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The N-cadherin cytoplasmic domain confers anchorage-independent growth and the loss of contact inhibition.

Ozawa M - Sci Rep (2015)

Bottom Line: Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated.An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth.Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

ABSTRACT
Tumor growth is characterized by anchorage independence and the loss of contact inhibition. Previously, we showed that either a red fluorescent protein (DsRed)-tagged N-cadherin or E-cadherin cytoplasmic domain (DNCT or DECT) could function as a dominant negative inhibitor by blocking the cell surface localization of endogenous E-cadherin and inducing cell dissociation. Here, we show that expression of DNCT abrogated contact inhibition of proliferation and conferred anchorage-independent growth. DNCT expression induced the relocation of the tumor suppressor Merlin from the cell surface to intracellular compartments. Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated. An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth. Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

No MeSH data available.


Related in: MedlinePlus

DNCT expression changes the intracellular localization of Merlin.(a) Cells were stained with anti–E-cadherin (E-cad), anti–β-catenin (β-cat), anti–α-catenin (α-cat), and anti-Merlin antibodies, and with phalloidin to detect actin. Although DNCT expression led to the intracellular localization of E-cadherin, β-catenin, and Merlin, repression of DNCT expression by Dox addition (+Dox) resulted in the establishment of cadherin-based junctions, including cortical actin filaments and the membrane localization of Merlin. (b) Immunoblot detection of Merlin and phospho-Merlin revealed that DNCT expression does not change the amounts of these proteins. Vinculin was used as a loading control. (c) Subcellular distribution of E-cadherin, DNCT, β-catenin, and Merlin in DNCT+ cells cultured for 2 d in the presence (+) or absence (−) of Dox. DNCT+ cells were cultured for 2 days in the presence (+) or absence (−) of Dox. The cells were homogenized and centrifuged to obtain cytosolic (C) and particulate (M) fractions. Each fraction was subjected to immunoblot analysis with the indicated antibodies. (d) Immunofluorescence staining with an anti-Merlin antibody revealed the co-localization of Merlin with DNCT. By contrast, Merlin did not co-localize with DsRed or with DNCTSA. (e) Merlin co-immunoprecipitated with DNCT but not with DsRed or DNCTSA. Immunoprecipitates obtained using an anti-FLAG antibody on lysates from DsRed+, DNCT+, or DNCTSA+ cells were blotted with the indicated antibodies. An asterisk indicates the position of the immunoglobulin heavy chain. (f) Merlin did not co-immunoprecipitate with ELA or ELAαC exprssed in DNCT+ cells. HA-tagged ELA or ELAαC were collected using an anti-HA antibody from lysates of DNCT+ELA, or DNCT+ELAαC cells and co-orecipitated materials were blotted with the indicated antibodies. Bars, 25 μm. (g) Expression of ELAαC restores intracellular localization of Merlin. Cells were stained with anti-Merlin antibodies. Although expression of ELA did not change the cytoplasmic localization of Merlin, ELAαC expression in DNCT+ cells resulted in membrane localization of Merlin.
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f4: DNCT expression changes the intracellular localization of Merlin.(a) Cells were stained with anti–E-cadherin (E-cad), anti–β-catenin (β-cat), anti–α-catenin (α-cat), and anti-Merlin antibodies, and with phalloidin to detect actin. Although DNCT expression led to the intracellular localization of E-cadherin, β-catenin, and Merlin, repression of DNCT expression by Dox addition (+Dox) resulted in the establishment of cadherin-based junctions, including cortical actin filaments and the membrane localization of Merlin. (b) Immunoblot detection of Merlin and phospho-Merlin revealed that DNCT expression does not change the amounts of these proteins. Vinculin was used as a loading control. (c) Subcellular distribution of E-cadherin, DNCT, β-catenin, and Merlin in DNCT+ cells cultured for 2 d in the presence (+) or absence (−) of Dox. DNCT+ cells were cultured for 2 days in the presence (+) or absence (−) of Dox. The cells were homogenized and centrifuged to obtain cytosolic (C) and particulate (M) fractions. Each fraction was subjected to immunoblot analysis with the indicated antibodies. (d) Immunofluorescence staining with an anti-Merlin antibody revealed the co-localization of Merlin with DNCT. By contrast, Merlin did not co-localize with DsRed or with DNCTSA. (e) Merlin co-immunoprecipitated with DNCT but not with DsRed or DNCTSA. Immunoprecipitates obtained using an anti-FLAG antibody on lysates from DsRed+, DNCT+, or DNCTSA+ cells were blotted with the indicated antibodies. An asterisk indicates the position of the immunoglobulin heavy chain. (f) Merlin did not co-immunoprecipitate with ELA or ELAαC exprssed in DNCT+ cells. HA-tagged ELA or ELAαC were collected using an anti-HA antibody from lysates of DNCT+ELA, or DNCT+ELAαC cells and co-orecipitated materials were blotted with the indicated antibodies. Bars, 25 μm. (g) Expression of ELAαC restores intracellular localization of Merlin. Cells were stained with anti-Merlin antibodies. Although expression of ELA did not change the cytoplasmic localization of Merlin, ELAαC expression in DNCT+ cells resulted in membrane localization of Merlin.

