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Numb regulates cell-cell adhesion and polarity in response to tyrosine kinase signalling.

Wang Z, Sandiford S, Wu C, Li SS - EMBO J. (2009)

Bottom Line: Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF-induced EMT.Knockdown of Numb by small hairpin RNA caused a basolateral-to-apicolateral translocation of E-cad and beta-catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF-induced cell scattering, a decrease in cell-cell adhesion, and an increase in cell migration.Our work identifies Numb as an important regulator of epithelial polarity and cell-cell adhesion and a sensor of HGF signalling or Src activity during EMT.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and the Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

ABSTRACT
Epithelial-mesenchymal transition (EMT), which can be caused by aberrant tyrosine kinase signalling, marks epithelial tumour progression and metastasis, yet the underlying molecular mechanism is not fully understood. Here, we report that Numb interacts with E-cadherin (E-cad) through its phosphotyrosine-binding domain (PTB) and thereby regulates the localization of E-cad to the lateral domain of epithelial cell-cell junction. Moreover, Numb engages the polarity complex Par3-aPKC-Par6 by binding to Par3 in polarized Madin-Darby canine kidney cells. Intriguingly, after Src activation or hepatocyte growth factor (HGF) treatment, Numb decouples from E-cad and Par3 and associates preferably with aPKC-Par6. Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF-induced EMT. Knockdown of Numb by small hairpin RNA caused a basolateral-to-apicolateral translocation of E-cad and beta-catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF-induced cell scattering, a decrease in cell-cell adhesion, and an increase in cell migration. Our work identifies Numb as an important regulator of epithelial polarity and cell-cell adhesion and a sensor of HGF signalling or Src activity during EMT.

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Related in: MedlinePlus

Numb binds to E-cadherin through its PTB domain and a conserved NVYY motif. (A) Reciprocal Co-IP of Numb and E-cadherin from lysate of MDCKII cells. Interaction of Numb with E-cadherin was disrupted by tyrosine phosphorylation of the latter induced by pervanadate treatment or v-Src expression. Ctrl, untreated MDCKII cells; E-cad, E-cadherin; Per, pervanadate; PP2, an Src kinase inhibitor. (B) The Numb PTB domain precipitated E-cadherin from MDCKII cell lysate in a GST pull-down assay. (C) Multiple sequence alignment revealed a highly conserved region in E-cadherins from vertebrates. The black box marks a predicted Numb PTB domain-binding motif N[V/I][Y/F][Y/F]. (D) Peptide array screening confirmed an NVYY motif in E-cadherin as a specific PTB-binding site. For clarity and space consideration, only a portion of the ‘peptide-walking' array of the E-cadherin cytoplasmic region is shown on the left strip (the remaining array exhibited no appreciable binding to Numb PTB). GST exhibited no binding to the same strip. The left column on the strip to the right was an array of a peptide corresponding to the known Numb PTB-binding site (sequence as shown) in LNX, used as a positive control for binding and a quality control for the peptide array synthesis. The right column represents results from substitution analysis of the YYY triad at the PTB-binding site in E-cadherin. Each Tyr in the triad was replaced by an Ala, a Phe or a pTyr to evaluate its importance in PTB binding. (E) Binding of E-cadherin mutants to Numb. The triad YYY in E-cadherin was replaced, through site-directed mutagenesis, by AAA or FFF to generate the mutants E-cad(3A) or E-cad(3F). Wild-type E-cad and the mutants were expressed as GFP fusions in MDCKII cells and evaluated for ability to bind Numb by western blot. The bar graphs below panels of (A), (B) and (E), respectively, are data from quantitative analysis of the corresponding western blots. The error bars represent mean±s.d.
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f1: Numb binds to E-cadherin through its PTB domain and a conserved NVYY motif. (A) Reciprocal Co-IP of Numb and E-cadherin from lysate of MDCKII cells. Interaction of Numb with E-cadherin was disrupted by tyrosine phosphorylation of the latter induced by pervanadate treatment or v-Src expression. Ctrl, untreated MDCKII cells; E-cad, E-cadherin; Per, pervanadate; PP2, an Src kinase inhibitor. (B) The Numb PTB domain precipitated E-cadherin from MDCKII cell lysate in a GST pull-down assay. (C) Multiple sequence alignment revealed a highly conserved region in E-cadherins from vertebrates. The black box marks a predicted Numb PTB domain-binding motif N[V/I][Y/F][Y/F]. (D) Peptide array screening confirmed an NVYY motif in E-cadherin as a specific PTB-binding site. For clarity and space consideration, only a portion of the ‘peptide-walking' array of the E-cadherin cytoplasmic region is shown on the left strip (the remaining array exhibited no appreciable binding to Numb PTB). GST exhibited no binding to the same strip. The left column on the strip to the right was an array of a peptide corresponding to the known Numb PTB-binding site (sequence as shown) in LNX, used as a positive control for binding and a quality control for the peptide array synthesis. The right column represents results from substitution analysis of the YYY triad at the PTB-binding site in E-cadherin. Each Tyr in the triad was replaced by an Ala, a Phe or a pTyr to evaluate its importance in PTB binding. (E) Binding of E-cadherin mutants to Numb. The triad YYY in E-cadherin was replaced, through site-directed mutagenesis, by AAA or FFF to generate the mutants E-cad(3A) or E-cad(3F). Wild-type E-cad and the mutants were expressed as GFP fusions in MDCKII cells and evaluated for ability to bind Numb by western blot. The bar graphs below panels of (A), (B) and (E), respectively, are data from quantitative analysis of the corresponding western blots. The error bars represent mean±s.d.

