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Cell-surface localization of Pellino antagonizes Toll-mediated innate immune signalling by controlling MyD88 turnover in Drosophila.

Ji S, Sun M, Zheng X, Li L, Sun L, Chen D, Sun Q - Nat Commun (2014)

Bottom Line: We show that Pellino accumulates at the plasma membrane upon the activation of Toll signalling in a MyD88-dependent manner.Moreover, we find that Pellino is associated with MyD88 via its CTE domain, which is necessary and sufficient to promote Pellino accumulation at the plasma membrane where it targets MyD88 for ubiquitination and degradation.Collectively, our study uncovers a mechanism by which a feedback regulatory loop involving MyD88 and Pellino controls Toll-mediated signalling, thereby maintaining homeostasis of host innate immunity.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China [2] State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China [3].

ABSTRACT
Innate immunity mediated by Toll signalling has been extensively studied, but how Toll signalling is precisely controlled in balancing innate immune responses remains poorly understood. It was reported that the plasma membrane localization of Drosophila MyD88 is necessary for the recruitment of cytosolic adaptor Tube to the cell surface, thus contributing to Toll signalling transduction. Here we demonstrate that Drosophila Pellino functions as a negative regulator in Toll-mediated signalling. We show that Pellino accumulates at the plasma membrane upon the activation of Toll signalling in a MyD88-dependent manner. Moreover, we find that Pellino is associated with MyD88 via its CTE domain, which is necessary and sufficient to promote Pellino accumulation at the plasma membrane where it targets MyD88 for ubiquitination and degradation. Collectively, our study uncovers a mechanism by which a feedback regulatory loop involving MyD88 and Pellino controls Toll-mediated signalling, thereby maintaining homeostasis of host innate immunity.

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Pellino-mediated MyD88 ubiquitination is dependent on CTE domain.(a,b) S2 cells were transfected with Myc-MyD88ΔCTE and HA-Ub with Flag-Pellino (a), or also pretreated with pellino dsRNA (b). Cell lysates were used to perform immunoprecipitation experiments with anti-Myc beads followed by immunoblot analysis with anti-HA antibodies to show ubiquitination patterns of MyD88ΔCTE. Expression levels of transfected proteins and Pellino knockdown efficiency in whole-cell lysates are shown in the bottom panel. (c,d) S2 cells transfected with Flag-Pellino alone or in combination with Myc-tagged MyD88 CTE domain were stained with Hoechst and anti-Flag antibody alone or with anti-Myc antibody as indicated and imaged by confocal microscopy (c). Scale bars, 10 μm. (d) Statistical assays of PM or CP localization of Pellino in transfected cells in panel (c). (e,f) S2 cells transfected with Flag-Tube alone or in combination with Myc-tagged MyD88 or MyD88 CTE domain were stained with Hoechst and anti-Flag antibody alone or with anti-Myc antibody as indicated and imaged by confocal microscopy (e). Scale bars, 10 μm. (f) Quantification assays of P.M. or C.P. localization of Tube in transfected cells in (e).
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f6: Pellino-mediated MyD88 ubiquitination is dependent on CTE domain.(a,b) S2 cells were transfected with Myc-MyD88ΔCTE and HA-Ub with Flag-Pellino (a), or also pretreated with pellino dsRNA (b). Cell lysates were used to perform immunoprecipitation experiments with anti-Myc beads followed by immunoblot analysis with anti-HA antibodies to show ubiquitination patterns of MyD88ΔCTE. Expression levels of transfected proteins and Pellino knockdown efficiency in whole-cell lysates are shown in the bottom panel. (c,d) S2 cells transfected with Flag-Pellino alone or in combination with Myc-tagged MyD88 CTE domain were stained with Hoechst and anti-Flag antibody alone or with anti-Myc antibody as indicated and imaged by confocal microscopy (c). Scale bars, 10 μm. (d) Statistical assays of PM or CP localization of Pellino in transfected cells in panel (c). (e,f) S2 cells transfected with Flag-Tube alone or in combination with Myc-tagged MyD88 or MyD88 CTE domain were stained with Hoechst and anti-Flag antibody alone or with anti-Myc antibody as indicated and imaged by confocal microscopy (e). Scale bars, 10 μm. (f) Quantification assays of P.M. or C.P. localization of Tube in transfected cells in (e).

