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Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells.

Larson DR, Gosse JA, Holowka DA, Baird BA, Webb WW - J. Cell Biol. (2005)

Bottom Line: During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity.Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI implicated in downstream signaling events.They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein-protein interactions in intact, living cells.

View Article: PubMed Central - PubMed

Affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcepsilonRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcepsilonRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcepsilonRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein-protein interactions in intact, living cells.

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Degranulation responses for RBL mast cells sensitized with various IgEs and stimulated at 21 or 37°C. Cells were sensitized with IgE derivatives as described in Materials and methods and stimulated with 1 μg/ml DNP-BSA for 1 h at 21 or at 37°C. Release of β-hexosaminidase into cell supernatants was measured as described in Materials and methods. Error bars represent the SD from three separate experiments.
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fig7: Degranulation responses for RBL mast cells sensitized with various IgEs and stimulated at 21 or 37°C. Cells were sensitized with IgE derivatives as described in Materials and methods and stimulated with 1 μg/ml DNP-BSA for 1 h at 21 or at 37°C. Release of β-hexosaminidase into cell supernatants was measured as described in Materials and methods. Error bars represent the SD from three separate experiments.

Mentions: Lyn has also been shown to play negative roles in regulating Fyn kinase, phosphoinositide 3-kinase, and degranulation (Parravicini et al., 2002; Odom et al., 2004), and the interactions that we detect may also be relevant to this function of Lyn. In Lyn−/− mast cells, stimulated tyrosine phosphorylation of FcɛRI and other proteins is more sustained, albeit reduced in magnitude (Hernandez-Hansen et al., 2004), so it is possible that the interaction between Lyn and FcɛRI that we detect is important for regulating the time course of the tyrosine phosphorylation response. Consistent with this idea, cytochalasin D reduces the magnitude of this stimulated interaction (Fig. 7) while facilitating a more sustained phosphorylation response to antigen (Holowka et al., 2000). Although ordered membrane lipids may play a role in facilitating the interactions detected, the targeting of EGFP by the PM sequence is clearly not sufficient for these interactions to occur.


Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells.

Larson DR, Gosse JA, Holowka DA, Baird BA, Webb WW - J. Cell Biol. (2005)

Degranulation responses for RBL mast cells sensitized with various IgEs and stimulated at 21 or 37°C. Cells were sensitized with IgE derivatives as described in Materials and methods and stimulated with 1 μg/ml DNP-BSA for 1 h at 21 or at 37°C. Release of β-hexosaminidase into cell supernatants was measured as described in Materials and methods. Error bars represent the SD from three separate experiments.
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Related In: Results  -  Collection

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fig7: Degranulation responses for RBL mast cells sensitized with various IgEs and stimulated at 21 or 37°C. Cells were sensitized with IgE derivatives as described in Materials and methods and stimulated with 1 μg/ml DNP-BSA for 1 h at 21 or at 37°C. Release of β-hexosaminidase into cell supernatants was measured as described in Materials and methods. Error bars represent the SD from three separate experiments.
Mentions: Lyn has also been shown to play negative roles in regulating Fyn kinase, phosphoinositide 3-kinase, and degranulation (Parravicini et al., 2002; Odom et al., 2004), and the interactions that we detect may also be relevant to this function of Lyn. In Lyn−/− mast cells, stimulated tyrosine phosphorylation of FcɛRI and other proteins is more sustained, albeit reduced in magnitude (Hernandez-Hansen et al., 2004), so it is possible that the interaction between Lyn and FcɛRI that we detect is important for regulating the time course of the tyrosine phosphorylation response. Consistent with this idea, cytochalasin D reduces the magnitude of this stimulated interaction (Fig. 7) while facilitating a more sustained phosphorylation response to antigen (Holowka et al., 2000). Although ordered membrane lipids may play a role in facilitating the interactions detected, the targeting of EGFP by the PM sequence is clearly not sufficient for these interactions to occur.

Bottom Line: During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity.Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI implicated in downstream signaling events.They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein-protein interactions in intact, living cells.

View Article: PubMed Central - PubMed

Affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcepsilonRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcepsilonRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcepsilonRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein-protein interactions in intact, living cells.

Show MeSH
Related in: MedlinePlus