Mentions: To understand the function of the Numb–E-cad interaction in epithelial cells, we first established by co-immunoprecipitation (co-IP) that endogenous Numb and E-cad interacted in MDCKII cells (Figure 1A). To determine whether the Numb–E-cad interaction was modulated by tyrosine kinase signalling, cells were either incubated with the phosphatase inhibitor pervanadate or transfected with a v-src-expressing vector, both of which significantly enhanced the tyrosine phosphorylation level of E-cad (Figure 1A). The phosphorylation of E-cad in v-src-transfected cells was not as pronounced as in pervanate-treated cells, likely due to the more specific effect of Src in comparison to pervanadate. Conversely, cells were treated with PP2, an Src-specific kinase inhibitor to suppress phosphorylation. Although the Numb–E-cad interaction was attenuated by pervanadate and completely abolished by v-Src expression, it was strengthened by PP2 treatment (Figure 1A). These data show that the Numb–E-cad interaction is negatively regulated by Src activity.


Numb regulates cell-cell adhesion and polarity in response to tyrosine kinase signalling.

Wang Z, Sandiford S, Wu C, Li SS - EMBO J. (2009)

Numb binds to E-cadherin through its PTB domain and a conserved NVYY motif. (A) Reciprocal Co-IP of Numb and E-cadherin from lysate of MDCKII cells. Interaction of Numb with E-cadherin was disrupted by tyrosine phosphorylation of the latter induced by pervanadate treatment or v-Src expression. Ctrl, untreated MDCKII cells; E-cad, E-cadherin; Per, pervanadate; PP2, an Src kinase inhibitor. (B) The Numb PTB domain precipitated E-cadherin from MDCKII cell lysate in a GST pull-down assay. (C) Multiple sequence alignment revealed a highly conserved region in E-cadherins from vertebrates. The black box marks a predicted Numb PTB domain-binding motif N[V/I][Y/F][Y/F]. (D) Peptide array screening confirmed an NVYY motif in E-cadherin as a specific PTB-binding site. For clarity and space consideration, only a portion of the ‘peptide-walking' array of the E-cadherin cytoplasmic region is shown on the left strip (the remaining array exhibited no appreciable binding to Numb PTB). GST exhibited no binding to the same strip. The left column on the strip to the right was an array of a peptide corresponding to the known Numb PTB-binding site (sequence as shown) in LNX, used as a positive control for binding and a quality control for the peptide array synthesis. The right column represents results from substitution analysis of the YYY triad at the PTB-binding site in E-cadherin. Each Tyr in the triad was replaced by an Ala, a Phe or a pTyr to evaluate its importance in PTB binding. (E) Binding of E-cadherin mutants to Numb. The triad YYY in E-cadherin was replaced, through site-directed mutagenesis, by AAA or FFF to generate the mutants E-cad(3A) or E-cad(3F). Wild-type E-cad and the mutants were expressed as GFP fusions in MDCKII cells and evaluated for ability to bind Numb by western blot. The bar graphs below panels of (A), (B) and (E), respectively, are data from quantitative analysis of the corresponding western blots. The error bars represent mean±s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2712596&req=5

