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This Article Full Text (PDF) All Versions of this Article: 19/1/115 04-1942fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kotelevets, L. Articles by Chastre, E. Search for Related Content PubMed PubMed Citation Articles by Kotelevets, L. Articles by Chastre, E.

Implication of the MAGI-1b/PTEN signalosome in stabilization of adherens junctions and suppression of invasiveness

Larissa Kotelevets^*, Jolanda van Hengel, Erik Bruyneel, Marc Mareel, Frans van Roy and Eric Chastre^*,1

^* INSERM U410, Faculté de Médecine Bichat, Paris, France;
Department for Molecular Biomedical Research, VIB-Ghent University, Ghent; and
Laboratory of Experimental Cancerology, Ghent University Hospital, Ghent, Belgium

¹Correspondence: INSERM U410, Faculté de Médecine X. Bichat, 16 rue Huchard, Paris 74018, France. E-mail: chastre{at}bichat.inserm.fr

SPECIFIC AIMS

We reported the critical role of lipid phosphatase activity of the PTEN tumor suppressor in stabilizing the E-cadherin junctional complexes and reverting invasiveness. Here we investigate the molecular partners and signaling pathways of E-cadherin junctional complexes and PTEN relative to the invasive phenotype to identify the molecular mechanisms involved.

PRINCIPAL FINDINGS

1. Interaction of PTEN and PI3K with E-cadherin/catenin complexes
We conducted coimmunoprecipitation studies with the kidney epithelial HEK 293 and MDCK cells transiently transfected with PTEN expression vectors. PTEN was identified in E-cadherin, ß-catenin (ß-ctn), and p120ctn immunocomplexes. ß-Catenin and p120ctn were identified in PTEN immunoprecipitates. The p85 regulatory subunit of PI3K coimmunoprecipitated with E-cadherin and catenins.

2. Implication of the MAGI-1b scaffolding molecule in the recruitment of PTEN to junctional complexes
To investigate whether PTEN binds directly to E-cadherin or catenins, we performed a yeast two-hybrid (Y2H) assay; no interaction of PTEN with E-cadherin, ß-ctn, E-catenin, or p120ctn was observed (Fig. 1 A), suggesting the involvement of adaptor proteins. We addressed this by analyzing the possible interaction with MAGI-1b, a scaffold molecule involved in the formation of specialized cell-cell junctions. The Y2H assay demonstrated that PTEN interacts indirectly with ß-ctn through MAGI-1b. We showed that PTEN binds selectively the 2nd PDZ domain of MAGI-1b whereas ß-ctn binds the 5th PDZ domain (Fig. 1A ). The association of MAGI-1b with PTEN and ß-ctn was corroborated by coimmunoprecipitation in kidney epithelial HEK 293 and MDCK cell lines and in MDCKts-src cells, MDCK derivatives transformed by a temperature-sensitive v-src (Fig. 1B-D ). The interaction of PTEN with E-cadherin junctional complexes involved the PDZ binding motifs of PTEN and ß-ctn, as evidenced by the inability of C-terminally truncated PTENStop399 to interact with MAGI-1b (Fig. 1B ) and by disruption of the PTEN/ß-ctn-E-cadherin complex after intracellular transfer of a peptide corresponding to the C terminus of ß-ctn and competing with ß-ctn for binding to MAGI-1b.

View larger version (88K): [in this window] [in a new window]   Figure 1. Characterization of PTEN molecular partners. A) Y2H assay. The yeast strain AH109 was transformed with pairs of pGBKT7-derived bait and pGADT7-derived prey vectors, and plated on yeast media containing X--Gal but lacking tryptophan, leucine, histidine, and adenine. After 7 days of incubation at 30°C, 2-hybrid interactions were scored. Interaction of E-catenin with the N terminus of ß-ctn as a positive control. B) Coimmunoprecipitation of PTEN and Flag-MAGI-1b in MDCK cells transiently transfected with GFP-PTEN wild-type or GFP-PTEN truncated upstream of the PDZ binding domain (Stop399). Antibodies used in immunoprecipitation (IP) and Western blot (WB) are indicated. C) Coimmunoprecipitation of PTEN, Flag-tagged wild-type MAGI-1b, or GFP-tagged MAGI-1b domains PDZ245 and PDZ2345 with components of the cadherin/catenin junctions in transiently transfected HEK293 cells. D) Characterization of MDCKts-src clones stably transfected with constructs encoding 2nd or 5th PDZ domain PDZ245, PDZ2345, or wild-type MAGI-1b. The corresponding cell lysates were used in IP experiments to evaluate interaction of MAGI-1b with ß-ctn or E-cadherin. Numbers above lanes refer to transfectant clone number; -, parental MDCKts-src. E) Confocal microscopy of subcellular localization of MAGI-1b-derived proteins to adherens junctions after stable transfection in MDCKts-src cells. Transformants grown at 40°C is shown. Results show that the PDZ domains 245 of MAGI-1b fused to GFP are targeted to E-cadherin-positive cell-cell contacts. Bar, 30 µm.

