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J. Cell Sci. 121 (3): 358-368

Overexpression of EPHA2 receptor destabilizes adherens junctions via a RhoA-dependent mechanism

Wei Bin Fang1, Reneé C. Ireton1, Guanglei Zhuang1, Takamune Takahashi2, Al Reynolds1,3, and Jin Chen1,3,4,5,*

1 Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
2 Department of Medicine, Division of Nephrology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
3 Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
4 Division of Rheumatology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
5 Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA


Figure 1
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Fig. 1. EPHA2 overexpression in MCF-10A cells impairs cell-cell adhesion. (A) A431, A431D and MCF-10A cells were subjected to a hanging-drop aggregation assay, as described in the Materials and Methods. A431D cells, which lack E-cadherin expression, exhibited a defect in cell-cell adhesion. Ephrin-A1 stimulation also induced dissociation of MCF-10A cells. (B) MCF-10A-EPHA2, but not MCF-10A-{Delta}C cells, exhibited decreased cell-cell adhesion. (C) Western blot analysis of EPHA2 and Myc-tagged mutant {Delta}C protein in MCF-10A cells.

 

Figure 2
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Fig. 2. Effect of EPHA2 expression on level and phosphorylation of cadherin or catenin proteins and the composition of adherens junction complexes. (A) Total levels of E-cadherin, p120 catenin, β-catenin and {alpha}-catenin were assayed by western blot analysis. No significant differences in expression of these proteins were detected in the respective cell types in the presence or absence of ephrin-A1 stimulation. (B) Association of the catenin proteins to E-cadherin was assessed by coimmunoprecipitation of p120 catenin, β-catenin or {alpha}-catenin with E-cadherin. The ability of these proteins to associate with E-cadherin was unchanged regardless of ephrin-A1 stimulation. (C-F) E-cadherin is localized at cell-cell junctions in confluent MCF-10A, vector control, MCF-10A-EPHA2 and MCF-10A-{Delta}C cells. (G) Association of catenin proteins with E-cadherin was assayed by coimmunoprecipitation in the presence of normal or low Ca2+ medium. Tyrosine phosphorylation of cadherin or catenin proteins was determined by western blot analysis.

 

Figure 3
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Fig. 3. EPHA2 overexpression destabilizes adherens junctions. MCF-10A cells were cultured in normal (A,C) or low Ca2+ (B) medium for 24 hours. Cells were subsequently fixed and stained for anti-E-cadherin (A,B) or secondary antibody alone (C). MCF-10A cells carrying LZRS control vector (D), LZRS-EPHA2 (E) or LZRS-{Delta}C (F) were subjected to Ca2+ depletion for 8 hours and stained for E-cadherin. EPHA2 overexpression destabilized adherens junctions: E-cadherin was barely detectable in MCF-10A-EPHA2 cells (E).

 

Figure 4
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Fig. 4. EPHA2 regulates adherens junction stability via modulation of RhoA activity. (A) Activated Rho and Rac GTPases in MCF-10A, MCF-10A-EPHA2, or MCF-10A-{Delta}C cells in response to ephrin-A1 stimulation were measured by GST-Rhotekin binding domain and GST-Pak binding domain pull-down assays, respectively. Total levels of Rho and Rac proteins were assay by western blot analysis. *P<0.05. (B-E) MCF-10A, MCF-10A-{Delta}C, MCF-10A-EPHA2 or MCF-10A-EPHA2 treated with ROCK kinase inhibitor, Y27632, were subjected to Ca2+ depletion for 8 hours, followed by detection of E-cadherin. (F-I) MCF-10A and MCF-10A-{Delta}C cells expressing a constitutively active Rho (Q63L) or control β-galactosidase (LacZ) were assayed for adherens junction stability by Ca2+-depletion assay. (J) The expression and activity of Q63L in control and MCF-10A-{Delta}C cells were confirmed by Rhotekin pull-down assays and western blot analysis.

