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ASPP2 links the apical lateral polarity complex to the regulation of YAP activity in epithelial cells.

Royer C, Koch S, Qin X, Zak J, Buti L, Dudziec E, Zhong S, Ratnayaka I, Srinivas S, Lu X - PLoS ONE (2014)

Bottom Line: The Hippo pathway, by tightly controlling the phosphorylation state and activity of the transcription cofactors YAP and TAZ is essential during development and tissue homeostasis whereas its deregulation may lead to cancer.ASPP2 thereby directly induces the dephosphorylation and activation of junctional YAP.We propose that this mechanism may commonly control YAP functions in epithelial tissues.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom; Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The Hippo pathway, by tightly controlling the phosphorylation state and activity of the transcription cofactors YAP and TAZ is essential during development and tissue homeostasis whereas its deregulation may lead to cancer. Recent studies have linked the apicobasal polarity machinery in epithelial cells to components of the Hippo pathway and YAP and TAZ themselves. However the molecular mechanism by which the junctional pool of YAP proteins is released and activated in epithelial cells remains unknown. Here we report that the tumour suppressor ASPP2 forms an apical-lateral polarity complex at the level of tight junctions in polarised epithelial cells, acting as a scaffold for protein phosphatase 1 (PP1) and junctional YAP via dedicated binding domains. ASPP2 thereby directly induces the dephosphorylation and activation of junctional YAP. Collectively, this study unearths a novel mechanistic paradigm revealing the critical role of the apical-lateral polarity complex in activating this localised pool of YAP in vitro, in epithelial cells, and in vivo, in the murine colonic epithelium. We propose that this mechanism may commonly control YAP functions in epithelial tissues.

