Limits...
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.

Show MeSH

Related in: MedlinePlus

WIP associates with Syk after FcεRI ligation and protects it from degradation. (A) Association of WIP and Syk after FcεRI ligation. WIP immunoprecipitates from WT BMMCs were probed with Syk before and after FcεRI ligation. (B) Syk protein levels in Western blots from lysates of WT and WIP−/− BMMCs. (C) Syk phosphorylation after FcεRI ligation in WT and WIP−/− BMMCs. Syk immunoprecipitates were probed with antiphosphotyrosine mAb 4G10 and with anti-Syk as loading control. Numbers represent densitometric ratio of phopshoSyk to Syk bands. (D) Northern blot analysis of Syk mRNA in WT and WIP−/− BMMCs. 28S RNA is shown as loading control. (E) Effect of pretreatment with calpeptin and MG132 for 6 h on Syk levels in WT and WIP−/− BMMCs.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211794&req=5

fig6: WIP associates with Syk after FcεRI ligation and protects it from degradation. (A) Association of WIP and Syk after FcεRI ligation. WIP immunoprecipitates from WT BMMCs were probed with Syk before and after FcεRI ligation. (B) Syk protein levels in Western blots from lysates of WT and WIP−/− BMMCs. (C) Syk phosphorylation after FcεRI ligation in WT and WIP−/− BMMCs. Syk immunoprecipitates were probed with antiphosphotyrosine mAb 4G10 and with anti-Syk as loading control. Numbers represent densitometric ratio of phopshoSyk to Syk bands. (D) Northern blot analysis of Syk mRNA in WT and WIP−/− BMMCs. 28S RNA is shown as loading control. (E) Effect of pretreatment with calpeptin and MG132 for 6 h on Syk levels in WT and WIP−/− BMMCs.

Mentions: Syk plays a critical role in transmitting signals from FcεRI. The multiple defects in FcεRI signaling in WIP−/− BMMCs prompted us to examine whether WIP is linked to Syk. Phosphorylated Syk is known to associate with the SH2 and SH3 domains containing adaptor protein CrkL via phosphotyrosine–SH2 domain interactions (37). We have recently shown that CrkL directly binds to WIP and that the Syk homologue ZAP-70 associates with WIP after TCR ligation, suggesting that CrkL bridges phosphorylated ZAP-70 to WIP (37). Therefore, we examined whether Syk and WIP associate after FcεRI ligation. WIP immunoprecipitates from lysates of BMMCs from WT mice were probed for Syk before and after FcεRI ligation. There was no detectable Syk in WIP immunoprecipitates from unstimulated cells. FcεRI ligation resulted in the association of Syk with WIP (Fig. 6 A). These results suggest that WIP and Syk are recruited to the same complex in activated BMMCs.


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)

WIP associates with Syk after FcεRI ligation and protects it from degradation. (A) Association of WIP and Syk after FcεRI ligation. WIP immunoprecipitates from WT BMMCs were probed with Syk before and after FcεRI ligation. (B) Syk protein levels in Western blots from lysates of WT and WIP−/− BMMCs. (C) Syk phosphorylation after FcεRI ligation in WT and WIP−/− BMMCs. Syk immunoprecipitates were probed with antiphosphotyrosine mAb 4G10 and with anti-Syk as loading control. Numbers represent densitometric ratio of phopshoSyk to Syk bands. (D) Northern blot analysis of Syk mRNA in WT and WIP−/− BMMCs. 28S RNA is shown as loading control. (E) Effect of pretreatment with calpeptin and MG132 for 6 h on Syk levels in WT and WIP−/− BMMCs.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: WIP associates with Syk after FcεRI ligation and protects it from degradation. (A) Association of WIP and Syk after FcεRI ligation. WIP immunoprecipitates from WT BMMCs were probed with Syk before and after FcεRI ligation. (B) Syk protein levels in Western blots from lysates of WT and WIP−/− BMMCs. (C) Syk phosphorylation after FcεRI ligation in WT and WIP−/− BMMCs. Syk immunoprecipitates were probed with antiphosphotyrosine mAb 4G10 and with anti-Syk as loading control. Numbers represent densitometric ratio of phopshoSyk to Syk bands. (D) Northern blot analysis of Syk mRNA in WT and WIP−/− BMMCs. 28S RNA is shown as loading control. (E) Effect of pretreatment with calpeptin and MG132 for 6 h on Syk levels in WT and WIP−/− BMMCs.
Mentions: Syk plays a critical role in transmitting signals from FcεRI. The multiple defects in FcεRI signaling in WIP−/− BMMCs prompted us to examine whether WIP is linked to Syk. Phosphorylated Syk is known to associate with the SH2 and SH3 domains containing adaptor protein CrkL via phosphotyrosine–SH2 domain interactions (37). We have recently shown that CrkL directly binds to WIP and that the Syk homologue ZAP-70 associates with WIP after TCR ligation, suggesting that CrkL bridges phosphorylated ZAP-70 to WIP (37). Therefore, we examined whether Syk and WIP associate after FcεRI ligation. WIP immunoprecipitates from lysates of BMMCs from WT mice were probed for Syk before and after FcεRI ligation. There was no detectable Syk in WIP immunoprecipitates from unstimulated cells. FcεRI ligation resulted in the association of Syk with WIP (Fig. 6 A). These results suggest that WIP and Syk are recruited to the same complex in activated BMMCs.

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