Mentions: We next tested whether MyD88 was sufficient to induce the plasma membrane accumulation of Pellino. Plasma membrane accumulation of Pellino significantly increased when S2 cells were co-expressed with Myc-Pellino and Flag-MyD88, compared with Pellino expressed alone in S2 cells (Fig. 5c,d). To investigate the molecular basis of how MyD88 promotes plasma membrane accumulation of Pellino, we identified the specific domain in MyD88 required for Pellino interactions and generated a series of mutant forms of MyD88 constructs (Fig. 5e). Co-immunoprecipitation demonstrated that although DD and TIR domains were not required for MyD88 interactions with Pellino, the MyD88 CTE domain (a phosphoinositide-binding domain at the C-terminal of MyD88)17 was essential since the mutant form of MyD88ΔCTE lacking CTE domain did not associate with Pellino (Fig. 5f). To test whether the CTE domain was required for MyD88 to promote the plasma membrane accumulation of Pellino, we co-overexpressed the mutant form of MyD88ΔCTE and Pellino in S2, and found that, unlike wild-type MyD88, MyD88ΔCTE failed to promote Pellino plasma membrane accumulation (Fig. 5c,d,g and h). Thus, the CTE domain is required for MyD88 plasma membrane localization, MyD88–Pellino interaction and MyD88 recruitment of Pellino plasma membrane accumulation. We next tested whether the MyD88 CTE domain was involved in Pellino-mediated MyD88 ubiquitination and degradation. Either overexpression or knockdown of Pellino did not affect the ubiquitination status and stability of MyD88ΔCTE, suggesting that Pellino regulates MyD88 ubiquitination through the CTE domain (Fig. 6a,b and Supplementary Fig. 7e). Given that the specific interaction of Pellino with the MyD88 CTE domain, we determined whether the MyD88 CTE domain was sufficient to promote plasma membrane localization by co-expression of Pellino and the MyD88 CTE domain in S2 cell cultures. Compared with the expression of Pellino alone, co-expression of Pellino with the MyD88 CTE domain resulted in significant cell-surface accumulation of Pellino (Fig. 6c,b). To determine the specificity of the CTE domain of MyD88 for the sub-cellular localization of Pellino, we tested whether MyD88 CTE was sufficient to recruit Tube onto the cell surface. Overexpression of full-length MyD88 resulted in the cell-surface accumulation of Tube, but MyD88 CTE alone did not (Fig. 6e,f). Taken together, our findings establish a model by which the activation of MyD88 promotes Pellino to accumulate onto the plasma membrane, where Pellino targets MyD88 for ubiquitination and degradation. This reciprocal regulation between MyD88 and Pellino appears to be mediated by the CTE domain of MyD88.


Cell-surface localization of Pellino antagonizes Toll-mediated innate immune signalling by controlling MyD88 turnover in Drosophila.

Ji S, Sun M, Zheng X, Li L, Sun L, Chen D, Sun Q - Nat Commun (2014)