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Related in: MedlinePlus

ASPP2 and YAP form a junctional complex in epithelial cells.(A) Immunostaining of ASPP2, Par3 and YAP in confluent monolayers of Caco-2 cells. White and green arrows point to tight junction localised ASPP2 and YAP respectively. Scale bars: 20 µm. (B) YAP and ASPP2 immunostaining in polarised MDCK cells. Sections SI and SII represent xy optical sections going through the apical-lateral domain and the middle of nuclei respectively. The bottom panel represents the xz section corresponding to the dashed line. SI and SII are shown with black arrowheads. White arrowheads show co-localisation of ASPP2 and YAP at the apical-lateral domain. Nuclei are counterstained with DAPI. Scale bar: 8 µm. (C) The localisation of YAP and ASPP2 was analysed by immunostaining of frozen sections obtained from wild type mice. YAP was apical (white arrows) and nuclear (yellow arrows) in the epithelial cells of colonic crypts. Nuclei are counterstained with DAPI. Scale bar: 20 µm. (D) The interaction between ASPP2 and Par3 was tested in Caco-2 cells. Endogenous ASPP2 was immunoprecipitated with an anti-ASPP2 mouse monoclonal antibody (DX50.13) and an anti-Gal4 mouse monoclonal antibody was used as a negative control. ASPP2, Par-3 and YAP were detected by SDS-Page/immunoblotting. White arrowheads point to different Par-3 isoforms. (E) ASPP2 and YAP co-immunoprecipitation in Caco-2 cells plated at different cell densities. Lysates were obtained from Caco-2 cells plated at various cell densities and endogenous ASPP2 was immunoprecipitated with an anti-ASPP2 mouse monoclonal antibody (DX50.13) and an anti-Gal4 mouse monoclonal antibody was used as a negative control. ASPP2, YAP and YAP phosphorylated at S127 were subsequently detected by SDS-Page/immunoblotting. Long and short exposures are shown for ASPP2. β-tubulin was used as loading control. LD: low density; MD: medium density; HD: high density. (F-G) The phosphorylation status of YAP regulates its subcellular localisation and interaction with ASPP2. Stable Caco-2 cells expressing either hYAP-myc or hYAP-S127A-Flag were used to test the requirement of YAP phosphorylation at serine 127 for its interaction with ASPP2 and its junctional localisation. (F) hYAP-myc, hYAP-S127A-Flag and endogenous ASPP2 were detected by immunostaining. DAPI was used to stain nuclei. Scale bars: 20 µm. (G) hYAP-myc and hYAP-S127A-Flag were immunoprecipitated using an anti-myc monoclonal (9E10) and anti-Flag monoclonal antibody respectively. ASPP2 and YAP were subsequently detected by SDS-Page/immunoblotting. Molecular markers are indicated in the figure (values in kDa).
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pone-0111384-g001: ASPP2 and YAP form a junctional complex in epithelial cells.(A) Immunostaining of ASPP2, Par3 and YAP in confluent monolayers of Caco-2 cells. White and green arrows point to tight junction localised ASPP2 and YAP respectively. Scale bars: 20 µm. (B) YAP and ASPP2 immunostaining in polarised MDCK cells. Sections SI and SII represent xy optical sections going through the apical-lateral domain and the middle of nuclei respectively. The bottom panel represents the xz section corresponding to the dashed line. SI and SII are shown with black arrowheads. White arrowheads show co-localisation of ASPP2 and YAP at the apical-lateral domain. Nuclei are counterstained with DAPI. Scale bar: 8 µm. (C) The localisation of YAP and ASPP2 was analysed by immunostaining of frozen sections obtained from wild type mice. YAP was apical (white arrows) and nuclear (yellow arrows) in the epithelial cells of colonic crypts. Nuclei are counterstained with DAPI. Scale bar: 20 µm. (D) The interaction between ASPP2 and Par3 was tested in Caco-2 cells. Endogenous ASPP2 was immunoprecipitated with an anti-ASPP2 mouse monoclonal antibody (DX50.13) and an anti-Gal4 mouse monoclonal antibody was used as a negative control. ASPP2, Par-3 and YAP were detected by SDS-Page/immunoblotting. White arrowheads point to different Par-3 isoforms. (E) ASPP2 and YAP co-immunoprecipitation in Caco-2 cells plated at different cell densities. Lysates were obtained from Caco-2 cells plated at various cell densities and endogenous ASPP2 was immunoprecipitated with an anti-ASPP2 mouse monoclonal antibody (DX50.13) and an anti-Gal4 mouse monoclonal antibody was used as a negative control. ASPP2, YAP and YAP phosphorylated at S127 were subsequently detected by SDS-Page/immunoblotting. Long and short exposures are shown for ASPP2. β-tubulin was used as loading control. LD: low density; MD: medium density; HD: high density. (F-G) The phosphorylation status of YAP regulates its subcellular localisation and interaction with ASPP2. Stable Caco-2 cells expressing either hYAP-myc or hYAP-S127A-Flag were used to test the requirement of YAP phosphorylation at serine 127 for its interaction with ASPP2 and its junctional localisation. (F) hYAP-myc, hYAP-S127A-Flag and endogenous ASPP2 were detected by immunostaining. DAPI was used to stain nuclei. Scale bars: 20 µm. (G) hYAP-myc and hYAP-S127A-Flag were immunoprecipitated using an anti-myc monoclonal (9E10) and anti-Flag monoclonal antibody respectively. ASPP2 and YAP were subsequently detected by SDS-Page/immunoblotting. Molecular markers are indicated in the figure (values in kDa).

Mentions: To understand how ASPP2 may control both apicobasal polarity and the activity of YAP, we examined their subcellular localisation in Caco-2 cells, a colorectal cancer cell line exhibiting strong epithelial characteristics and retaining the ability to polarise. Similar to previous observations in epithelial cells and tissues, ASPP2 co-localised with Par3 at tight junctions. Interestingly, in addition to its nuclear localisation, YAP was found to co-localise with ASPP2 at tight junctions, suggesting that they may form a complex at this level (Figure 1A). A similar observation could be made in polarised MDCK cells, as YAP co-localised with ASPP2 at the level of the apical-lateral domain where tight junctions reside (Figure 1B and Figure S1A). In addition, in colonic crypt cells, YAP was also expressed apically towards the lumen, in a localisation pattern reminiscent of ASPP2's, suggesting that, ASPP2 and YAP may also interact at tight junctions in vivo (Figure 1C). Importantly, reduced junctional and nuclear YAP signal following YAP knockdown could be observed, demonstrating the specificity of the antibody used. Of note, YAP depletion did not affect the localisation of ASPP2 at tight junctions, suggesting that YAP is not important for its subcellular localisation pattern (Figure S1B).