Mentions: Merlin plays an important role in contact inhibition of proliferation3031. Upon cell–cell contact, Merlin interacts with the cadherin–catenin complex32 and attenuates downstream signaling from EGFR30. Merlin is also known to be an upstream regulator of the Hippo signaling pathway and functions through the YAP/TAZ33. Importantly, association with the membrane is important for Merlin to function as a growth regulator3435. Therefore, we assessed the localization of Merlin in DNCT+ cells with or without of Dox (Fig. 4a). In the presence of Dox, when the cell–cell junctions were established, Merlin was mainly detected at the membrane. By contrast, Merlin was predominantly observed in the cytoplasm in the absence of Dox. Therefore, DNCT expression changed the intracellular localization of Merlin. DNCT did not affect the amounts of phospho-Merlin and Merlin (Fig. 4b); therefore it was not Merlin phosphorylation at S518 that determined its localization. We next examined the membrane versus cytosolic distribution of Merlin, E-cadherin, and β-catenin in DNCT+ cells (Fig. 4c). In DNCT+ cells cultured in the presence of Dox, E-cadherin, β-catenin, and Merlin were primarily found in the membrane (particulate) fraction (Fig. 4c, right panels). When the cells were cultured in the absence of Dox, DNCT was present in both the cytosolic and membrane fractions (Fig. 4c, left panels). Although DNCT expression did not change the distribution of E-cadherin and Merlin, a significant portion (~50%) of β-catenin was present in the cytosolic fraction. Because the membrane fraction also contains cytoskeletons components, including actin and vinculin, it is possible that DNCT may be recovered in this fraction because of its interaction with β-catenin and α-catenin.


The N-cadherin cytoplasmic domain confers anchorage-independent growth and the loss of contact inhibition.

Ozawa M - Sci Rep (2015)

