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Lulu2 regulates the circumferential actomyosin tensile system in epithelial cells through p114RhoGEF.

Nakajima H, Tanoue T - J. Cell Biol. (2011)

Bottom Line: In its regulation of the belt, Lulu2 interacts with and activates p114RhoGEF, a Rho-specific guanine nucleotide exchanging factor (GEF), at apical cell-cell junctions.This interaction is negatively regulated via phosphorylation events in the FERM-adjacent domain of Lulu2 catalyzed by atypical protein kinase C.We further found that Patj, an apical cell polarity regulator, recruits p114RhoGEF to apical cell-cell boundaries via PDZ (PSD-95/Dlg/ZO-1) domain-mediated interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Global Centers of Excellence Program for Integrative Membrane Biology, Graduate School of Medicine, Kobe University, Chuo-ku, Kobe 650-0017, Japan.

ABSTRACT
Myosin II-driven mechanical forces control epithelial cell shape and morphogenesis. In particular, the circumferential actomyosin belt, which is located along apical cell-cell junctions, regulates many cellular processes. Despite its importance, the molecular mechanisms regulating the belt are not fully understood. In this paper, we characterize Lulu2, a FERM (4.1 protein, ezrin, radixin, moesin) domain-containing molecule homologous to Drosophila melanogaster Yurt, as an important regulator. In epithelial cells, Lulu2 is localized along apical cell-cell boundaries, and Lulu2 depletion by ribonucleic acid interference results in disorganization of the circumferential actomyosin belt. In its regulation of the belt, Lulu2 interacts with and activates p114RhoGEF, a Rho-specific guanine nucleotide exchanging factor (GEF), at apical cell-cell junctions. This interaction is negatively regulated via phosphorylation events in the FERM-adjacent domain of Lulu2 catalyzed by atypical protein kinase C. We further found that Patj, an apical cell polarity regulator, recruits p114RhoGEF to apical cell-cell boundaries via PDZ (PSD-95/Dlg/ZO-1) domain-mediated interaction. These findings therefore reveal a novel molecular system regulating the circumferential actomyosin belt in epithelial cells.

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Lulu2 is negatively regulated by aPKC. (A) Quantification of apical areas defined by ZO-1 staining signals in EGFP-Lulu2–, EGFP-Lulu2 4A–, or EGFP-Lulu2 4E–expressing cells. Relative apical areas in Lulu2-expressing cells normalized by those in neighboring cells were quantified as in Fig. 4 B. MDCK cells were used. N, N terminus; C, C terminus. (B and C) DLD-1 cells transfected with T7-tagged wild-type aPKC-ζ or dominant-negative aPKC (aPKC DN) were stained for T7 and ZO-1 (B). Parental or Lulu2-expressing MDCK cells were transfected with T7–aPKC DN and stained for T7 and ZO-1 (C). Relative apical areas in aPKC-expressing cells normalized by those in neighboring cells were quantified as in Fig. 4 B. Singly locating aPKC-expressing cells surrounded by nonexpressing cells were measured. Arrows show T7-aPKC–expressing cells. (D) GST-FERM-FA phosphorylated by aPKC-ζ was examined for binding to p114RhoGEF by GST pull-down assays. Phosphorylated (ATP+) or unphosphorylated (ATP−) GST-FERM-FA was incubated with lysates of MDCK cells expressing Flag-p114RhoGEF (Lysate) or with eluted Flag-p114RhoGEF (Eluted). Coprecipitated Flag-p114RhoGEF was detected by anti-Flag antibody. See Materials and methods for details. (E) In vitro guanine nucleotide exchange reaction of p114RhoGEF toward RhoA. aPKC-phosphorylated Lulu2 was incubated with p114RhoGEF (red). See Materials and methods for details. (F) Flag-tagged wild-type Lulu2, Lulu2 4A, and Lulu2 4E were examined for binding to HA-tagged p114RhoGEF in coimmunoprecipitation (IP) assays. Coprecipitated HA-p114RhoGEF was detected (HA). (G) DLD-1 cells treated with control siRNA or PKC-λ siRNA-1 were stained for ZO-1, F-actin, or myosin IIA. (H) Parental MDCK cells or MDCK cells overexpressing Myc-Lulu2 treated with control siRNA or PKC-λ siRNA-1 were stained for ZO-1. n = 3 independent experiments, in each of which >50 cells were measured. Error bars indicate SD. *, P < 0.05 by Student’s t test. Bars, 20 µm.
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fig8: Lulu2 is negatively regulated by aPKC. (A) Quantification of apical areas defined by ZO-1 staining signals in EGFP-Lulu2–, EGFP-Lulu2 4A–, or EGFP-Lulu2 4E–expressing cells. Relative apical areas in Lulu2-expressing cells normalized by those in neighboring cells were quantified as in Fig. 4 B. MDCK cells were used. N, N terminus; C, C terminus. (B and C) DLD-1 cells transfected with T7-tagged wild-type aPKC-ζ or dominant-negative aPKC (aPKC DN) were stained for T7 and ZO-1 (B). Parental or Lulu2-expressing MDCK cells were transfected with T7–aPKC DN and stained for T7 and ZO-1 (C). Relative apical areas in aPKC-expressing cells normalized by those in neighboring cells were quantified as in Fig. 4 B. Singly locating aPKC-expressing cells surrounded by nonexpressing cells were measured. Arrows show T7-aPKC–expressing cells. (D) GST-FERM-FA phosphorylated by aPKC-ζ was examined for binding to p114RhoGEF by GST pull-down assays. Phosphorylated (ATP+) or unphosphorylated (ATP−) GST-FERM-FA was incubated with lysates of MDCK cells expressing Flag-p114RhoGEF (Lysate) or with eluted Flag-p114RhoGEF (Eluted). Coprecipitated Flag-p114RhoGEF was detected by anti-Flag antibody. See Materials and methods for details. (E) In vitro guanine nucleotide exchange reaction of p114RhoGEF toward RhoA. aPKC-phosphorylated Lulu2 was incubated with p114RhoGEF (red). See Materials and methods for details. (F) Flag-tagged wild-type Lulu2, Lulu2 4A, and Lulu2 4E were examined for binding to HA-tagged p114RhoGEF in coimmunoprecipitation (IP) assays. Coprecipitated HA-p114RhoGEF was detected (HA). (G) DLD-1 cells treated with control siRNA or PKC-λ siRNA-1 were stained for ZO-1, F-actin, or myosin IIA. (H) Parental MDCK cells or MDCK cells overexpressing Myc-Lulu2 treated with control siRNA or PKC-λ siRNA-1 were stained for ZO-1. n = 3 independent experiments, in each of which >50 cells were measured. Error bars indicate SD. *, P < 0.05 by Student’s t test. Bars, 20 µm.

