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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 binds to the C-terminal portion of p114RhoGEF. (A) Amino acid numbers of human p114RhoGEF are indicated. EGFP-tagged full-length (FL) or various truncated forms of p114RhoGEF were expressed in MDCK cells and examined for binding to GST or GST-FERM by GST pull-down assays. C4ΔPBM: C4 without last six amino acid residues. See Materials and methods for the details of the truncated forms. DH, Dbl homology domain; PH, pleckstrin homology domain; CC, coiled-coil region. (B) Myc-tagged full-length Lulu2 was expressed in MDCK cells and examined for binding to GST-tagged truncated forms of p114RhoGEF by GST pull-down assays. GST fusion proteins used are shown in Fig. S4 B. (C) GST-fused C4ΔPBM or GST was mixed and incubated with MBP-fused FERM or MBP in vitro and precipitated with amylose resin (MBP pull-down). Precipitated MBP or GST proteins (pull-down) were detected using anti-MBP or -GST antibodies. Proteins used are shown in Fig. S4 B. See Materials and methods for details. (D) The Lulu2 binding region (red) in p114RhoGEF is poorly conserved among four Dbl family GEFs. Numbers indicate similarity between p114RhoGEF and the others. N, N terminus; C, C terminus.
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fig3: Lulu2 binds to the C-terminal portion of p114RhoGEF. (A) Amino acid numbers of human p114RhoGEF are indicated. EGFP-tagged full-length (FL) or various truncated forms of p114RhoGEF were expressed in MDCK cells and examined for binding to GST or GST-FERM by GST pull-down assays. C4ΔPBM: C4 without last six amino acid residues. See Materials and methods for the details of the truncated forms. DH, Dbl homology domain; PH, pleckstrin homology domain; CC, coiled-coil region. (B) Myc-tagged full-length Lulu2 was expressed in MDCK cells and examined for binding to GST-tagged truncated forms of p114RhoGEF by GST pull-down assays. GST fusion proteins used are shown in Fig. S4 B. (C) GST-fused C4ΔPBM or GST was mixed and incubated with MBP-fused FERM or MBP in vitro and precipitated with amylose resin (MBP pull-down). Precipitated MBP or GST proteins (pull-down) were detected using anti-MBP or -GST antibodies. Proteins used are shown in Fig. S4 B. See Materials and methods for details. (D) The Lulu2 binding region (red) in p114RhoGEF is poorly conserved among four Dbl family GEFs. Numbers indicate similarity between p114RhoGEF and the others. N, N terminus; C, C terminus.

Mentions: We next determined the Lulu2 binding region in p114RhoGEF. p114RhoGEF has Dbl homology and pleckstrin homology (PH) domains, which are necessary for its catalytic activity, followed by a coiled-coil region and a potential PDZ (PSD-95/Dlg/ZO-1) domain–binding motif (PBM) in its C-terminal tail (Fig. 3 A). From sequence similarity, p114RhoGEF and three other Dbl RhoGEFs, p190 RhoGEF, AKAP13/Lbc, and GEF-H1, form a subfamily (Fig. 3 D; Schmidt and Hall, 2002; García-Mata and Burridge, 2007). To narrow down the region of p114RhoGEF interacting with Lulu2, we tested various truncated mutants of p114RhoGEF for binding to Lulu2 by GST pull-down assays using GST-FERM and identified that the region C terminal to the coiled-coil region without the PBM (C4ΔPBM) was necessary and sufficient for interacting with Lulu2 (Fig. 3 A). We confirmed this result by conducting additional pull-down assays using GST-fused truncated mutants of p114RhoGEF. Myc-tagged full-length Lulu2 expressed in the cells was efficiently pulled down by GST-C2, -C4, or -C4ΔPBM but not by GST, GST-N, or -C1 of p114RhoGEF (Fig. 3 B and Fig. S4 B). Furthermore, p114RhoGEF C4ΔPBM proteins bound to Lulu2 FERM proteins in vitro (Fig. 3 C and Fig. S4, B and C). These results together indicate that Lulu2 might bind to the C4ΔPBM of p114RhoGEF.