Colocalization of MAGI-1b with E-cadherin was further confirmed immunohistochemically (Fig. 1E ). In the human colonic adenocarcinoma HT-29 and Caco-2 cell lines, which express higher amounts of PTEN than MDCK cells, we noted the interaction of endogenous PTEN with MAGI-1b and identified MAGI-1b in E-cadherin junctional complexes. In Caco-2 cells, we confirmed by confocal microscopy that MAGI-1b and PTEN were colocalized with ß-ctn/E-cadherin adhesion complexes at cell-cell contacts. These studies demonstrate that MAGI-1b localizes to the plasma membrane and assembles signaling complexes involving E-cadherin, ß-ctn, and PTEN under physiological conditions.

3. Invasion suppressor activity of MAGI-1b/PTEN complexes
Invasion assays in type-I collagen gels using the MDCKts-src derivatives confirmed that overexpression of wild-type MAGI-1b or MAGI-1b domains PDZ245 results in near complete reversion of Src-induced invasiveness, implicating this adaptor molecule in the control of cell invasiveness (Fig. 2 A). Individual 2nd and 5th PDZ domains of MAGI-1b did not cause reversion. The suppressive effects of MAGI-1b on the Src-induced invasiveness required its interaction with ß-ctn, since it was reverted by the C-terminal ß-ctn competing peptide but not by a ß-ctn control peptide (Fig. 2A ). In PTEN-deficient human prostate PC-3 carcinoma cells, ectopic expression of MAGI-1b did not suppress invasiveness. The invasive phenotype of PC-3 cells depends on the PI3K pathways, as it is blocked by the specific PI3K antagonist LY294002 and by restoration of wild-type PTEN expression. These results suggest that recruitment of PTEN to junctional complexes through its interaction with ß-ctn via MAGI-1b plays a critical role in the control of PI3K effector systems and the invasive phenotype.

View larger version (32K): [in this window] [in a new window]   Figure 2. Effects of MAGI-1b expression on invasiveness and aggregation of MDCKts-src cells. A) In vitro invasion assay. MDCKts-src cells and their derivatives stably transfected with constructs expressing wild-type MAGI-1b (wt, clones 7 and 9), MAGI-1b-PDZ245 (clones 1, 3), or individual 2nd (clones 7, 9) or 5th (clones 1, 7) PDZ domains of MAGI-1b were seeded on type-I collagen at the temperature permissive for Src activity (35°C). MDCKts-srcMAGI-1bwt7 and MDCKts-srcMAGI-1bwt9 cells were incubated for 3 h before the invasion assay in the absence or presence of the ß-ctn C-term (residues 772-781) or the control ß-ctn (residues 769-778) peptides combined with the protein transduction domain of Drosophila antennapedia. The number and the depth of cells inside the gel were measured after 24 h. MDCKts-srcPTENwt11 cells stably transfected with wild-type PTEN were used as a control for reversion of invasiveness. B) MDCKts-src cells and their derivatives expressing the individual 2nd or 5th MAGI-1b PDZ domain, MAGI-1b-PDZ245, MAGI-1b-PDZ2345, or wild-type MAGI-1b were subjected to fast aggregation assay for 30 min (t30) at a temperature permissive for Src activity (35°C). Absence of cell aggregates was confirmed at t0 for all clones. Treatment with the E-cadherin neutralizing antibody DECMA-1 abolished aggregation in all cell types at restrictive and permissive temperatures. Aggregation of MDCKts-src cells at 40°C served as positive control.

