Limits...
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.

Show MeSH

Related in: MedlinePlus

Src or HGF signalling regulates Numb binding to E-cadherin and the Par complex. (A) Co-IP of Numb with E-cadherin, aPKC, Par6 or Par3 in ts-src-MDCKI cells in the absence (40°C) or presence of Src (35°C), or in MDCKII cells without or with HGF treatment. (B) Tyrosine phosphorylation of E-cadherin, aPKC, Par6 or Par3 was examined with a specific anti-phospho-tyrosine antibody (4G10) under conditions specified in (A). E-cadhrin, aPKC, Par6 and Par3 (100 kDa, 150 kD and 180 kDa isoforms) were phosphorylated when Src was activated. In contrast, under HGF treatment, only Par6 and the 150 kD Par3 isoform were significantly phosphorylated while E-cadherin was weakly phosphorylated. (C) Co-IP of aPKC with Par6, Par3 and Numb, respectively, under conditions specified in (A). Both Src activation and HGF treatment promoted aPKC binding to Numb, but attenuated its interaction with Par3. (D) Co-IP of E-cadherin with p120-catenin and β-catenin, respectively, under conditions specified in (A). Tyrosine phosphorylation of E-cadherin enhanced its binding to p120-, but not to β-catenin. The corresponding quantification analysis of (A), (B), (C) and (D) are shown below each western blot panel. The error bars represent mean±s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2712596&req=5

f2: Src or HGF signalling regulates Numb binding to E-cadherin and the Par complex. (A) Co-IP of Numb with E-cadherin, aPKC, Par6 or Par3 in ts-src-MDCKI cells in the absence (40°C) or presence of Src (35°C), or in MDCKII cells without or with HGF treatment. (B) Tyrosine phosphorylation of E-cadherin, aPKC, Par6 or Par3 was examined with a specific anti-phospho-tyrosine antibody (4G10) under conditions specified in (A). E-cadhrin, aPKC, Par6 and Par3 (100 kDa, 150 kD and 180 kDa isoforms) were phosphorylated when Src was activated. In contrast, under HGF treatment, only Par6 and the 150 kD Par3 isoform were significantly phosphorylated while E-cadherin was weakly phosphorylated. (C) Co-IP of aPKC with Par6, Par3 and Numb, respectively, under conditions specified in (A). Both Src activation and HGF treatment promoted aPKC binding to Numb, but attenuated its interaction with Par3. (D) Co-IP of E-cadherin with p120-catenin and β-catenin, respectively, under conditions specified in (A). Tyrosine phosphorylation of E-cadherin enhanced its binding to p120-, but not to β-catenin. The corresponding quantification analysis of (A), (B), (C) and (D) are shown below each western blot panel. The error bars represent mean±s.d.

Mentions: As E-cad can be phosphorylated at the triad tyrosines during an EMT induced by the activation of Src or a receptor tyrosine kinase (Fujita et al, 2002), it is likely that a dynamic Numb–E-cad interaction has a function in this process. To explore this notion, we examined, by reciprocal co-IP and western blotting, the complex of Numb and E-cad in MDCKI cells transformed by a temperature-sensitive allele of src (Behrens et al, 1993). The ts-src-MDCKI cells formed normal cell–cell junctions when cultured as a monolayer at the non-permissive temperature of 40°C but acquired characteristics of mesenchymal cells at the permissive temperature of 35°C when Src was expressed (Supplementary Figure S1A). Parallel experiments were carried out in MDCKII cells treated with HGF to mimic EMT (Balkovetz et al, 1997). Although both MDCKI and MDCKII are epithelial cells derived from renal tubules, the MDCKII strain exhibits less clonal variation than MDCKI (Richardson et al, 1981; Matter and Balda, 2003). As the MDCKII strain is used more widely in the literature, we felt it necessary to repeat the experiments done in ts-src-MDCKI cells in this strain. As shown in Figure 2A, Numb bound strongly to E-cad in ts-src-MDCKI cells cultured at 40°C in the absence of Src, but the interaction was abolished at 35°C when Src was expressed. Similarly, Numb was decoupled from E-cad after HGF treatment of MDCKII cells (Figure 2A). As E-cad is tyrosine phosphorylated on Src expression or HGF stimulation (Figure 2B), it is likely that phosphorylation of one or more Tyr residue at the YYY triad in E-cad abrogated its binding to Numb.


