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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 phosphorylated by aPKC. (A) Amino acid sequence of the FA domain (378–428 aa; mouse Lulu2) is shown. Potential PKA or PKC phosphorylation sites (NetPhosK program) are in blue or red, respectively. N, N terminus; C, C terminus. (B) GST-FERM-FA, GST-FERM domain and GST-FA domain were subjected to in vitro kinase assays with PKC-ζ. Phosphorylation was detected by mobility shift patterns (bars) in Mn2+–Phos-tag SDS-PAGE followed by immunoblotting with anti-GST antibody. PKC-ζ used is shown in Fig. S4 E. See Materials and methods for details. (C, left) Lysates of MDCK cells expressing Myc-Lulu2 were immunoprecipitated (IP) with anti-Myc antibody. (left, top, PKC-λ/ζ) Coprecipitated endogenous PKC-λ/ζ was detected. (right) Lysates of MDCK cells expressing Myc-Lulu2 were immunoprecipitated with rabbit control IgG or anti-Par6 antibody. (top right, Myc) Coprecipitated Myc-Lulu2 was detected. (D) GST-fused mutant forms of the FA domain, in which Ser385, Thr408, Ser414, Ser419, or Thr424 was replaced by alanine, were prepared and examined for phosphorylation by aPKC-ζ in vitro (right, HA–PKC-ζ), or left untreated (left, −). Phosphorylated GST-FA was detected by mobility shift patterns (bar) in Mn2+–Phos-tag SDS-PAGE followed by immunoblotting with anti-GST antibody. (E) MDCK cells expressing Myc-Lulu2 were treated with control siRNA or PKC-λ siRNA-1. Phosphorylated Lulu2 was detected by mobility shift patterns (bar) in Mn2+–Phos-tag SDS-PAGE followed by immunoblotting with anti-Myc antibody.
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fig7: Lulu2 is phosphorylated by aPKC. (A) Amino acid sequence of the FA domain (378–428 aa; mouse Lulu2) is shown. Potential PKA or PKC phosphorylation sites (NetPhosK program) are in blue or red, respectively. N, N terminus; C, C terminus. (B) GST-FERM-FA, GST-FERM domain and GST-FA domain were subjected to in vitro kinase assays with PKC-ζ. Phosphorylation was detected by mobility shift patterns (bars) in Mn2+–Phos-tag SDS-PAGE followed by immunoblotting with anti-GST antibody. PKC-ζ used is shown in Fig. S4 E. See Materials and methods for details. (C, left) Lysates of MDCK cells expressing Myc-Lulu2 were immunoprecipitated (IP) with anti-Myc antibody. (left, top, PKC-λ/ζ) Coprecipitated endogenous PKC-λ/ζ was detected. (right) Lysates of MDCK cells expressing Myc-Lulu2 were immunoprecipitated with rabbit control IgG or anti-Par6 antibody. (top right, Myc) Coprecipitated Myc-Lulu2 was detected. (D) GST-fused mutant forms of the FA domain, in which Ser385, Thr408, Ser414, Ser419, or Thr424 was replaced by alanine, were prepared and examined for phosphorylation by aPKC-ζ in vitro (right, HA–PKC-ζ), or left untreated (left, −). Phosphorylated GST-FA was detected by mobility shift patterns (bar) in Mn2+–Phos-tag SDS-PAGE followed by immunoblotting with anti-GST antibody. (E) MDCK cells expressing Myc-Lulu2 were treated with control siRNA or PKC-λ siRNA-1. Phosphorylated Lulu2 was detected by mobility shift patterns (bar) in Mn2+–Phos-tag SDS-PAGE followed by immunoblotting with anti-Myc antibody.

Mentions: Although we showed that the FERM domain of Lulu2 is sufficient for interaction with p114RhoGEF, the role of the FA domain in the regulation of p114RhoGEF remained obscure and was investigated. We noticed that the FA domain of Lulu2 has several potential PKA and PKC phosphorylation sites (Fig. 7 A) and hypothesized that Lulu2 might be regulated by phosphorylation of these sites. To examine whether PKA and PKC phosphorylate Lulu2, we performed phosphate affinity gel electrophoresis of GST-fused proteins subjected to in vitro kinase assays using the acrylamide-pendant Phos-tag ligand followed by immunoblotting with an antibody for GST. In this way, we can detect a phosphorylated protein by retarding its mobility in electrophoresis (Kinoshita et al., 2006). Using this method, it was found that aPKC (PKC-ζ), conventional PKC (PKC-α), and PKA phosphorylate the FA domain. These kinases could efficiently phosphorylate the GST-fused FA domain and GST-fused FERM-FA domain but not the GST-fused FERM domain in vitro (Fig. 7 B and Fig. S4, B and D–G).