Because MAGI-1b interfered with cell invasiveness, we investigated by a fast aggregation assay whether MAGI-1b affects the E-cadherin-dependent adhesion. Parental MDCKts-src cells are characterized by an inability to aggregate at the temperature permissive for Src activity (35°C), though they form aggregates at 40°C in an E-cadherin-dependent manner as demonstrated by inhibition with the E-cadherin neutralizing antibody DECMA-1 (Fig. 2B ). In contrast, cells transfected with wild-type MAGI-1b, PDZ245 or PDZ2345 domains, but not with the individual 2nd or 5th PDZ domains, formed aggregates at the temperature permissive for Src activity.

4. Requirement of AKT kinase activity in Src-induced invasiveness
We show that ectopic expression of MAGI-1b in MDCKts-src derivatives decreases the activity of AKT, a downstream effector of PI3K and demonstrate that stable transfection of constitutively active (myristilated), but not wild-type AKT, induces invasiveness of MDCKts-src at a temperature restricting Src activity. These effects were not associated with notable changes in the accumulation of E-cadherin or ß-ctn. Conversely, dominant negative AKT reverted Src-induced invasiveness. Thus, AKT activity is required and sufficient to trigger invasiveness of MDCKts-src cells. To address a putative effect of MAGI-1b on AKT activity independent of PTEN, we performed complementary experiments in the human PTEN-defective PC-3 cells. Ectopic expression of MAGI-1b in PC-3 cell derivatives did not affect AKT activity. AKT activity was decreased by transient transfection of wild-type PTEN, and this effect was potentiated by cotransfection of MAGI-1b, indicating that PTEN is required for the MAGI-1b-induced down-regulation of AKT activity.

Taken together, our data indicate that Src-induced migration requires AKT activity, whereas the destabilization of junctional complexes involved both AKT-dependent and AKT-independent pathways.

CONCLUSIONS AND SIGNIFICANCE

Cadherin junctional complexes are critical to the control of many cellular functions, including cell survival, proliferation, and differentiation. Disruption of these junctional complexes hallmarks neoplastic progression and is associated with invasiveness and metastasis.

Our results demonstrate that E-cadherin and catenins are components of large signaling complexes involving PI3K and the MAGI-1b/PTEN signalosome. It is becoming clearer that scaffold molecules play a critical role in organizing signaling complexes that control cell growth, differentiation, and function. An emerging group of proteins with PDZ domains (e.g., MAGI-1b) triggers the assembly and subcellular localization of multiprotein complexes, generally at the membrane-cytoplasm interface. Our study extends this to cell adhesion molecules and demonstrates that MAGI-1b plays a critical role in maintaining cell-cell contacts and reversing invasiveness. According to this signalosome model (Fig. 3 ), we propose that PTEN is recruited to specific subcellular microenvironments (e.g., adherens junctions) via binding of its PDZ binding motif to scaffolding molecules such as MAGI-1b, which in turn bind to the cadherin/catenin complex via ß-ctn. As ß-ctn also binds the PI3K enzyme, adherens junctions function as focal points to regulate PIP₃ pools and control recruitment and activation of downstream effector systems. Our results demonstrate that Src-induced invasiveness involves both AKT-dependent and -independent pathways. A future challenge is to elucidate the spatiotemporal regulation of these macromolecular complexes.

View larger version (74K): [in this window] [in a new window]   Figure 3. Model for stabilization of junctional complexes containing E-cadherin by the MAGI-1b/PTEN signalosome. PTEN is targeted to the plasma membrane via its C2 domain; its PDZ binding motif anchors it at adherens junctions via interaction of MAGI-1b with ß-ctn. Activated Src recruits PI3K to the plasma membrane, where its regulatory p85 subunit interacts with ß-ctn. Antagonistic activities of PTEN and PI3K allow regulation of second messenger PIP3 and recruitment of proteins containing a PH domain, such as AKT and GTP exchange factors (GEFs) for the Rho subfamily of GTPases. AKT activity is necessary and sufficient for Src-induced cell migration; destabilization of junctional complexes by Src might involve other pathways, such as activation of small GTPases. Cytoplasmic p120ctn is an efficient substrate for Src kinase activity, modulates the activation state of small GTPases of the Rho family, and stimulates cell migration. PM, plasma membrane.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1942fje;

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This Article Full Text (PDF) All Versions of this Article: 19/1/115 04-1942fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kotelevets, L. Articles by Chastre, E. Search for Related Content PubMed PubMed Citation Articles by Kotelevets, L. Articles by Chastre, E.