Pellino-mediated MyD88 ubiquitination is dependent on CTE domain.(a,b) S2 cells were transfected with Myc-MyD88ΔCTE and HA-Ub with Flag-Pellino (a), or also pretreated with pellino dsRNA (b). Cell lysates were used to perform immunoprecipitation experiments with anti-Myc beads followed by immunoblot analysis with anti-HA antibodies to show ubiquitination patterns of MyD88ΔCTE. Expression levels of transfected proteins and Pellino knockdown efficiency in whole-cell lysates are shown in the bottom panel. (c,d) S2 cells transfected with Flag-Pellino alone or in combination with Myc-tagged MyD88 CTE domain were stained with Hoechst and anti-Flag antibody alone or with anti-Myc antibody as indicated and imaged by confocal microscopy (c). Scale bars, 10 μm. (d) Statistical assays of PM or CP localization of Pellino in transfected cells in panel (c). (e,f) S2 cells transfected with Flag-Tube alone or in combination with Myc-tagged MyD88 or MyD88 CTE domain were stained with Hoechst and anti-Flag antibody alone or with anti-Myc antibody as indicated and imaged by confocal microscopy (e). Scale bars, 10 μm. (f) Quantification assays of P.M. or C.P. localization of Tube in transfected cells in (e).
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f6: Pellino-mediated MyD88 ubiquitination is dependent on CTE domain.(a,b) S2 cells were transfected with Myc-MyD88ΔCTE and HA-Ub with Flag-Pellino (a), or also pretreated with pellino dsRNA (b). Cell lysates were used to perform immunoprecipitation experiments with anti-Myc beads followed by immunoblot analysis with anti-HA antibodies to show ubiquitination patterns of MyD88ΔCTE. Expression levels of transfected proteins and Pellino knockdown efficiency in whole-cell lysates are shown in the bottom panel. (c,d) S2 cells transfected with Flag-Pellino alone or in combination with Myc-tagged MyD88 CTE domain were stained with Hoechst and anti-Flag antibody alone or with anti-Myc antibody as indicated and imaged by confocal microscopy (c). Scale bars, 10 μm. (d) Statistical assays of PM or CP localization of Pellino in transfected cells in panel (c). (e,f) S2 cells transfected with Flag-Tube alone or in combination with Myc-tagged MyD88 or MyD88 CTE domain were stained with Hoechst and anti-Flag antibody alone or with anti-Myc antibody as indicated and imaged by confocal microscopy (e). Scale bars, 10 μm. (f) Quantification assays of P.M. or C.P. localization of Tube in transfected cells in (e).
Mentions: We next tested whether MyD88 was sufficient to induce the plasma membrane accumulation of Pellino. Plasma membrane accumulation of Pellino significantly increased when S2 cells were co-expressed with Myc-Pellino and Flag-MyD88, compared with Pellino expressed alone in S2 cells (Fig. 5c,d). To investigate the molecular basis of how MyD88 promotes plasma membrane accumulation of Pellino, we identified the specific domain in MyD88 required for Pellino interactions and generated a series of mutant forms of MyD88 constructs (Fig. 5e). Co-immunoprecipitation demonstrated that although DD and TIR domains were not required for MyD88 interactions with Pellino, the MyD88 CTE domain (a phosphoinositide-binding domain at the C-terminal of MyD88)17 was essential since the mutant form of MyD88ΔCTE lacking CTE domain did not associate with Pellino (Fig. 5f). To test whether the CTE domain was required for MyD88 to promote the plasma membrane accumulation of Pellino, we co-overexpressed the mutant form of MyD88ΔCTE and Pellino in S2, and found that, unlike wild-type MyD88, MyD88ΔCTE failed to promote Pellino plasma membrane accumulation (Fig. 5c,d,g and h). Thus, the CTE domain is required for MyD88 plasma membrane localization, MyD88–Pellino interaction and MyD88 recruitment of Pellino plasma membrane accumulation. We next tested whether the MyD88 CTE domain was involved in Pellino-mediated MyD88 ubiquitination and degradation. Either overexpression or knockdown of Pellino did not affect the ubiquitination status and stability of MyD88ΔCTE, suggesting that Pellino regulates MyD88 ubiquitination through the CTE domain (Fig. 6a,b and Supplementary Fig. 7e). Given that the specific interaction of Pellino with the MyD88 CTE domain, we determined whether the MyD88 CTE domain was sufficient to promote plasma membrane localization by co-expression of Pellino and the MyD88 CTE domain in S2 cell cultures. Compared with the expression of Pellino alone, co-expression of Pellino with the MyD88 CTE domain resulted in significant cell-surface accumulation of Pellino (Fig. 6c,b). To determine the specificity of the CTE domain of MyD88 for the sub-cellular localization of Pellino, we tested whether MyD88 CTE was sufficient to recruit Tube onto the cell surface. Overexpression of full-length MyD88 resulted in the cell-surface accumulation of Tube, but MyD88 CTE alone did not (Fig. 6e,f). Taken together, our findings establish a model by which the activation of MyD88 promotes Pellino to accumulate onto the plasma membrane, where Pellino targets MyD88 for ubiquitination and degradation. This reciprocal regulation between MyD88 and Pellino appears to be mediated by the CTE domain of MyD88.

Bottom Line: We show that Pellino accumulates at the plasma membrane upon the activation of Toll signalling in a MyD88-dependent manner.Moreover, we find that Pellino is associated with MyD88 via its CTE domain, which is necessary and sufficient to promote Pellino accumulation at the plasma membrane where it targets MyD88 for ubiquitination and degradation.Collectively, our study uncovers a mechanism by which a feedback regulatory loop involving MyD88 and Pellino controls Toll-mediated signalling, thereby maintaining homeostasis of host innate immunity.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China [2] State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China [3].

ABSTRACT
Innate immunity mediated by Toll signalling has been extensively studied, but how Toll signalling is precisely controlled in balancing innate immune responses remains poorly understood. It was reported that the plasma membrane localization of Drosophila MyD88 is necessary for the recruitment of cytosolic adaptor Tube to the cell surface, thus contributing to Toll signalling transduction. Here we demonstrate that Drosophila Pellino functions as a negative regulator in Toll-mediated signalling. We show that Pellino accumulates at the plasma membrane upon the activation of Toll signalling in a MyD88-dependent manner. Moreover, we find that Pellino is associated with MyD88 via its CTE domain, which is necessary and sufficient to promote Pellino accumulation at the plasma membrane where it targets MyD88 for ubiquitination and degradation. Collectively, our study uncovers a mechanism by which a feedback regulatory loop involving MyD88 and Pellino controls Toll-mediated signalling, thereby maintaining homeostasis of host innate immunity.

Show MeSH
Related in: MedlinePlus