Figure 4
PTP1B directly dephosphorylates PITX1, which leads to destabilization of PITX1 protein.
(A) PTP1B directly associated with PITX-1. The interactions between PITX-1 and PTP1B were detected in DLD1 endogenous immunoprecipitation (left panel) and Hct-116 overexpression with PTP1B immunoprecipitation (right panel). Representative images of triplicated experiments were shown here. (B) Modulation of PTP1B levels influenced the phosphorylation status of endogenous PITX-1 in DLD1 cells. Ectopic expression of PTP1B induced tyrosine dephosphorylation of PITX-1 (left panel) and silencing of PTP1B upregulated the phospho-PITX-1 level (right panel). Representative images of triplicated experiments were shown here. (C) Specific PTP1B inhibitor treatment increased the phospho-tyrosine status of PITX1 and augmented PITX1/p120RasGAP expression in CRC cells. The phospho-tyrosine status of PITX1 in PITX1-overexpressed DLD1 cells were determined by immunoprecipitation assay after exposing to PTP1B inhibitor treatment at the indicated doses for 4 hours (left panel). The expression of PITX1/p120RasGAP after PTP1B treatment described above were also examined by western blot (right panel). Representative images of triplicated experiments were shown here. (D) PTP1B interacted with PITX-1 at three tyrosine residues, namely Y160, Y175 and Y179. PTP1B could only induced PITX-1 phosphorylation change in wild-type PITX-1-expressing cells. Phospho-(T)-PITX1 in DLD1 expressing wild-type, Y160F-, Y175F-, and Y79F-mutant PITX1 were determined by immuneprecipitation and immunebloting. Representative images of triplicated experiments were shown here. (E) PTP1B-dependent dephosphorylation reduced the stability of PITX-1. DLD1 cells with different PITX-1-phospho-mutants were treated with 50 μg/ml cycloheximide (CHX) for the indicated times and analyzed by western blot. Representative images of triplicated experiments were shown over the left panel. The quantified results of PITX1 expression in each experiment were shown over the right panel (n = 3). (F) Degradation of wild-type and mutant PITX-1 protein were affected by lactacystin treatment. Effects of proteasome inhibitor, Lacacystin, on wild-type or the three mutants PITX-1 were examined by western blot (N = 3).
