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The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness.

Kotelevets L, van Hengel J, Bruyneel E, Mareel M, van Roy F, Chastre E - J. Cell Biol. (2001)

Bottom Line: In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin.PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins.Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) U410, Faculté de Médecine Bichat, 75018 Paris, France.

ABSTRACT
To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell-cell aggregation, and suppressed the invasive phenotype in an E-cadherin-dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.

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Effect of functional transfer of wild-type or mutant PTEN expression vectors on the morphology of MDCKts-src. (A) Analysis of PTEN expression. The ectopic expression of wild-type and PTEN mutants deficient in either the lipid phosphatase activity (PTENΔ237–239) or both the protein and lipid phosphatase activities (PTEN Δ55–70) was assessed by Western blot using antibodies directed against PTEN, or against the Xpress or HA epitopes. Cell lines are indicated as parental (p) or as transfected clone numbers. (B) Analysis of Akt activity. Akt phosphorylation, which is a measurement of Akt activity, was analyzed with anti–phospho-Akt(Ser 473) antibody in cell lysates from parental MDCKts-src cells (p) and their derivatives transfected with either wild-type (clone 18) or mutant PTEN. Growth was either at the nonpermissive temperature for Src activity (40°C) or after transfer to 35°C for 1 or 18 h as indicated. The total amount of Akt was assessed using anti-Akt antibody. (C) Morphology of parental MDCKts-src cells and their derivatives transfected with either wild-type or mutant PTEN. MDCKts-src cells exhibited an epithelial morphology at 40°C and a fibroblast-like morphology when grown for 14 h at the permissive temperature for Src activity (35°C). Neither lipid phosphatase–deficient (PTENΔ237–239) nor the phosphatase-inactive (PTEN Δ55–70) PTEN mutants preserved the epithelial morphology at the permissive temperature as the wild-type PTEN did. Bar, 20 μm.
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fig1: Effect of functional transfer of wild-type or mutant PTEN expression vectors on the morphology of MDCKts-src. (A) Analysis of PTEN expression. The ectopic expression of wild-type and PTEN mutants deficient in either the lipid phosphatase activity (PTENΔ237–239) or both the protein and lipid phosphatase activities (PTEN Δ55–70) was assessed by Western blot using antibodies directed against PTEN, or against the Xpress or HA epitopes. Cell lines are indicated as parental (p) or as transfected clone numbers. (B) Analysis of Akt activity. Akt phosphorylation, which is a measurement of Akt activity, was analyzed with anti–phospho-Akt(Ser 473) antibody in cell lysates from parental MDCKts-src cells (p) and their derivatives transfected with either wild-type (clone 18) or mutant PTEN. Growth was either at the nonpermissive temperature for Src activity (40°C) or after transfer to 35°C for 1 or 18 h as indicated. The total amount of Akt was assessed using anti-Akt antibody. (C) Morphology of parental MDCKts-src cells and their derivatives transfected with either wild-type or mutant PTEN. MDCKts-src cells exhibited an epithelial morphology at 40°C and a fibroblast-like morphology when grown for 14 h at the permissive temperature for Src activity (35°C). Neither lipid phosphatase–deficient (PTENΔ237–239) nor the phosphatase-inactive (PTEN Δ55–70) PTEN mutants preserved the epithelial morphology at the permissive temperature as the wild-type PTEN did. Bar, 20 μm.

Mentions: To investigate the cross-talk between the PTEN and the Src and Ras oncogenic pathways, we expressed wild-type PTEN and lipid phosphatase– or phosphatase-deficient PTEN mutants in MDCK cells, transformed by either a temperature-sensitive mutant of v-Src (MDCKts-src cells) (Behrens et al., 1989, 1993; Takeda et al., 1995) or v-Ras (MDCKras-f) (Vleminckx et al., 1991). It was previously demonstrated that PTEN mutation Δ237–239 results in a 10-fold decrease in phosphatase activity toward phosphatidylinositol (1,3,4,5)P4 without affecting protein phosphatase activity, whereas PTEN mutations Δ55–70 and C124A affect both the lipid and protein phosphatase activities of PTEN (Furnari et al., 1998; Tamura et al., 1998). PTEN expression in transformants was assessed by immunoblotting using anti-tag antibodies (anti-Xpress or anti-HA) as well as anti-PTEN antibody (Fig. 1 A). In MDCKts-src cells, PTEN overexpression affected neither the phosphorylation of focal adhesion kinase nor the MAPK and PI3-kinase activities (unpublished data). In contrast, the ectopic expression of wild-type PTEN, but not PTEN mutants, reduced the phosphorylation of Akt, a downstream target of PI3-kinase, as shown in Fig. 1 B and in line with previous reports for other cell lines (Myers et al., 1998).


The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness.

