PA 824 was shown to inhibit biosynthesis of proteins and lipids in a

Phosphorylation has been reported to promote Bad translocation from mitochondria into cytosol, interaction with the scaffold protein 14 3 3 and dissociation Celecoxib from Bcl XL. To test whether rapamycin stimulated Bad phosphorylation affects its subcellular localization and interactions with 14 3 3 or Bcl XL, human lung cancer H460 cells were treated with rapamycin for 45 min, and subcellular distributions of total Bad, pBad, 14 3 3 and Bcl XL were examined by subcellular fractionation analysis as previously described. After treatment with rapamycin, Bad was translocated from mitochondria into the cytosol. Since rapamycin increases the phosphorylated forms of Bad in the cytosol and only the phosphorylated Bad could be observed in the cytosolic fraction, this indicates that rapamycin mediated sequestration of Bad from mitochondria may occur through its phosphorylation. By contrast, rapamycin has no significant effect on the subcellular Eumycetoma localization of 14 3 3 or Bcl XL. To determine the purity of the subcellular fractions obtained, fraction specific proteins were assessed by probing the same filters. Prohibitin, an exclusively mitochondrial protein, was detected only in the mitochondrial fraction whereas proliferating cell nuclear antigen, a nuclear marker, was detected exclusively in the nuclear fraction, and GAPDH, the cytosol marker, was only observed in the cytosolic fraction. This determination further confirmed the purity of these subcellular fractions without artifactual cross contamination. In addition to accumulation of Bad in the cytosol, rapamycin also enhances Bad/14 3 3 interaction in association with decreased Bad/Bcl XL binding. These findings indicate that rapamycin induced Bad phosphorylation BAY 11-7082 in sequestering Bad from the mitochondria and functionally blocking its proapoptotic function. Decreased Bad/Bcl XL binding induced by rapamycin can render Bad less able to suppress the antiapoptotic function of Bcl XL. Rapamycin promotes Bad ubiquitination and degradation Phosphorylation has been demonstrated to regulate ubiquitination and degradation of the Bcl2 family proteins. To test whether rapamycin induced Bad phosphorylation affects its stability in human lung cancer cells, the half life of Bad was measured using cycloheximide blocking methods as described. H460 cells were treated with 100 ug/ml cycloheximide in the absence or presence of rapamycin for various times as indicated. Levels of Bad were analyzed by Western blot and further quantified by the ImageJ software for calculating the half life as described. reveal that rapamycin significantly reduces the half life of Bad from 53. 3 h to 37. 5 h, indicating that rapamycin induced Bad phosphorylation may promote Bad degradation. To further uncover the mechanism by which rapamycin reduces Bad stability, ubiquitination was measured following rapamycin treatment as described.