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Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells.

Seveau S, Bierne H, Giroux S, Prévost MC, Cossart P - J. Cell Biol. (2004)

Bottom Line: We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry.In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent.Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

View Article: PubMed Central - PubMed

Affiliation: Unité des Interactions Bactéries-Cellules, INSERM U604, Institut Pasteur, 75015 Paris Cedex 15, France.

ABSTRACT
Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

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Cholesterol depletion does not affect HGF-R phosphorylation and association of PI 3-kinase with phosphotyrosine proteins. (A) Control (−) and cholesterol-depleted (+) cells were stimulated or not (NS) with soluble InlB (8 nM) for 1 min at 37°C. Cells were lysed and lysates were immunoprecipitated with anti–HGF-R antibodies. Immunoprecipitated proteins were analyzed by Western blotting with anti-phosphotyrosine and anti–HGF-R. (B) Control (−) and cholesterol-depleted (+) cells were stimulated or not (NS) with soluble InlB (8 nM) or HGF (0.6 nM) for 1 min at 37°C. Cells were lysed and lysates were immunoprecipitated with anti-phosphotyrosine antibodies. Total cell lysates and immunoprecipitated proteins were analyzed by Western blotting with anti–p85-α antibodies.
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fig9: Cholesterol depletion does not affect HGF-R phosphorylation and association of PI 3-kinase with phosphotyrosine proteins. (A) Control (−) and cholesterol-depleted (+) cells were stimulated or not (NS) with soluble InlB (8 nM) for 1 min at 37°C. Cells were lysed and lysates were immunoprecipitated with anti–HGF-R antibodies. Immunoprecipitated proteins were analyzed by Western blotting with anti-phosphotyrosine and anti–HGF-R. (B) Control (−) and cholesterol-depleted (+) cells were stimulated or not (NS) with soluble InlB (8 nM) or HGF (0.6 nM) for 1 min at 37°C. Cells were lysed and lysates were immunoprecipitated with anti-phosphotyrosine antibodies. Total cell lysates and immunoprecipitated proteins were analyzed by Western blotting with anti–p85-α antibodies.

Mentions: Some of the InlB- and HGF-signaling events occurring downstream from the initial interaction with HGF-R have been identified, including HGF-R tyrosine phosphorylation, recruitment and activation of Gab1, Cbl and Shc, and formation of signaling complexes containing these adapters and the p85 subunit of PI 3-kinase (Ireton et al., 1999). We investigated whether InlB could still induce HGF-R tyrosine phosphorylation and PI 3-kinase recruitment after cholesterol depletion. Vero cells incubated at 37°C for 1 min with soluble InlB (8 nM) were lysed and cell lysates were immunoprecipitated with anti–HGF-R or with anti-phosphotyrosine antibodies as described previously (Ireton et al., 1999). As shown in Fig. 9, MβCD treatment did not induce HGF-R phosphorylation or p85α recruitment in Vero cells. This control was important as it has been reported that MβCD treatment could induce by itself PI 3-Kinase activation and the phosphorylation of the epidermal growth factor (Chen and Resh, 2002). As previously reported, in control cells (not incubated with MβCD), soluble InlB induced HGF-R tyrosine phosphorylation and coimmunoprecipitation of the p85α subunit of PI 3-kinase with phosphotyrosine proteins (Ireton et al., 1999). After cholesterol depletion, soluble InlB and HGF still induced tyrosine phosphorylation of HGF-R (Fig. 9 A) as well as the coimmunoprecipitation of the p85α subunit of PI 3-kinase with phosphotyrosine proteins (Fig. 9 B).


Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells.

Seveau S, Bierne H, Giroux S, Prévost MC, Cossart P - J. Cell Biol. (2004)

Cholesterol depletion does not affect HGF-R phosphorylation and association of PI 3-kinase with phosphotyrosine proteins. (A) Control (−) and cholesterol-depleted (+) cells were stimulated or not (NS) with soluble InlB (8 nM) for 1 min at 37°C. Cells were lysed and lysates were immunoprecipitated with anti–HGF-R antibodies. Immunoprecipitated proteins were analyzed by Western blotting with anti-phosphotyrosine and anti–HGF-R. (B) Control (−) and cholesterol-depleted (+) cells were stimulated or not (NS) with soluble InlB (8 nM) or HGF (0.6 nM) for 1 min at 37°C. Cells were lysed and lysates were immunoprecipitated with anti-phosphotyrosine antibodies. Total cell lysates and immunoprecipitated proteins were analyzed by Western blotting with anti–p85-α antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172418&req=5

fig9: Cholesterol depletion does not affect HGF-R phosphorylation and association of PI 3-kinase with phosphotyrosine proteins. (A) Control (−) and cholesterol-depleted (+) cells were stimulated or not (NS) with soluble InlB (8 nM) for 1 min at 37°C. Cells were lysed and lysates were immunoprecipitated with anti–HGF-R antibodies. Immunoprecipitated proteins were analyzed by Western blotting with anti-phosphotyrosine and anti–HGF-R. (B) Control (−) and cholesterol-depleted (+) cells were stimulated or not (NS) with soluble InlB (8 nM) or HGF (0.6 nM) for 1 min at 37°C. Cells were lysed and lysates were immunoprecipitated with anti-phosphotyrosine antibodies. Total cell lysates and immunoprecipitated proteins were analyzed by Western blotting with anti–p85-α antibodies.
Mentions: Some of the InlB- and HGF-signaling events occurring downstream from the initial interaction with HGF-R have been identified, including HGF-R tyrosine phosphorylation, recruitment and activation of Gab1, Cbl and Shc, and formation of signaling complexes containing these adapters and the p85 subunit of PI 3-kinase (Ireton et al., 1999). We investigated whether InlB could still induce HGF-R tyrosine phosphorylation and PI 3-kinase recruitment after cholesterol depletion. Vero cells incubated at 37°C for 1 min with soluble InlB (8 nM) were lysed and cell lysates were immunoprecipitated with anti–HGF-R or with anti-phosphotyrosine antibodies as described previously (Ireton et al., 1999). As shown in Fig. 9, MβCD treatment did not induce HGF-R phosphorylation or p85α recruitment in Vero cells. This control was important as it has been reported that MβCD treatment could induce by itself PI 3-Kinase activation and the phosphorylation of the epidermal growth factor (Chen and Resh, 2002). As previously reported, in control cells (not incubated with MβCD), soluble InlB induced HGF-R tyrosine phosphorylation and coimmunoprecipitation of the p85α subunit of PI 3-kinase with phosphotyrosine proteins (Ireton et al., 1999). After cholesterol depletion, soluble InlB and HGF still induced tyrosine phosphorylation of HGF-R (Fig. 9 A) as well as the coimmunoprecipitation of the p85α subunit of PI 3-kinase with phosphotyrosine proteins (Fig. 9 B).

Bottom Line: We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry.In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent.Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

View Article: PubMed Central - PubMed

Affiliation: Unité des Interactions Bactéries-Cellules, INSERM U604, Institut Pasteur, 75015 Paris Cedex 15, France.

ABSTRACT
Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

Show MeSH
Related in: MedlinePlus