DNCT expression changes the intracellular localization of Merlin.(a) Cells were stained with anti–E-cadherin (E-cad), anti–β-catenin (β-cat), anti–α-catenin (α-cat), and anti-Merlin antibodies, and with phalloidin to detect actin. Although DNCT expression led to the intracellular localization of E-cadherin, β-catenin, and Merlin, repression of DNCT expression by Dox addition (+Dox) resulted in the establishment of cadherin-based junctions, including cortical actin filaments and the membrane localization of Merlin. (b) Immunoblot detection of Merlin and phospho-Merlin revealed that DNCT expression does not change the amounts of these proteins. Vinculin was used as a loading control. (c) Subcellular distribution of E-cadherin, DNCT, β-catenin, and Merlin in DNCT+ cells cultured for 2 d in the presence (+) or absence (−) of Dox. DNCT+ cells were cultured for 2 days in the presence (+) or absence (−) of Dox. The cells were homogenized and centrifuged to obtain cytosolic (C) and particulate (M) fractions. Each fraction was subjected to immunoblot analysis with the indicated antibodies. (d) Immunofluorescence staining with an anti-Merlin antibody revealed the co-localization of Merlin with DNCT. By contrast, Merlin did not co-localize with DsRed or with DNCTSA. (e) Merlin co-immunoprecipitated with DNCT but not with DsRed or DNCTSA. Immunoprecipitates obtained using an anti-FLAG antibody on lysates from DsRed+, DNCT+, or DNCTSA+ cells were blotted with the indicated antibodies. An asterisk indicates the position of the immunoglobulin heavy chain. (f) Merlin did not co-immunoprecipitate with ELA or ELAαC exprssed in DNCT+ cells. HA-tagged ELA or ELAαC were collected using an anti-HA antibody from lysates of DNCT+ELA, or DNCT+ELAαC cells and co-orecipitated materials were blotted with the indicated antibodies. Bars, 25 μm. (g) Expression of ELAαC restores intracellular localization of Merlin. Cells were stained with anti-Merlin antibodies. Although expression of ELA did not change the cytoplasmic localization of Merlin, ELAαC expression in DNCT+ cells resulted in membrane localization of Merlin.
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f4: DNCT expression changes the intracellular localization of Merlin.(a) Cells were stained with anti–E-cadherin (E-cad), anti–β-catenin (β-cat), anti–α-catenin (α-cat), and anti-Merlin antibodies, and with phalloidin to detect actin. Although DNCT expression led to the intracellular localization of E-cadherin, β-catenin, and Merlin, repression of DNCT expression by Dox addition (+Dox) resulted in the establishment of cadherin-based junctions, including cortical actin filaments and the membrane localization of Merlin. (b) Immunoblot detection of Merlin and phospho-Merlin revealed that DNCT expression does not change the amounts of these proteins. Vinculin was used as a loading control. (c) Subcellular distribution of E-cadherin, DNCT, β-catenin, and Merlin in DNCT+ cells cultured for 2 d in the presence (+) or absence (−) of Dox. DNCT+ cells were cultured for 2 days in the presence (+) or absence (−) of Dox. The cells were homogenized and centrifuged to obtain cytosolic (C) and particulate (M) fractions. Each fraction was subjected to immunoblot analysis with the indicated antibodies. (d) Immunofluorescence staining with an anti-Merlin antibody revealed the co-localization of Merlin with DNCT. By contrast, Merlin did not co-localize with DsRed or with DNCTSA. (e) Merlin co-immunoprecipitated with DNCT but not with DsRed or DNCTSA. Immunoprecipitates obtained using an anti-FLAG antibody on lysates from DsRed+, DNCT+, or DNCTSA+ cells were blotted with the indicated antibodies. An asterisk indicates the position of the immunoglobulin heavy chain. (f) Merlin did not co-immunoprecipitate with ELA or ELAαC exprssed in DNCT+ cells. HA-tagged ELA or ELAαC were collected using an anti-HA antibody from lysates of DNCT+ELA, or DNCT+ELAαC cells and co-orecipitated materials were blotted with the indicated antibodies. Bars, 25 μm. (g) Expression of ELAαC restores intracellular localization of Merlin. Cells were stained with anti-Merlin antibodies. Although expression of ELA did not change the cytoplasmic localization of Merlin, ELAαC expression in DNCT+ cells resulted in membrane localization of Merlin.
Mentions: Merlin plays an important role in contact inhibition of proliferation3031. Upon cell–cell contact, Merlin interacts with the cadherin–catenin complex32 and attenuates downstream signaling from EGFR30. Merlin is also known to be an upstream regulator of the Hippo signaling pathway and functions through the YAP/TAZ33. Importantly, association with the membrane is important for Merlin to function as a growth regulator3435. Therefore, we assessed the localization of Merlin in DNCT+ cells with or without of Dox (Fig. 4a). In the presence of Dox, when the cell–cell junctions were established, Merlin was mainly detected at the membrane. By contrast, Merlin was predominantly observed in the cytoplasm in the absence of Dox. Therefore, DNCT expression changed the intracellular localization of Merlin. DNCT did not affect the amounts of phospho-Merlin and Merlin (Fig. 4b); therefore it was not Merlin phosphorylation at S518 that determined its localization. We next examined the membrane versus cytosolic distribution of Merlin, E-cadherin, and β-catenin in DNCT+ cells (Fig. 4c). In DNCT+ cells cultured in the presence of Dox, E-cadherin, β-catenin, and Merlin were primarily found in the membrane (particulate) fraction (Fig. 4c, right panels). When the cells were cultured in the absence of Dox, DNCT was present in both the cytosolic and membrane fractions (Fig. 4c, left panels). Although DNCT expression did not change the distribution of E-cadherin and Merlin, a significant portion (~50%) of β-catenin was present in the cytosolic fraction. Because the membrane fraction also contains cytoskeletons components, including actin and vinculin, it is possible that DNCT may be recovered in this fraction because of its interaction with β-catenin and α-catenin.

Bottom Line: Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated.An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth.Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

ABSTRACT
Tumor growth is characterized by anchorage independence and the loss of contact inhibition. Previously, we showed that either a red fluorescent protein (DsRed)-tagged N-cadherin or E-cadherin cytoplasmic domain (DNCT or DECT) could function as a dominant negative inhibitor by blocking the cell surface localization of endogenous E-cadherin and inducing cell dissociation. Here, we show that expression of DNCT abrogated contact inhibition of proliferation and conferred anchorage-independent growth. DNCT expression induced the relocation of the tumor suppressor Merlin from the cell surface to intracellular compartments. Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated. An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth. Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

No MeSH data available.


Related in: MedlinePlus