Figure 5. NDRG2 recruits PP2A to PTEN leading to its dephosphorylation at pSer380/pThr382/pThr383.

(a) KK1-NDRG2 cell lysates were incubated with a pSer380/pThr382/pThr383 PTEN phosphopeptide in the presence or absence of different concentrations of OA. The amount of released phosphate was quantitated using the malachite green assay. The mean±s.d. is shown; **P<0.05 compared with untreated control (Student’s t-test). The data are representative of three experiments. (b) KK1-Mock and KK1-NDRG2 cells were treated with increasing concentrations of OA for 2 h and subjected to western blot analysis. Asterisk, nonspecific band. The data are representative of three experiments. (c) After immunoprecipitating lysates from 293T cells transfected with the indicated vectors, the beads were incubated with a pSer380/pThr382/pThr383 phosphopeptide in the presence or absence of 10 nM OA, and phosphate release was determined. The mean±s.d. is shown; **P<0.05 (Student’s t-test). The data are representative of three experiments. (d) After immunoprecipitating the NIH3T3 lysates with an anti-PP2Ac antibody, the beads were incubated with a pSer380/pThr382/pThr383 phosphopeptide in the presence or absence of 10 nM OA, and phosphate release was determined. The mean±s.d. is shown; **P<0.05 (Student’s t-test). The data are representative of three experiments. (e) 0.5 unit of recombinant PP2A was incubated with 200 μg ml⁻¹ of either PTEN peptide or the phosphopeptides containing either pSer380, pThr382, pThr383 or pSer380/pThr382/pThr383, and phosphate release was determined. The mean±s.d. is shown; **P<0.05 (Student’s t-test). The data are representative of three experiments. (f) The beads were incubated with or without recombinant PP2A after immunoprecipitating the KK1-Mock lysates with an anti-PTEN antibody. Western blot analysis of the reaction mixtures was performed to determine the degree of phosphorylation of PTEN. The data are representative of two experiments. (g) The lysates of HUT102-NDRG2 or HUT102-Mock cells treated with the crosslinker DTBP were immunoprecipitated with an anti-FLAG antibody and subsequently probed for co-precipitated PP2Ac by Western blotting. The data are representative of three experiments. (h) The lysates from KK1-NDRG2 or KK1-Mock cells treated with DTBP were immunoprecipitated with an anti-PTEN antibody, and the Western blots were probed with the indicated antibodies. Asterisk, nonspecific band. The data are representative of three experiments.