Fig. 7. LPP3 regulates ß-catenin-mediated TCF transcriptional activity. (A) ß-catenin transcriptional activity in wild-type and LPP3 null ES cells, as measured by luciferase levels produced from the transfected reporter construct TOPFlash. ß-catenin-mediated TCF activity is upregulated approx. 10- to 15-fold in the LPP3 null ES cells. (B) Transfection of HEK293 cells (which lack endogenous LPP3 activity) with the NH₂ truncated (stabilized) ß-catenin results in high levels of ß-catenin-mediated transcription. These levels are attenuated by co-transfection of increasing levels of LPP3. As a control, a ß-catenin unresponsive construct (FOPFlash) was used in these experiments. LPP3 activity in the transfected cells was verified by the release of ³²P from labeled LPA. (C) Increasing levels of transfected LPP3 also inhibits endogenous ß-catenin-mediated transcription in the HEK293 cells. (D) Phosphatase-deficient LPP3 also inhibits ß-catenin-mediated TCF transcription. HEK 293 cells transfected with TOPFlash reporter construct and an LPP3 expression cassette carrying the Ser197Thr mutation that inactivates the phosphatase site inhibited TCF/ß-catenin transcription. (E) Western analysis of extracts from the transfected cells in B show that higher concentrations of LPP3 decreased phosphorylation at Ser9 in GSK-3, which correlates with GSK-3 having an increased inhibitory effect on ß-catenin. This coincides with the levels of ß-catenin dephosphorylated at Ser37/Thr41 (the stabilized form) being reduced by increasing LPP3 levels.

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