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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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The Rho–ROCK pathway is involved in uropod formation. (A) Y-27632 abolishes uropod but not polar cap formation (arrows). Y-27632–treated parental EL4.G8 cells were stained for p-ERM and CD44 (top panels). GFP-tagged WT and T567D ezrin were also colocalized with CD44 in Y-27632–treated stable transfectants (middle and bottom panels). Bar, 10 μm. (B) Percentage of cells exhibiting the polar cap in A (n > 150). (C) Y-27632 does not alter the phosphorylation status of ERM proteins. The total amount of ezrin and the p-ERM level were determined by Western blotting using lysates from control or Y-27632–treated EL4.G8 cells. (D) WT RhoA shows a diffuse distribution in the cytoplasm, including the leading edge (arrowhead). Constitutively active (Q63L) RhoA is preferentially localized at the rear part of the cells (asterisk), and a dominant-negative form (T19N) abolishes uropod formation but not polar cap formation (arrow). EL4.G8 cells were transiently transfected with WT, Q63L, or T19N RhoA and examined for GFP and CD44 24 h after the transfection. Bar, 10 μm. (E) Percentage of cells exhibiting the uropod in D (n > 75).
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fig4: The Rho–ROCK pathway is involved in uropod formation. (A) Y-27632 abolishes uropod but not polar cap formation (arrows). Y-27632–treated parental EL4.G8 cells were stained for p-ERM and CD44 (top panels). GFP-tagged WT and T567D ezrin were also colocalized with CD44 in Y-27632–treated stable transfectants (middle and bottom panels). Bar, 10 μm. (B) Percentage of cells exhibiting the polar cap in A (n > 150). (C) Y-27632 does not alter the phosphorylation status of ERM proteins. The total amount of ezrin and the p-ERM level were determined by Western blotting using lysates from control or Y-27632–treated EL4.G8 cells. (D) WT RhoA shows a diffuse distribution in the cytoplasm, including the leading edge (arrowhead). Constitutively active (Q63L) RhoA is preferentially localized at the rear part of the cells (asterisk), and a dominant-negative form (T19N) abolishes uropod formation but not polar cap formation (arrow). EL4.G8 cells were transiently transfected with WT, Q63L, or T19N RhoA and examined for GFP and CD44 24 h after the transfection. Bar, 10 μm. (E) Percentage of cells exhibiting the uropod in D (n > 75).

Mentions: Because several Ser/Thr kinases (including the Rho effector ROCK and PKCs) have been shown to phosphorylate ERM proteins (Mangeat et al., 1999; Tsukita and Yonemura, 1999; Bretscher et al., 2002), we next treated EL4.G8 cells with an inhibitor for ROCK (Y-27632) or for PKCs (bisindolylmaleimide I [BIM]). BIM had no remarkable effect on the morphology of the cells (not depicted). In contrast, Y-27632 completely eliminated the uropod, although the CD44/p-ERM polar cap was maintained (Fig. 4, A and B). The preservation of p-ERM was confirmed by Western blotting (Fig. 4 C). The phosphorylation status was also unchanged in Y-27632–treated BW5147 cells, despite the fact that the uropod was completely disrupted (unpublished data). These experiments demonstrate that endogenous p-ERM proteins are also capable of constructing the polar cap in uropod-less cells. Likewise, transfectants of WT as well as T567D ezrin exhibited a polar cap but not a uropod under the ROCK-inhibited condition (Fig. 4, A and B). Thus, ERM phosphorylation is independent of ROCK as well as of PKCs in EL4.G8, but ROCK activity is indispensable for the formation of the mature uropod.


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)

The Rho–ROCK pathway is involved in uropod formation. (A) Y-27632 abolishes uropod but not polar cap formation (arrows). Y-27632–treated parental EL4.G8 cells were stained for p-ERM and CD44 (top panels). GFP-tagged WT and T567D ezrin were also colocalized with CD44 in Y-27632–treated stable transfectants (middle and bottom panels). Bar, 10 μm. (B) Percentage of cells exhibiting the polar cap in A (n > 150). (C) Y-27632 does not alter the phosphorylation status of ERM proteins. The total amount of ezrin and the p-ERM level were determined by Western blotting using lysates from control or Y-27632–treated EL4.G8 cells. (D) WT RhoA shows a diffuse distribution in the cytoplasm, including the leading edge (arrowhead). Constitutively active (Q63L) RhoA is preferentially localized at the rear part of the cells (asterisk), and a dominant-negative form (T19N) abolishes uropod formation but not polar cap formation (arrow). EL4.G8 cells were transiently transfected with WT, Q63L, or T19N RhoA and examined for GFP and CD44 24 h after the transfection. Bar, 10 μm. (E) Percentage of cells exhibiting the uropod in D (n > 75).
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fig4: The Rho–ROCK pathway is involved in uropod formation. (A) Y-27632 abolishes uropod but not polar cap formation (arrows). Y-27632–treated parental EL4.G8 cells were stained for p-ERM and CD44 (top panels). GFP-tagged WT and T567D ezrin were also colocalized with CD44 in Y-27632–treated stable transfectants (middle and bottom panels). Bar, 10 μm. (B) Percentage of cells exhibiting the polar cap in A (n > 150). (C) Y-27632 does not alter the phosphorylation status of ERM proteins. The total amount of ezrin and the p-ERM level were determined by Western blotting using lysates from control or Y-27632–treated EL4.G8 cells. (D) WT RhoA shows a diffuse distribution in the cytoplasm, including the leading edge (arrowhead). Constitutively active (Q63L) RhoA is preferentially localized at the rear part of the cells (asterisk), and a dominant-negative form (T19N) abolishes uropod formation but not polar cap formation (arrow). EL4.G8 cells were transiently transfected with WT, Q63L, or T19N RhoA and examined for GFP and CD44 24 h after the transfection. Bar, 10 μm. (E) Percentage of cells exhibiting the uropod in D (n > 75).
Mentions: Because several Ser/Thr kinases (including the Rho effector ROCK and PKCs) have been shown to phosphorylate ERM proteins (Mangeat et al., 1999; Tsukita and Yonemura, 1999; Bretscher et al., 2002), we next treated EL4.G8 cells with an inhibitor for ROCK (Y-27632) or for PKCs (bisindolylmaleimide I [BIM]). BIM had no remarkable effect on the morphology of the cells (not depicted). In contrast, Y-27632 completely eliminated the uropod, although the CD44/p-ERM polar cap was maintained (Fig. 4, A and B). The preservation of p-ERM was confirmed by Western blotting (Fig. 4 C). The phosphorylation status was also unchanged in Y-27632–treated BW5147 cells, despite the fact that the uropod was completely disrupted (unpublished data). These experiments demonstrate that endogenous p-ERM proteins are also capable of constructing the polar cap in uropod-less cells. Likewise, transfectants of WT as well as T567D ezrin exhibited a polar cap but not a uropod under the ROCK-inhibited condition (Fig. 4, A and B). Thus, ERM phosphorylation is independent of ROCK as well as of PKCs in EL4.G8, but ROCK activity is indispensable for the formation of the mature uropod.

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