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

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

Src or HGF signalling regulates Numb binding to E-cadherin and the Par complex. (A) Co-IP of Numb with E-cadherin, aPKC, Par6 or Par3 in ts-src-MDCKI cells in the absence (40°C) or presence of Src (35°C), or in MDCKII cells without or with HGF treatment. (B) Tyrosine phosphorylation of E-cadherin, aPKC, Par6 or Par3 was examined with a specific anti-phospho-tyrosine antibody (4G10) under conditions specified in (A). E-cadhrin, aPKC, Par6 and Par3 (100 kDa, 150 kD and 180 kDa isoforms) were phosphorylated when Src was activated. In contrast, under HGF treatment, only Par6 and the 150 kD Par3 isoform were significantly phosphorylated while E-cadherin was weakly phosphorylated. (C) Co-IP of aPKC with Par6, Par3 and Numb, respectively, under conditions specified in (A). Both Src activation and HGF treatment promoted aPKC binding to Numb, but attenuated its interaction with Par3. (D) Co-IP of E-cadherin with p120-catenin and β-catenin, respectively, under conditions specified in (A). Tyrosine phosphorylation of E-cadherin enhanced its binding to p120-, but not to β-catenin. The corresponding quantification analysis of (A), (B), (C) and (D) are shown below each western blot panel. The error bars represent mean±s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Src or HGF signalling regulates Numb binding to E-cadherin and the Par complex. (A) Co-IP of Numb with E-cadherin, aPKC, Par6 or Par3 in ts-src-MDCKI cells in the absence (40°C) or presence of Src (35°C), or in MDCKII cells without or with HGF treatment. (B) Tyrosine phosphorylation of E-cadherin, aPKC, Par6 or Par3 was examined with a specific anti-phospho-tyrosine antibody (4G10) under conditions specified in (A). E-cadhrin, aPKC, Par6 and Par3 (100 kDa, 150 kD and 180 kDa isoforms) were phosphorylated when Src was activated. In contrast, under HGF treatment, only Par6 and the 150 kD Par3 isoform were significantly phosphorylated while E-cadherin was weakly phosphorylated. (C) Co-IP of aPKC with Par6, Par3 and Numb, respectively, under conditions specified in (A). Both Src activation and HGF treatment promoted aPKC binding to Numb, but attenuated its interaction with Par3. (D) Co-IP of E-cadherin with p120-catenin and β-catenin, respectively, under conditions specified in (A). Tyrosine phosphorylation of E-cadherin enhanced its binding to p120-, but not to β-catenin. The corresponding quantification analysis of (A), (B), (C) and (D) are shown below each western blot panel. The error bars represent mean±s.d.
Mentions: As E-cad can be phosphorylated at the triad tyrosines during an EMT induced by the activation of Src or a receptor tyrosine kinase (Fujita et al, 2002), it is likely that a dynamic Numb–E-cad interaction has a function in this process. To explore this notion, we examined, by reciprocal co-IP and western blotting, the complex of Numb and E-cad in MDCKI cells transformed by a temperature-sensitive allele of src (Behrens et al, 1993). The ts-src-MDCKI cells formed normal cell–cell junctions when cultured as a monolayer at the non-permissive temperature of 40°C but acquired characteristics of mesenchymal cells at the permissive temperature of 35°C when Src was expressed (Supplementary Figure S1A). Parallel experiments were carried out in MDCKII cells treated with HGF to mimic EMT (Balkovetz et al, 1997). Although both MDCKI and MDCKII are epithelial cells derived from renal tubules, the MDCKII strain exhibits less clonal variation than MDCKI (Richardson et al, 1981; Matter and Balda, 2003). As the MDCKII strain is used more widely in the literature, we felt it necessary to repeat the experiments done in ts-src-MDCKI cells in this strain. As shown in Figure 2A, Numb bound strongly to E-cad in ts-src-MDCKI cells cultured at 40°C in the absence of Src, but the interaction was abolished at 35°C when Src was expressed. Similarly, Numb was decoupled from E-cad after HGF treatment of MDCKII cells (Figure 2A). As E-cad is tyrosine phosphorylated on Src expression or HGF stimulation (Figure 2B), it is likely that phosphorylation of one or more Tyr residue at the YYY triad in E-cad abrogated its binding to Numb.

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