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

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

Lulu2 binds to the C-terminal portion of p114RhoGEF. (A) Amino acid numbers of human p114RhoGEF are indicated. EGFP-tagged full-length (FL) or various truncated forms of p114RhoGEF were expressed in MDCK cells and examined for binding to GST or GST-FERM by GST pull-down assays. C4ΔPBM: C4 without last six amino acid residues. See Materials and methods for the details of the truncated forms. DH, Dbl homology domain; PH, pleckstrin homology domain; CC, coiled-coil region. (B) Myc-tagged full-length Lulu2 was expressed in MDCK cells and examined for binding to GST-tagged truncated forms of p114RhoGEF by GST pull-down assays. GST fusion proteins used are shown in Fig. S4 B. (C) GST-fused C4ΔPBM or GST was mixed and incubated with MBP-fused FERM or MBP in vitro and precipitated with amylose resin (MBP pull-down). Precipitated MBP or GST proteins (pull-down) were detected using anti-MBP or -GST antibodies. Proteins used are shown in Fig. S4 B. See Materials and methods for details. (D) The Lulu2 binding region (red) in p114RhoGEF is poorly conserved among four Dbl family GEFs. Numbers indicate similarity between p114RhoGEF and the others. N, N terminus; C, C terminus.
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fig3: Lulu2 binds to the C-terminal portion of p114RhoGEF. (A) Amino acid numbers of human p114RhoGEF are indicated. EGFP-tagged full-length (FL) or various truncated forms of p114RhoGEF were expressed in MDCK cells and examined for binding to GST or GST-FERM by GST pull-down assays. C4ΔPBM: C4 without last six amino acid residues. See Materials and methods for the details of the truncated forms. DH, Dbl homology domain; PH, pleckstrin homology domain; CC, coiled-coil region. (B) Myc-tagged full-length Lulu2 was expressed in MDCK cells and examined for binding to GST-tagged truncated forms of p114RhoGEF by GST pull-down assays. GST fusion proteins used are shown in Fig. S4 B. (C) GST-fused C4ΔPBM or GST was mixed and incubated with MBP-fused FERM or MBP in vitro and precipitated with amylose resin (MBP pull-down). Precipitated MBP or GST proteins (pull-down) were detected using anti-MBP or -GST antibodies. Proteins used are shown in Fig. S4 B. See Materials and methods for details. (D) The Lulu2 binding region (red) in p114RhoGEF is poorly conserved among four Dbl family GEFs. Numbers indicate similarity between p114RhoGEF and the others. N, N terminus; C, C terminus.
Mentions: We next determined the Lulu2 binding region in p114RhoGEF. p114RhoGEF has Dbl homology and pleckstrin homology (PH) domains, which are necessary for its catalytic activity, followed by a coiled-coil region and a potential PDZ (PSD-95/Dlg/ZO-1) domain–binding motif (PBM) in its C-terminal tail (Fig. 3 A). From sequence similarity, p114RhoGEF and three other Dbl RhoGEFs, p190 RhoGEF, AKAP13/Lbc, and GEF-H1, form a subfamily (Fig. 3 D; Schmidt and Hall, 2002; García-Mata and Burridge, 2007). To narrow down the region of p114RhoGEF interacting with Lulu2, we tested various truncated mutants of p114RhoGEF for binding to Lulu2 by GST pull-down assays using GST-FERM and identified that the region C terminal to the coiled-coil region without the PBM (C4ΔPBM) was necessary and sufficient for interacting with Lulu2 (Fig. 3 A). We confirmed this result by conducting additional pull-down assays using GST-fused truncated mutants of p114RhoGEF. Myc-tagged full-length Lulu2 expressed in the cells was efficiently pulled down by GST-C2, -C4, or -C4ΔPBM but not by GST, GST-N, or -C1 of p114RhoGEF (Fig. 3 B and Fig. S4 B). Furthermore, p114RhoGEF C4ΔPBM proteins bound to Lulu2 FERM proteins in vitro (Fig. 3 C and Fig. S4, B and C). These results together indicate that Lulu2 might bind to the C4ΔPBM of p114RhoGEF.

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