PHOSPHORYLATION OF BCL-2 IS A MARKER OF M-PHASE EVENTS AND NOT A DETERMINANT OF APOPTOSIS

Phosphorylation of Bcl-2 protein is a post-translational modification of unclear functional consequences. We studied the correlation between Bcl-2 phosphorylation, mitotic arrest, and apoptosis induced by the a ntitubulin agent paclitaxel, Continuous exposure of human cervical car cinoma HeLa cells to 50 ng/ml paclitaxel resulted in mitotic arrest wi th a symmetrical bell-shaped curve over time. The number of mitotic ce lls was highest at 24 h (82%), then declined as arrested cells progres sed into apoptosis, and barely no mitotic cells were present at 48-60 h. The time curves of paclitaxel-induced cyclin B1 accumulation and st imulation of Cdc2/cyclin B1 kinase activity were identical and superim posable to that of M phase arrest. In contrast, apoptosis was first de tected at 12 h and steadily increased thereafter until the termination of the experiments at 48-60 h, when about 80-96% of cells were apopto tic. Bcl-2 phosphorylation was closely associated in time with M phase arrest, accumulation of cyclin B1, and activation of Cdc2/cyclin B1 k inase, but not with apoptosis. At 24 h, when about 82% of the cells we re in mitosis, almost all Bcl-2 protein was phosphorylated, whereas at 48 h, when 70-90% of the cells were apoptotic, all Bcl-2 protein was unphosphorylated, Similar results were obtained with SKOV3 cells, indi cating that the association of paclitaxel-induced M phase arrest and B cl-2 phosphorylation is not restricted to HeLa cells, We used short ex posure to nocodazole and double thymidine to synchronize HeLa cells an d investigate the association of Bcl-2 phosphorylation with mitosis, T hese studies demonstrated that Bcl-2 phosphorylation occurs in tight a ssociation with the number of mitotic cells in experimental conditions that do not lead to apoptosis. However, a continuous exposure to noco dazole resulted in a pattern of Bcl-2 phosphorylation, M phase arrest, and apoptosis similar to that observed with paclitaxel, The phosphata se inhibitor okadaic acid was found to inhibit the dephosphorylation o f phosphorylated Bcl-2 and to delay the progression of nocodazole M ph ase-arrested cells into interphase, In contrast, the serine/threonine kinase inhibitor staurosporine, but not the tyrosine kinase inhibitor genistein, led to rapid dephosphorylation of phosphorylated Bcl-2 and accelerated the progression of nocodazole M phase-arrested cells into interphase, Immune complex kinase assays in cell-free systems demonstr ated that Bcl-2 protein can be a substrate of Cdc2/cyclin B1 kinase is olated from paclitaxel-treated cells arrested in M phase. Taken togeth er, these studies suggest that Bcl-2 phosphorylation is tightly associ ated with mitotic arrest and fail to demonstrate that it is a determin ant of progression into apoptosis after mitotic arrest induced by anti -tubulin agents.