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Figure 6


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.





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