FIG. 6.
Calyculin A increases 14-3-3 association and enhances survival of cells expressing wild-type BAD and 112A BAD with intact Ser155. (A to D) Cells expressing wild-type BAD (A), phosphorylation-defective BAD mutant 112A (B), 112A155A BAD (C), or 112A136A155A BAD (D) were treated with 5 nM calyculin A or dimethyl sulfoxide (DMSO) for 3 h and deprived of IL-3. BAD was immunoprecipitated at the indicated times after IL-3 withdrawal and immunoblotted for 14-3-3. Graphs show percent viability as measured by trypan blue exclusion in the absence (−) or presence (+) of calyculin A at 18 h after IL-3 withdrawal. Standard deviations are derived from three experiments. (E) 14-3-3 is absent from BAD/BCL-XL complexes. BCL-XL was immunoprecipitated from FL5.12 cells expressing BCL-XL or BCL-XL and BAD with monoclonal antibody 7B2.5 and immunoblotted for BCL-XL (H-5), BAD (C-20), and 14-3-3 (K19). One-tenth of the supernatant after the immunoprecipitations was loaded as a control. (F) Calyculin A inhibits dephosphorylation of pSer112, pSer136, and pSer155. Lysates of NIH 3T3 cells expressing wild-type BAD were subjected to dephosphorylation in vitro in the absence and presence of calyculin A and immunoblotted for pSer112, pSer136, pSer155, and BAD, as described earlier. (G) FL5.12 cells expressing wild-type BAD were untreated or treated with 5 μM fostriecin for 3 h and deprived of IL-3. Graphs show percent viability as measured by trypan blue exclusion at 12 h after IL-3 withdrawal, normalized to viability in the presence of IL-3. Data are means ± standard deviations of triplicate assays and are representative of two experiments. The viability difference between assays with IL-3 and without IL-3 in the presence and absence of fostriecin has a P value of <0.001 by analysis of variance.