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Roles of p-ERM and Rho-ROCK signaling in lymphocyte polarity and uropod formation.

Lee JH, Katakai T, Hara T, Gonda H, Sugai M, Shimizu A - J. Cell Biol. (2004)

Bottom Line: T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton-dependent manner.Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation.Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Biology and Genetics, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.

ABSTRACT
Front-rear asymmetry in motile cells is crucial for efficient directional movement. The uropod in migrating lymphocytes is a posterior protrusion in which several proteins, including CD44 and ezrin/radixin/moesin (ERM), are concentrated. In EL4.G8 T-lymphoma cells, Thr567 phosphorylation in the COOH-terminal domain of ezrin regulates the selective localization of ezrin in the uropod. Overexpression of the phosphorylation-mimetic T567D ezrin enhances uropod size and cell migration. T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton-dependent manner. Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor Y-27632 disrupts the uropod but not the polar cap, indicating that Rho-ROCK signaling is required for posterior protrusion but not for ERM phosphorylation. Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation. Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells. Thus, phosphorylated ERM has a potential function in establishing plasma membrane "posteriority" in the induction of the uropod in T lymphocytes.

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Rho-GEF activity associated with p-ERM and interaction of Dbl and open-form ezrin in EL4.G8 cells. (A) Protein complex containing p-ERM has Rho-GEF activity. p-ERM was immunoprecipitated from EL4.G8 cell lysate, and the associated Rho-GEF activity was measured compared with that when the immunoprecipitation was performed with control antibody. Rho-GTP in the reaction was pulled down and detected by Western blotting (top). The presence of p-ERM was also confirmed by Western blotting (bottom). (B) Ezrin interacts with Dbl. Dbl coimmunoprecipitated with ezrin was detected by Western blotting. (C) Open-form ezrin specifically binds to Dbl by its NT domain. Ezrin mutants were immunoprecipitated with anti-GFP antibody from lysates of stable transfectants and the interaction with Dbl was analyzed. (D) Most of the Rho-GEF activity associated with ezrin is associated with the phosphorylated form. p-ERM was immunodepleted from EL4.G8 cell lysate, and Rho-GEF activity associated with ezrin was measured compared with that when the immunodepletion was performed with control antibody. (D and E) The relative activity compared with the control level was normalized by the amount of immunoprecipitated ezrin and is shown as a percent of control (histograms). (E) Dbl mediates a major part of the association of Rho-GEF activity with ezrin. Dbl was immunodepleted and Rho-GEF activity associated with ezrin was analyzed. (F) Rho-GEF activity is increased in lysates containing T567D compared with WT ezrin and the activity is associated with the NT but not the CT domain. Rho-GEF activity was analyzed in lysates containing ezrin mutants. The activity in lysates containing T567D relative to that in lysates containing WT ezrin is shown (histogram). (G) Overexpression of Dbl-AID significantly inhibits uropod formation in EL4.G8 cells. EL4.G8 cells were transiently transfected with a construct for GFP-tagged Dbl-AID, and the percentage of cells exhibiting the uropod is shown (n > 180).
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fig5: Rho-GEF activity associated with p-ERM and interaction of Dbl and open-form ezrin in EL4.G8 cells. (A) Protein complex containing p-ERM has Rho-GEF activity. p-ERM was immunoprecipitated from EL4.G8 cell lysate, and the associated Rho-GEF activity was measured compared with that when the immunoprecipitation was performed with control antibody. Rho-GTP in the reaction was pulled down and detected by Western blotting (top). The presence of p-ERM was also confirmed by Western blotting (bottom). (B) Ezrin interacts with Dbl. Dbl coimmunoprecipitated with ezrin was detected by Western blotting. (C) Open-form ezrin specifically binds to Dbl by its NT domain. Ezrin mutants were immunoprecipitated with anti-GFP antibody from lysates of stable transfectants and the interaction with Dbl was analyzed. (D) Most of the Rho-GEF activity associated with ezrin is associated with the phosphorylated form. p-ERM was immunodepleted from EL4.G8 cell lysate, and Rho-GEF activity associated with ezrin was measured compared with that when the immunodepletion was performed with control antibody. (D and E) The relative activity compared with the control level was normalized by the amount of immunoprecipitated ezrin and is shown as a percent of control (histograms). (E) Dbl mediates a major part of the association of Rho-GEF activity with ezrin. Dbl was immunodepleted and Rho-GEF activity associated with ezrin was analyzed. (F) Rho-GEF activity is increased in lysates containing T567D compared with WT ezrin and the activity is associated with the NT but not the CT domain. Rho-GEF activity was analyzed in lysates containing ezrin mutants. The activity in lysates containing T567D relative to that in lysates containing WT ezrin is shown (histogram). (G) Overexpression of Dbl-AID significantly inhibits uropod formation in EL4.G8 cells. EL4.G8 cells were transiently transfected with a construct for GFP-tagged Dbl-AID, and the percentage of cells exhibiting the uropod is shown (n > 180).

Mentions: To address the possibility that p-ERM is involved in Rho–ROCK signaling, we checked the level of Rho-GDP/GTP exchange activity associated with p-ERM in EL4.G8 cells. Using immunoprecipitated p-ERM protein complex from the cell lysate, the exchange activity was assessed by an in vitro nucleotide exchange reaction for recombinant Rho-GDP, followed by RBD (Rho-binding domain)-GST–mediated pull-down detection of Rho-GTP. Strikingly, we detected a substantial nucleotide exchange activity for Rho in p-ERM–containing precipitate, whereas no activity was detected using control antibody (Fig. 5 A). When p-ERM was depleted previously from the cell lysate, the exchange activity associated with ezrin was markedly reduced (Fig. 5 D).


