PTPRK binds to STAT3 and directly dephosphorylates phospho-STAT3 at Tyr705. (A) Immunoblot showing GST alone and GST fusion proteins (WT/mutant types) eluted from the indicated beads before incubation with cell lysates. An antibody against GST was used. The arrow indicates the fusion proteins. (B) Phosphatase substrate-trapping mutant assays. Cell lysates of NKYS (top) and KHYG (bottom) cells were incubated with beads bound to the indicated GST-fusion proteins. Proteins bound to the beads were resolved on a 7.5% sodium dodecyl sulfate/polyacrylamide gel electrophoresis gel, and western blots were performed with the indicated antibodies. (C) In vitro phosphatase assay. The immunoprecipitated STAT3/phospho-STAT3^Tyr705 protein was incubated in vitro with purified WT and mutant GST-PTPRK in the absence or presence of a phosphatase inhibitor (Na₃VO₄). The levels of STAT3/phospho-STAT3^Tyr705 proteins were analyzed by western blot using STAT3 and phospho-STAT3^Tyr705 antibodies. The purified GST protein alone without the phosphatase domain of PTPRK was used as the control. The incubation of WT protein but not mutant 2 or the control, in the absence of Na₃VO₄ but not in the presence of Na₃VO₄, resulted in a significant decrease in phospho-STAT3^Tyr705 levels. This result indicated that phospho-STAT3^Tyr705 was a direct substrate of PTPRK.