Mentions: To examine the effect of phosphorylation on Lulu2 activity, we prepared a phosphorylation-mimicking (Lulu2 4E) and a phosphorylation-deficient (Lulu2 4A) form of full-length Lulu2, in which the aforementioned four aPKC phosphorylation sites were replaced by glutamic acids or alanines and tested them for the ability to induce apical constriction (Fig. 8 A). It was found that whereas wild-type Lulu2 and Lulu2 4A induced strong apical constriction in MDCK cells, Lulu2 4E did not (Fig. 8 A), suggesting that the phosphorylation of these sites might negatively regulate Lulu2 activity.


Lulu2 regulates the circumferential actomyosin tensile system in epithelial cells through p114RhoGEF.

Nakajima H, Tanoue T - J. Cell Biol. (2011)

Lulu2 is negatively regulated by aPKC. (A) Quantification of apical areas defined by ZO-1 staining signals in EGFP-Lulu2–, EGFP-Lulu2 4A–, or EGFP-Lulu2 4E–expressing cells. Relative apical areas in Lulu2-expressing cells normalized by those in neighboring cells were quantified as in Fig. 4 B. MDCK cells were used. N, N terminus; C, C terminus. (B and C) DLD-1 cells transfected with T7-tagged wild-type aPKC-ζ or dominant-negative aPKC (aPKC DN) were stained for T7 and ZO-1 (B). Parental or Lulu2-expressing MDCK cells were transfected with T7–aPKC DN and stained for T7 and ZO-1 (C). Relative apical areas in aPKC-expressing cells normalized by those in neighboring cells were quantified as in Fig. 4 B. Singly locating aPKC-expressing cells surrounded by nonexpressing cells were measured. Arrows show T7-aPKC–expressing cells. (D) GST-FERM-FA phosphorylated by aPKC-ζ was examined for binding to p114RhoGEF by GST pull-down assays. Phosphorylated (ATP+) or unphosphorylated (ATP−) GST-FERM-FA was incubated with lysates of MDCK cells expressing Flag-p114RhoGEF (Lysate) or with eluted Flag-p114RhoGEF (Eluted). Coprecipitated Flag-p114RhoGEF was detected by anti-Flag antibody. See Materials and methods for details. (E) In vitro guanine nucleotide exchange reaction of p114RhoGEF toward RhoA. aPKC-phosphorylated Lulu2 was incubated with p114RhoGEF (red). See Materials and methods for details. (F) Flag-tagged wild-type Lulu2, Lulu2 4A, and Lulu2 4E were examined for binding to HA-tagged p114RhoGEF in coimmunoprecipitation (IP) assays. Coprecipitated HA-p114RhoGEF was detected (HA). (G) DLD-1 cells treated with control siRNA or PKC-λ siRNA-1 were stained for ZO-1, F-actin, or myosin IIA. (H) Parental MDCK cells or MDCK cells overexpressing Myc-Lulu2 treated with control siRNA or PKC-λ siRNA-1 were stained for ZO-1. n = 3 independent experiments, in each of which >50 cells were measured. Error bars indicate SD. *, P < 0.05 by Student’s t test. Bars, 20 µm.
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fig8: Lulu2 is negatively regulated by aPKC. (A) Quantification of apical areas defined by ZO-1 staining signals in EGFP-Lulu2–, EGFP-Lulu2 4A–, or EGFP-Lulu2 4E–expressing cells. Relative apical areas in Lulu2-expressing cells normalized by those in neighboring cells were quantified as in Fig. 4 B. MDCK cells were used. N, N terminus; C, C terminus. (B and C) DLD-1 cells transfected with T7-tagged wild-type aPKC-ζ or dominant-negative aPKC (aPKC DN) were stained for T7 and ZO-1 (B). Parental or Lulu2-expressing MDCK cells were transfected with T7–aPKC DN and stained for T7 and ZO-1 (C). Relative apical areas in aPKC-expressing cells normalized by those in neighboring cells were quantified as in Fig. 4 B. Singly locating aPKC-expressing cells surrounded by nonexpressing cells were measured. Arrows show T7-aPKC–expressing cells. (D) GST-FERM-FA phosphorylated by aPKC-ζ was examined for binding to p114RhoGEF