 

Figure 5
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Fig. 5. EPHA2 regulates adherens junction stability through p190 RhoGAP. (A) p190 RhoGAP was immunoprecipitated from MCF-10A, MCF-10A-{Delta}C, or MCF-10A-EPHA2 cell lysates and blotted for tyrosine phosphorylation. Tyrosine phosphorylation of p190 was decreased in MCF-10A-EPHA2 cells. (B-I) MCF-10A, MCF-10A-EPHA2 and MCF-10A-{Delta}C cells expressing p190 or the dominant negative p190 mutant 30-1 were assayed for adherens junction stability by Ca2+-depletion assay. Expression of wild-type p190 rescued the adherens junctions in MCF-10A-EPHA2 cells (I), whereas expression of the 30-1 mutant destabilized cell-cell adhesion in parental MCF-10A cells and MCF-10A-{Delta}C cells (D,G).

 

Figure 6
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Fig. 6. LMW-PTP interacts with EPHA2 and regulates adherens junction stability. (A) LMW-PTP was immunoprecipitated from MCF-10A, MCF-10A-EPHA2 or MCF-10A-{Delta}C cells and incubated with the phosphatase substrate DiFMUP following a time course (as described in the Materials and Methods). LMW-PTP phosphatase activity was elevated in MCF-10A-EPHA2 cells, compared with the levels in MCF-10A-{Delta}C cells. (B) Average fold increase of LMW-PTP phosphatase activity in different cell types after incubation with substrate for 2.5 hours. (C) The association of LMW-PTP and EPHA2 was assessed by GST-LMW-PTP pull-down assay. The levels of EPHA2 that bound to GST-LMW-PTP were significantly higher in cells overexpressing EPHA2 than those expressing {Delta}C or vector control. (D) MCF-10A-EPHA2 cells were transduced with control retrovirus pBABE (vector) or pBABE-LMW-PTP-C12S (C12S), a dominant negative mutant of LMW-PTP. Tyrosine phosphorylation of p190 RhoGAP was decreased in cells expressing EPHA2 and EPHA2 vector cells. Expression of C12S restored phosphorylated tyrosine levels in MCF-10A-EPHA2 cells, compared with parental or cells expressing {Delta}C. (E,F) MCF-10A-EPHA2 cells were infected with retrovirus carrying either vector or the C12S mutant. Stability of the adherens junctions in these cells was determined by Ca2+ depletion. C12S rescued cell-cell contact in MCF-10A-EPHA2 cells.

 

Figure 7
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Fig. 7. EPHA2 recruits Src kinase to regulate adherens junction stability. Association of EPHA2 with Src kinase was measured by an MBP-EPHA2 pull-down assay (A) and coimmunoprecipitation and western blot analysis (B). (C,D) Inhibition of Src activity restores adherens junction stability in MCF-10A-EPHA2 cells. (E) Co-immunoprecipitation of EPHA2 and Src from COS7 cells transfected with Src and wild-type or mutant EPHA2. (F,G) Overexpression of EPHA2-Y812F and EPHA2-Y816F mutants in MCF-10A cells fails to destabilize adherens junction, even after 8 hours of Ca2+ depletion. (I-L) Overexpression of wild-type LMW-PTP is sufficient to destabilize adherens junctions in MCF-10A-Y812F and MCF-10A-Y816F cells.

 

Figure 8
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Fig. 8. Level of EPHA2 overexpression determines rate of adherens junction disassembly. (A) Expression and tyrosine phosphorylation of EPHA2 receptor in various MCF-10A cells and two breast cancer cell lines, BT-549 and MDA-MB-231, were determined by immunoprecipitation and western blot analysis. (B) MCF-10A, MCF-10A-LZRS-EPHA2, MCF-10A-LacZ, or MCF-10A-Ad/EPHA2 cells were immunostained for E-cadherin localization in the presence of normal or low Ca2+ medium for 2 hours.

 

Figure 9
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Fig. 9. A model showing how EPHA2 overexpression promotes destabilization of adherens junctions. Overexpression of EPHA2 increases the recruitment of LMW-PTP and Src kinase. Increased LMW-PTP phosphatase activity dephosphorylates p190 RhoGAP and inhibits p190 RhoGAP activity. Decreased p190 RhoGAP activity in turn upregulates activated Rho-GTP levels. Rho signaling through ROCK destabilizes the adherens junction.

 


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