PP-1α and BAD in parental and BCR/ABL-expressing cells.

(A) Association of PP-1α and BAD in parental and BCR/ABL-expressing 32D cells. The association of HA-BAD and PP-1α was assessed in cytoplasm extracts of exponentially growing and IL-3–starved (2 hours) parental and BCR/ABL-expressing cells (left). As control, the interaction of BAD with PP-2α (middle, lane 2) and that of BAX with PP-1α (right, lanes 1 and 2) were also assessed in BAD- and BAX- expressing cells. Levels of HA-BAD (left and middle panels), and PP-1α (right) were measured as immunoprecipitation controls (left panel, lane 2 of middle panel, lanes 1 and 2 of right panel); levels of PP-2α (lane 1, middle) and BAX (lane 3, right) were measured in whole cell lysate as Western blot controls. Arrows indicate PP-1α HA-BAD, and PP-2α proteins. Asterisks indicate immunoglobulin chains. (B) PP-1α–dependent dephosphorylation of [³²P]-labeled GST-BAD. Constitutively active HA-MyrAkt was immunoprecipitated from transiently transfected 293T cells and incubated with 1 μg of GST-BAD and 0.37 MBq (10 μCi) [³²P]-γATP for 30 minutes at 30°C. Phosphorylated GST-BAD was then incubated with glutathione sepharose beads for 1 hour at 4°C. Phosphatase reactions were carried out in the presence of 2 units of recombinant PP-1α for 30 minutes at 30°C, and stopped by adding 2 × SDS-sample buffer. Levels of BAD were measured as control of loading. Two independent experiments are shown. (C) Inhibition of PP-1α activity by anti-BCR/ABL immunoprecipitates. Histograms show the percentage of inhibition (mean + SD) of PP-1α activity by anti-ABL immunoprecipitates from BCR/ABL-expressing cells, compared with parental cells, in the presence or in the absence of 10 μmol/L ATP. PP-1α activity was assessed using a malachite green dye solution to detect phosphates released from the synthetic substrate. Samples were also assessed for PP-1α levels by Western blotting with anti-PP-1α antibody.