by GST pull-down assays. Phosphorylated (ATP+) or unphosphorylated (ATP−) GST-FERM-FA was incubated with lysates of MDCK cells expressing Flag-p114RhoGEF (Lysate) or with eluted Flag-p114RhoGEF (Eluted). Coprecipitated Flag-p114RhoGEF was detected by anti-Flag antibody. See Materials and methods for details. (E) In vitro guanine nucleotide exchange reaction of p114RhoGEF toward RhoA. aPKC-phosphorylated Lulu2 was incubated with p114RhoGEF (red). See Materials and methods for details. (F) Flag-tagged wild-type Lulu2, Lulu2 4A, and Lulu2 4E were examined for binding to HA-tagged p114RhoGEF in coimmunoprecipitation (IP) assays. Coprecipitated HA-p114RhoGEF was detected (HA). (G) DLD-1 cells treated with control siRNA or PKC-λ siRNA-1 were stained for ZO-1, F-actin, or myosin IIA. (H) Parental MDCK cells or MDCK cells overexpressing Myc-Lulu2 treated with control siRNA or PKC-λ siRNA-1 were stained for ZO-1. n = 3 independent experiments, in each of which >50 cells were measured. Error bars indicate SD. *, P < 0.05 by Student’s t test. Bars, 20 µm.
Mentions: To examine the effect of phosphorylation on Lulu2 activity, we prepared a phosphorylation-mimicking (Lulu2 4E) and a phosphorylation-deficient (Lulu2 4A) form of full-length Lulu2, in which the aforementioned four aPKC phosphorylation sites were replaced by glutamic acids or alanines and tested them for the ability to induce apical constriction (Fig. 8 A). It was found that whereas wild-type Lulu2 and Lulu2 4A induced strong apical constriction in MDCK cells, Lulu2 4E did not (Fig. 8 A), suggesting that the phosphorylation of these sites might negatively regulate Lulu2 activity.

Bottom Line: In its regulation of the belt, Lulu2 interacts with and activates p114RhoGEF, a Rho-specific guanine nucleotide exchanging factor (GEF), at apical cell-cell junctions.This interaction is negatively regulated via phosphorylation events in the FERM-adjacent domain of Lulu2 catalyzed by atypical protein kinase C.We further found that Patj, an apical cell polarity regulator, recruits p114RhoGEF to apical cell-cell boundaries via PDZ (PSD-95/Dlg/ZO-1) domain-mediated interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Global Centers of Excellence Program for Integrative Membrane Biology, Graduate School of Medicine, Kobe University, Chuo-ku, Kobe 650-0017, Japan.

ABSTRACT
Myosin II-driven mechanical forces control epithelial cell shape and morphogenesis. In particular, the circumferential actomyosin belt, which is located along apical cell-cell junctions, regulates many cellular processes. Despite its importance, the molecular mechanisms regulating the belt are not fully understood. In this paper, we characterize Lulu2, a FERM (4.1 protein, ezrin, radixin, moesin) domain-containing molecule homologous to Drosophila melanogaster Yurt, as an important regulator. In epithelial cells, Lulu2 is localized along apical cell-cell boundaries, and Lulu2 depletion by ribonucleic acid interference results in disorganization of the circumferential actomyosin belt. In its regulation of the belt, Lulu2 interacts with and activates p114RhoGEF, a Rho-specific guanine nucleotide exchanging factor (GEF), at apical cell-cell junctions. This interaction is negatively regulated via phosphorylation events in the FERM-adjacent domain of Lulu2 catalyzed by atypical protein kinase C. We further found that Patj, an apical cell polarity regulator, recruits p114RhoGEF to apical cell-cell boundaries via PDZ (PSD-95/Dlg/ZO-1) domain-mediated interaction. These findings therefore reveal a novel molecular system regulating the circumferential actomyosin belt in epithelial cells.

Show MeSH
Related in: MedlinePlus