Binding of phosphatase activity to α4 in cells. (A) Cell lysates of Jurkat were immunoprecipitated by either anti-hα4 Ab, anti-PP2Ac Ab, or normal rabbit serum (NRS). Phosphatase activities in the complex were assayed using 32P-labeled MBP as a substrate. Results are shown as the percentage of input 32P dephosphorylated from MBP. Optimal enzyme and substrate ratio and the incubation time were determined with the rat PP2A purified as described previously.33 Data are the mean of duplicate samples + standard deviations. (B) Jurkat (▪) or Raji (□) cells were treated with various concentrations of rapamycin for 48 hours. The cells were lysed and assayed for their phosphatase activity using 32P-labeled MBP as a substrate. The activity was measured in the presence or absence of 50 nmol/L of OA and the value without OA subtracted from that with OA was calculated. For comparison, the value of lysate without rapamycin was set as 100%. Protein concentrations of samples were adjusted and the amounts of PP2Ac protein were shown in (C). (C) Western blot analysis to demonstrate the amounts of PP2Ac in all samples. Results indicate that proteins are equally adjusted before measuring the phosphatase activities. (D) COS-7 cells were transfected with the mα4 cDNA in pCDM8 or control vector. Cells were lysed after 72 hours of culture and the phosphatase assay was performed as in (B). The effect of rapamycin (10 nmol/L) was measured on mα4-COS transfectant (R+/F−) in comparison to the culture without rapamycin (R−/F−). Recovery of phosphatase activity was measured by the addition of FK506 (100 nmol/L) (R+/F+). (E) Upregulation of phosphatase activity by α4. Effect of GST-α4 (▨) or GST alone (□) was measured in the in vitro phosphatase assay using the phosphorylated MBP or histone H1 with purified PP2A. Regulatory activity on the PP2A was shown as the percentage of control measured only with substrate and enzyme.