ASPP2 links the apical lateral polarity complex to the regulation of YAP activity in epithelial cells.

Royer C, Koch S, Qin X, Zak J, Buti L, Dudziec E, Zhong S, Ratnayaka I, Srinivas S, Lu X - PLoS ONE (2014)

ASPP2 and YAP form a junctional complex in epithelial cells.(A) Immunostaining of ASPP2, Par3 and YAP in confluent monolayers of Caco-2 cells. White and green arrows point to tight junction localised ASPP2 and YAP respectively. Scale bars: 20 µm. (B) YAP and ASPP2 immunostaining in polarised MDCK cells. Sections SI and SII represent xy optical sections going through the apical-lateral domain and the middle of nuclei respectively. The bottom panel represents the xz section corresponding to the dashed line. SI and SII are shown with black arrowheads. White arrowheads show co-localisation of ASPP2 and YAP at the apical-lateral domain. Nuclei are counterstained with DAPI. Scale bar: 8 µm. (C) The localisation of YAP and ASPP2 was analysed by immunostaining of frozen sections obtained from wild type mice. YAP was apical (white arrows) and nuclear (yellow arrows) in the epithelial cells of colonic crypts. Nuclei are counterstained with DAPI. Scale bar: 20 µm. (D) The interaction between ASPP2 and Par3 was tested in Caco-2 cells. Endogenous ASPP2 was immunoprecipitated with an anti-ASPP2 mouse monoclonal antibody (DX50.13) and an anti-Gal4 mouse monoclonal antibody was used as a negative control. ASPP2, Par-3 and YAP were detected by SDS-Page/immunoblotting. White arrowheads point to different Par-3 isoforms. (E) ASPP2 and YAP co-immunoprecipitation in Caco-2 cells plated at different cell densities. Lysates were obtained from Caco-2 cells plated at various cell densities and endogenous ASPP2 was immunoprecipitated with an anti-ASPP2 mouse monoclonal antibody (DX50.13) and an anti-Gal4 mouse monoclonal antibody was used as a negative control. ASPP2, YAP and YAP phosphorylated at S127 were subsequently detected by SDS-Page/immunoblotting. Long and short exposures are shown for ASPP2. β-tubulin was used as loading control. LD: low density; MD: medium density; HD: high density. (F-G) The phosphorylation status of YAP regulates its subcellular localisation and interaction with ASPP2. Stable Caco-2 cells expressing either hYAP-myc or hYAP-S127A-Flag were used to test the requirement of YAP phosphorylation at serine 127 for its interaction with ASPP2 and its junctional localisation. (F) hYAP-myc, hYAP-S127A-Flag and endogenous ASPP2 were detected by immunostaining. DAPI was used to stain nuclei. Scale bars: 20 µm. (G) hYAP-myc and hYAP-S127A-Flag were immunoprecipitated using an anti-myc monoclonal (9E10) and anti-Flag monoclonal antibody respectively. ASPP2 and YAP were subsequently detected by SDS-Page/immunoblotting. Molecular markers are indicated in the figure (values in kDa).
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Related In: Results  -  Collection