Kotelevets L, van Hengel J, Bruyneel E, Mareel M, van Roy F, Chastre E - J. Cell Biol. (2001)

Effect of functional transfer of wild-type or mutant PTEN expression vectors on the morphology of MDCKts-src. (A) Analysis of PTEN expression. The ectopic expression of wild-type and PTEN mutants deficient in either the lipid phosphatase activity (PTENΔ237–239) or both the protein and lipid phosphatase activities (PTEN Δ55–70) was assessed by Western blot using antibodies directed against PTEN, or against the Xpress or HA epitopes. Cell lines are indicated as parental (p) or as transfected clone numbers. (B) Analysis of Akt activity. Akt phosphorylation, which is a measurement of Akt activity, was analyzed with anti–phospho-Akt(Ser 473) antibody in cell lysates from parental MDCKts-src cells (p) and their derivatives transfected with either wild-type (clone 18) or mutant PTEN. Growth was either at the nonpermissive temperature for Src activity (40°C) or after transfer to 35°C for 1 or 18 h as indicated. The total amount of Akt was assessed using anti-Akt antibody. (C) Morphology of parental MDCKts-src cells and their derivatives transfected with either wild-type or mutant PTEN. MDCKts-src cells exhibited an epithelial morphology at 40°C and a fibroblast-like morphology when grown for 14 h at the permissive temperature for Src activity (35°C). Neither lipid phosphatase–deficient (PTENΔ237–239) nor the phosphatase-inactive (PTEN Δ55–70) PTEN mutants preserved the epithelial morphology at the permissive temperature as the wild-type PTEN did. Bar, 20 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199329&req=5

fig1: Effect of functional transfer of wild-type or mutant PTEN expression vectors on the morphology of MDCKts-src. (A) Analysis of PTEN expression. The ectopic expression of wild-type and PTEN mutants deficient in either the lipid phosphatase activity (PTENΔ237–239) or both the protein and lipid phosphatase activities (PTEN Δ55–70) was assessed by Western blot using antibodies directed against PTEN, or against the Xpress or HA epitopes. Cell lines are indicated as parental (p) or as transfected clone numbers. (B) Analysis of Akt activity. Akt phosphorylation, which is a measurement of Akt activity, was analyzed with anti–phospho-Akt(Ser 473) antibody in cell lysates from parental MDCKts-src cells (p) and their derivatives transfected with either wild-type (clone 18) or mutant PTEN. Growth was either at the nonpermissive temperature for Src activity (40°C) or after transfer to 35°C for 1 or 18 h as indicated. The total amount of Akt was assessed using anti-Akt antibody. (C) Morphology of parental MDCKts-src cells and their derivatives transfected with either wild-type or mutant PTEN. MDCKts-src cells exhibited an epithelial morphology at 40°C and a fibroblast-like morphology when grown for 14 h at the permissive temperature for Src activity (35°C). Neither lipid phosphatase–deficient (PTENΔ237–239) nor the phosphatase-inactive (PTEN Δ55–70) PTEN mutants preserved the epithelial morphology at the permissive temperature as the wild-type PTEN did. Bar, 20 μm.
Mentions: To investigate the cross-talk between the PTEN and the Src and Ras oncogenic pathways, we expressed wild-type PTEN and lipid phosphatase– or phosphatase-deficient PTEN mutants in MDCK cells, transformed by either a temperature-sensitive mutant of v-Src (MDCKts-src cells) (Behrens et al., 1989, 1993; Takeda et al., 1995) or v-Ras (MDCKras-f) (Vleminckx et al., 1991). It was previously demonstrated that PTEN mutation Δ237–239 results in a 10-fold decrease in phosphatase activity toward phosphatidylinositol (1,3,4,5)P4 without affecting protein phosphatase activity, whereas PTEN mutations Δ55–70 and C124A affect both the lipid and protein phosphatase activities of PTEN (Furnari et al., 1998; Tamura et al., 1998). PTEN expression in transformants was assessed by immunoblotting using anti-tag antibodies (anti-Xpress or anti-HA) as well as anti-PTEN antibody (Fig. 1 A). In MDCKts-src cells, PTEN overexpression affected neither the phosphorylation of focal adhesion kinase nor the MAPK and PI3-kinase activities (unpublished data). In contrast, the ectopic expression of wild-type PTEN, but not PTEN mutants, reduced the phosphorylation of Akt, a downstream target of PI3-kinase, as shown in Fig. 1 B and in line with previous reports for other cell lines (Myers et al., 1998).

Bottom Line: In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin.PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins.Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) U410, Faculté de Médecine Bichat, 75018 Paris, France.

ABSTRACT
To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell-cell aggregation, and suppressed the invasive phenotype in an E-cadherin-dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.

Show MeSH
Related in: MedlinePlus