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

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

Lulu2 is phosphorylated by aPKC. (A) Amino acid sequence of the FA domain (378–428 aa; mouse Lulu2) is shown. Potential PKA or PKC phosphorylation sites (NetPhosK program) are in blue or red, respectively. N, N terminus; C, C terminus. (B) GST-FERM-FA, GST-FERM domain and GST-FA domain were subjected to in vitro kinase assays with PKC-ζ. Phosphorylation was detected by mobility shift patterns (bars) in Mn2+–Phos-tag SDS-PAGE followed by immunoblotting with anti-GST antibody. PKC-ζ used is shown in Fig. S4 E. See Materials and methods for details. (C, left) Lysates of MDCK cells expressing Myc-Lulu2 were immunoprecipitated (IP) with anti-Myc antibody. (left, top, PKC-λ/ζ) Coprecipitated endogenous PKC-λ/ζ was detected. (right) Lysates of MDCK cells expressing Myc-Lulu2 were immunoprecipitated with rabbit control IgG or anti-Par6 antibody. (top right, Myc) Coprecipitated Myc-Lulu2 was detected. (D) GST-fused mutant forms of the FA domain, in which Ser385, Thr408, Ser414, Ser419, or Thr424 was replaced by alanine, were prepared and examined for phosphorylation by aPKC-ζ in vitro (right, HA–PKC-ζ), or left untreated (left, −). Phosphorylated GST-FA was detected by mobility shift patterns (bar) in Mn2+–Phos-tag SDS-PAGE followed by immunoblotting with anti-GST antibody. (E) MDCK cells expressing Myc-Lulu2 were treated with control siRNA or PKC-λ siRNA-1. Phosphorylated Lulu2 was detected by mobility shift patterns (bar) in Mn2+–Phos-tag SDS-PAGE followed by immunoblotting with anti-Myc antibody.
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Related In: Results  -  Collection

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fig7: Lulu2 is phosphorylated by aPKC. (A) Amino acid sequence of the FA domain (378–428 aa; mouse Lulu2) is shown. Potential PKA or PKC phosphorylation sites (NetPhosK program) are in blue or red, respectively. N, N terminus; C, C terminus. (B) GST-FERM-FA, GST-FERM domain and GST-FA domain were subjected to in vitro kinase assays with PKC-ζ. Phosphorylation was detected by mobility shift patterns (bars) in Mn2+–Phos-tag SDS-PAGE followed by immunoblotting with anti-GST antibody. PKC-ζ used is shown in Fig. S4 E. See Materials and methods for details. (C, left) Lysates of MDCK cells expressing Myc-Lulu2 were immunoprecipitated (IP) with anti-Myc antibody. (left, top, PKC-λ/ζ) Coprecipitated endogenous PKC-λ/ζ was detected. (right) Lysates of MDCK cells expressing Myc-Lulu2 were immunoprecipitated with rabbit control IgG or anti-Par6 antibody. (top right, Myc) Coprecipitated Myc-Lulu2 was detected. (D) GST-fused mutant forms of the FA domain, in which Ser385, Thr408, Ser414, Ser419, or Thr424 was replaced by alanine, were prepared and examined for phosphorylation by aPKC-ζ in vitro (right, HA–PKC-ζ), or left untreated (left, −). Phosphorylated GST-FA was detected by mobility shift patterns (bar) in Mn2+–Phos-tag SDS-PAGE followed by immunoblotting with anti-GST antibody. (E) MDCK cells expressing Myc-Lulu2 were treated with control siRNA or PKC-λ siRNA-1. Phosphorylated Lulu2 was detected by mobility shift patterns (bar) in Mn2+–Phos-tag SDS-PAGE followed by immunoblotting with anti-Myc antibody.
Mentions: Although we showed that the FERM domain of Lulu2 is sufficient for interaction with p114RhoGEF, the role of the FA domain in the regulation of p114RhoGEF remained obscure and was investigated. We noticed that the FA domain of Lulu2 has several potential PKA and PKC phosphorylation sites (Fig. 7 A) and hypothesized that Lulu2 might be regulated by phosphorylation of these sites. To examine whether PKA and PKC phosphorylate Lulu2, we performed phosphate affinity gel electrophoresis of GST-fused proteins subjected to in vitro kinase assays using the acrylamide-pendant Phos-tag ligand followed by immunoblotting with an antibody for GST. In this way, we can detect a phosphorylated protein by retarding its mobility in electrophoresis (Kinoshita et al., 2006). Using this method, it was found that aPKC (PKC-ζ), conventional PKC (PKC-α), and PKA phosphorylate the FA domain. These kinases could efficiently phosphorylate the GST-fused FA domain and GST-fused FERM-FA domain but not the GST-fused FERM domain in vitro (Fig. 7 B and Fig. S4, B and D–G).

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