotting with antiphosphotyrosine antibody. The membrane was reprobed with anti–PLC-γ2 to control for loading. (C) Tyrosine phosphorylation of SLP-76. Cells were stimulated as for PLC-γ2 tyrosine phosphorylation and cell lysates were immunoprecipitated with anti–SLP-76 antibody. The membrane was reprobed with anti–SLP-76 to control for loading.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211794&req=5

fig4: Calcium mobilization and tyrosine phosphorylation of PLC-γ2 in response to FcεRI ligation. (A) Change of fluorescence of the calcium-sensitive dye indo 1-AM was monitored for the indicated time. IgE-sensitized cells were stimulated with F(ab′)2 anti–rat Ig at the indicated time points. Values were plotted as ratio of fluorescence at 405 and 485 nm. (B) Tyrosine phosphorylation of PLC-γ2 in WIP−/− BMMCs. IgE-sensitized BMMCs were activated by FcεRI cross-linking with F(ab′)2 anti–rat Ig for the indicated time at 37°C. Cell lysates were immunoprecipitated with anti–PLC-γ2. PLC-γ2 tyrosine phosphorylation was analyzed by immunoblotting with antiphosphotyrosine antibody. The membrane was reprobed with anti–PLC-γ2 to control for loading. (C) Tyrosine phosphorylation of SLP-76. Cells were stimulated as for PLC-γ2 tyrosine phosphorylation and cell lysates were immunoprecipitated with anti–SLP-76 antibody. The membrane was reprobed with anti–SLP-76 to control for loading.
Mentions: Upon FcεRI cross-linking, PLC-γ becomes rapidly phosphorylated and activated to hydrolyze membrane phosphoinositides to generate IP3, which initiates an increase in intracellular calcium. A rapid sustained increase in intracellular calcium levels is important for FcεRI-mediated degranulation and cytokine release in mast cells (33, 34). After FcεRI ligation, intracellular calcium levels in WIP−/− BMMCs rapidly increased but remained significantly below those observed in WT BMMCs throughout the 6-min observation period (Fig. 4 A). Similarly decreased calcium fluxes were still observed under conditions of higher FcεRI cross-linking (10 mg/ml rat IgE and 50 mg F(ab′)2 anti–rat Ig).

Bottom Line: Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs.However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs.These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Wiskott-Aldrich syndrome protein-interacting protein (WIP) stabilizes actin filaments and is important for immunoreceptor-mediated signal transduction leading to actin cytoskeleton rearrangement in T and B cells. Here we report a role for WIP in signaling pathways downstream of the high affinity receptor for immunoglobulin (Ig)E (FcepsilonRI) in mast cells. WIP-deficient bone marrow-derived mast cells (BMMCs) were impaired in their capacity to degranulate and secrete interleukin 6 after FcepsilonRI ligation. Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs. WIP was found to associate with Syk after FcepsilonRI ligation and to inhibit Syk degradation as evidenced by markedly diminished Syk levels in WIP-deficient BMMCs. WIP-deficient BMMCs exhibited no apparent defect in their subcortical actin network and were normal in their ability to form protrusions when exposed to an IgE-coated surface. However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs. These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.

Show MeSH
Related in: MedlinePlus