Roles of p-ERM and Rho-ROCK signaling in lymphocyte polarity and uropod formation.

Lee JH, Katakai T, Hara T, Gonda H, Sugai M, Shimizu A - J. Cell Biol. (2004)

Rho-GEF activity associated with p-ERM and interaction of Dbl and open-form ezrin in EL4.G8 cells. (A) Protein complex containing p-ERM has Rho-GEF activity. p-ERM was immunoprecipitated from EL4.G8 cell lysate, and the associated Rho-GEF activity was measured compared with that when the immunoprecipitation was performed with control antibody. Rho-GTP in the reaction was pulled down and detected by Western blotting (top). The presence of p-ERM was also confirmed by Western blotting (bottom). (B) Ezrin interacts with Dbl. Dbl coimmunoprecipitated with ezrin was detected by Western blotting. (C) Open-form ezrin specifically binds to Dbl by its NT domain. Ezrin mutants were immunoprecipitated with anti-GFP antibody from lysates of stable transfectants and the interaction with Dbl was analyzed. (D) Most of the Rho-GEF activity associated with ezrin is associated with the phosphorylated form. p-ERM was immunodepleted from EL4.G8 cell lysate, and Rho-GEF activity associated with ezrin was measured compared with that when the immunodepletion was performed with control antibody. (D and E) The relative activity compared with the control level was normalized by the amount of immunoprecipitated ezrin and is shown as a percent of control (histograms). (E) Dbl mediates a major part of the association of Rho-GEF activity with ezrin. Dbl was immunodepleted and Rho-GEF activity associated with ezrin was analyzed. (F) Rho-GEF activity is increased in lysates containing T567D compared with WT ezrin and the activity is associated with the NT but not the CT domain. Rho-GEF activity was analyzed in lysates containing ezrin mutants. The activity in lysates containing T567D relative to that in lysates containing WT ezrin is shown (histogram). (G) Overexpression of Dbl-AID significantly inhibits uropod formation in EL4.G8 cells. EL4.G8 cells were transiently transfected with a construct for GFP-tagged Dbl-AID, and the percentage of cells exhibiting the uropod is shown (n > 180).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172551&req=5

fig5: Rho-GEF activity associated with p-ERM and interaction of Dbl and open-form ezrin in EL4.G8 cells. (A) Protein complex containing p-ERM has Rho-GEF activity. p-ERM was immunoprecipitated from EL4.G8 cell lysate, and the associated Rho-GEF activity was measured compared with that when the immunoprecipitation was performed with control antibody. Rho-GTP in the reaction was pulled down and detected by Western blotting (top). The presence of p-ERM was also confirmed by Western blotting (bottom). (B) Ezrin interacts with Dbl. Dbl coimmunoprecipitated with ezrin was detected by Western blotting. (C) Open-form ezrin specifically binds to Dbl by its NT domain. Ezrin mutants were immunoprecipitated with anti-GFP antibody from lysates of stable transfectants and the interaction with Dbl was analyzed. (D) Most of the Rho-GEF activity associated with ezrin is associated with the phosphorylated form. p-ERM was immunodepleted from EL4.G8 cell lysate, and Rho-GEF activity associated with ezrin was measured compared with that when the immunodepletion was performed with control antibody. (D and E) The relative activity compared with the control level was normalized by the amount of immunoprecipitated ezrin and is shown as a percent of control (histograms). (E) Dbl mediates a major part of the association of Rho-GEF activity with ezrin. Dbl was immunodepleted and Rho-GEF activity associated with ezrin was analyzed. (F) Rho-GEF activity is increased in lysates containing T567D compared with WT ezrin and the activity is associated with the NT but not the CT domain. Rho-GEF activity was analyzed in lysates containing ezrin mutants. The activity in lysates containing T567D relative to that in lysates containing WT ezrin is shown (histogram). (G) Overexpression of Dbl-AID significantly inhibits uropod formation in EL4.G8 cells. EL4.G8 cells were transiently transfected with a construct for GFP-tagged Dbl-AID, and the percentage of cells exhibiting the uropod is shown (n > 180).
Mentions: To address the possibility that p-ERM is involved in Rho–ROCK signaling, we checked the level of Rho-GDP/GTP exchange activity associated with p-ERM in EL4.G8 cells. Using immunoprecipitated p-ERM protein complex from the cell lysate, the exchange activity was assessed by an in vitro nucleotide exchange reaction for recombinant Rho-GDP, followed by RBD (Rho-binding domain)-GST–mediated pull-down detection of Rho-GTP. Strikingly, we detected a substantial nucleotide exchange activity for Rho in p-ERM–containing precipitate, whereas no activity was detected using control antibody (Fig. 5 A). When p-ERM was depleted previously from the cell lysate, the exchange activity associated with ezrin was markedly reduced (Fig. 5 D).

Bottom Line: T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton-dependent manner.Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation.Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Biology and Genetics, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.

ABSTRACT
Front-rear asymmetry in motile cells is crucial for efficient directional movement. The uropod in migrating lymphocytes is a posterior protrusion in which several proteins, including CD44 and ezrin/radixin/moesin (ERM), are concentrated. In EL4.G8 T-lymphoma cells, Thr567 phosphorylation in the COOH-terminal domain of ezrin regulates the selective localization of ezrin in the uropod. Overexpression of the phosphorylation-mimetic T567D ezrin enhances uropod size and cell migration. T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton-dependent manner. Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor Y-27632 disrupts the uropod but not the polar cap, indicating that Rho-ROCK signaling is required for posterior protrusion but not for ERM phosphorylation. Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation. Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells. Thus, phosphorylated ERM has a potential function in establishing plasma membrane "posteriority" in the induction of the uropod in T lymphocytes.

Show MeSH
Related in: MedlinePlus