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pone-0111384-g001: ASPP2 and YAP form a junctional complex in epithelial cells.(A) Immunostaining of ASPP2, Par3 and YAP in confluent monolayers of Caco-2 cells. White and green arrows point to tight junction localised ASPP2 and YAP respectively. Scale bars: 20 µm. (B) YAP and ASPP2 immunostaining in polarised MDCK cells. Sections SI and SII represent xy optical sections going through the apical-lateral domain and the middle of nuclei respectively. The bottom panel represents the xz section corresponding to the dashed line. SI and SII are shown with black arrowheads. White arrowheads show co-localisation of ASPP2 and YAP at the apical-lateral domain. Nuclei are counterstained with DAPI. Scale bar: 8 µm. (C) The localisation of YAP and ASPP2 was analysed by immunostaining of frozen sections obtained from wild type mice. YAP was apical (white arrows) and nuclear (yellow arrows) in the epithelial cells of colonic crypts. Nuclei are counterstained with DAPI. Scale bar: 20 µm. (D) The interaction between ASPP2 and Par3 was tested in Caco-2 cells. Endogenous ASPP2 was immunoprecipitated with an anti-ASPP2 mouse monoclonal antibody (DX50.13) and an anti-Gal4 mouse monoclonal antibody was used as a negative control. ASPP2, Par-3 and YAP were detected by SDS-Page/immunoblotting. White arrowheads point to different Par-3 isoforms. (E) ASPP2 and YAP co-immunoprecipitation in Caco-2 cells plated at different cell densities. Lysates were obtained from Caco-2 cells plated at various cell densities and endogenous ASPP2 was immunoprecipitated with an anti-ASPP2 mouse monoclonal antibody (DX50.13) and an anti-Gal4 mouse monoclonal antibody was used as a negative control. ASPP2, YAP and YAP phosphorylated at S127 were subsequently detected by SDS-Page/immunoblotting. Long and short exposures are shown for ASPP2. β-tubulin was used as loading control. LD: low density; MD: medium density; HD: high density. (F-G) The phosphorylation status of YAP regulates its subcellular localisation and interaction with ASPP2. Stable Caco-2 cells expressing either hYAP-myc or hYAP-S127A-Flag were used to test the requirement of YAP phosphorylation at serine 127 for its interaction with ASPP2 and its junctional localisation. (F) hYAP-myc, hYAP-S127A-Flag and endogenous ASPP2 were detected by immunostaining. DAPI was used to stain nuclei. Scale bars: 20 µm. (G) hYAP-myc and hYAP-S127A-Flag were immunoprecipitated using an anti-myc monoclonal (9E10) and anti-Flag monoclonal antibody respectively. ASPP2 and YAP were subsequently detected by SDS-Page/immunoblotting. Molecular markers are indicated in the figure (values in kDa).
Mentions: To understand how ASPP2 may control both apicobasal polarity and the activity of YAP, we examined their subcellular localisation in Caco-2 cells, a colorectal cancer cell line exhibiting strong epithelial characteristics and retaining the ability to polarise. Similar to previous observations in epithelial cells and tissues, ASPP2 co-localised with Par3 at tight junctions. Interestingly, in addition to its nuclear localisation, YAP was found to co-localise with ASPP2 at tight junctions, suggesting that they may form a complex at this level (Figure 1A). A similar observation could be made in polarised MDCK cells, as YAP co-localised with ASPP2 at the level of the apical-lateral domain where tight junctions reside (Figure 1B and Figure S1A). In addition, in colonic crypt cells, YAP was also expressed apically towards the lumen, in a localisation pattern reminiscent of ASPP2's, suggesting that, ASPP2 and YAP may also interact at tight junctions in vivo (Figure 1C). Importantly, reduced junctional and nuclear YAP signal following YAP knockdown could be observed, demonstrating the specificity of the antibody used. Of note, YAP depletion did not affect the localisation of ASPP2 at tight junctions, suggesting that YAP is not important for its subcellular localisation pattern (Figure S1B).

Bottom Line: The Hippo pathway, by tightly controlling the phosphorylation state and activity of the transcription cofactors YAP and TAZ is essential during development and tissue homeostasis whereas its deregulation may lead to cancer.ASPP2 thereby directly induces the dephosphorylation and activation of junctional YAP.We propose that this mechanism may commonly control YAP functions in epithelial tissues.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom; Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The Hippo pathway, by tightly controlling the phosphorylation state and activity of the transcription cofactors YAP and TAZ is essential during development and tissue homeostasis whereas its deregulation may lead to cancer. Recent studies have linked the apicobasal polarity machinery in epithelial cells to components of the Hippo pathway and YAP and TAZ themselves. However the molecular mechanism by which the junctional pool of YAP proteins is released and activated in epithelial cells remains unknown. Here we report that the tumour suppressor ASPP2 forms an apical-lateral polarity complex at the level of tight junctions in polarised epithelial cells, acting as a scaffold for protein phosphatase 1 (PP1) and junctional YAP via dedicated binding domains. ASPP2 thereby directly induces the dephosphorylation and activation of junctional YAP. Collectively, this study unearths a novel mechanistic paradigm revealing the critical role of the apical-lateral polarity complex in activating this localised pool of YAP in vitro, in epithelial cells, and in vivo, in the murine colonic epithelium. We propose that this mechanism may commonly control YAP functions in epithelial tissues.

Show MeSH
Related in: MedlinePlus