f1: Numb binds to E-cadherin through its PTB domain and a conserved NVYY motif. (A) Reciprocal Co-IP of Numb and E-cadherin from lysate of MDCKII cells. Interaction of Numb with E-cadherin was disrupted by tyrosine phosphorylation of the latter induced by pervanadate treatment or v-Src expression. Ctrl, untreated MDCKII cells; E-cad, E-cadherin; Per, pervanadate; PP2, an Src kinase inhibitor. (B) The Numb PTB domain precipitated E-cadherin from MDCKII cell lysate in a GST pull-down assay. (C) Multiple sequence alignment revealed a highly conserved region in E-cadherins from vertebrates. The black box marks a predicted Numb PTB domain-binding motif N[V/I][Y/F][Y/F]. (D) Peptide array screening confirmed an NVYY motif in E-cadherin as a specific PTB-binding site. For clarity and space consideration, only a portion of the ‘peptide-walking' array of the E-cadherin cytoplasmic region is shown on the left strip (the remaining array exhibited no appreciable binding to Numb PTB). GST exhibited no binding to the same strip. The left column on the strip to the right was an array of a peptide corresponding to the known Numb PTB-binding site (sequence as shown) in LNX, used as a positive control for binding and a quality control for the peptide array synthesis. The right column represents results from substitution analysis of the YYY triad at the PTB-binding site in E-cadherin. Each Tyr in the triad was replaced by an Ala, a Phe or a pTyr to evaluate its importance in PTB binding. (E) Binding of E-cadherin mutants to Numb. The triad YYY in E-cadherin was replaced, through site-directed mutagenesis, by AAA or FFF to generate the mutants E-cad(3A) or E-cad(3F). Wild-type E-cad and the mutants were expressed as GFP fusions in MDCKII cells and evaluated for ability to bind Numb by western blot. The bar graphs below panels of (A), (B) and (E), respectively, are data from quantitative analysis of the corresponding western blots. The error bars represent mean±s.d.
Mentions: To understand the function of the Numb–E-cad interaction in epithelial cells, we first established by co-immunoprecipitation (co-IP) that endogenous Numb and E-cad interacted in MDCKII cells (Figure 1A). To determine whether the Numb–E-cad interaction was modulated by tyrosine kinase signalling, cells were either incubated with the phosphatase inhibitor pervanadate or transfected with a v-src-expressing vector, both of which significantly enhanced the tyrosine phosphorylation level of E-cad (Figure 1A). The phosphorylation of E-cad in v-src-transfected cells was not as pronounced as in pervanate-treated cells, likely due to the more specific effect of Src in comparison to pervanadate. Conversely, cells were treated with PP2, an Src-specific kinase inhibitor to suppress phosphorylation. Although the Numb–E-cad interaction was attenuated by pervanadate and completely abolished by v-Src expression, it was strengthened by PP2 treatment (Figure 1A). These data show that the Numb–E-cad interaction is negatively regulated by Src activity.

Bottom Line: Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF-induced EMT.Knockdown of Numb by small hairpin RNA caused a basolateral-to-apicolateral translocation of E-cad and beta-catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF-induced cell scattering, a decrease in cell-cell adhesion, and an increase in cell migration.Our work identifies Numb as an important regulator of epithelial polarity and cell-cell adhesion and a sensor of HGF signalling or Src activity during EMT.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and the Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

ABSTRACT
Epithelial-mesenchymal transition (EMT), which can be caused by aberrant tyrosine kinase signalling, marks epithelial tumour progression and metastasis, yet the underlying molecular mechanism is not fully understood. Here, we report that Numb interacts with E-cadherin (E-cad) through its phosphotyrosine-binding domain (PTB) and thereby regulates the localization of E-cad to the lateral domain of epithelial cell-cell junction. Moreover, Numb engages the polarity complex Par3-aPKC-Par6 by binding to Par3 in polarized Madin-Darby canine kidney cells. Intriguingly, after Src activation or hepatocyte growth factor (HGF) treatment, Numb decouples from E-cad and Par3 and associates preferably with aPKC-Par6. Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF-induced EMT. Knockdown of Numb by small hairpin RNA caused a basolateral-to-apicolateral translocation of E-cad and beta-catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF-induced cell scattering, a decrease in cell-cell adhesion, and an increase in cell migration. Our work identifies Numb as an important regulator of epithelial polarity and cell-cell adhesion and a sensor of HGF signalling or Src activity during EMT.

Show MeSH
Related in: MedlinePlus