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Phosphorylation of Bcl-2 is a marker of M phase events and not a determinant of apoptosis.

Authors:
Ling YH, Tornos C, Perez-Soler R
Affiliation:
Journal:
The Journal of biological chemistry

Abstract

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 anti-tubulin agent paclitaxel. Continuous exposure of human cervical carcinoma HeLa cells to 50 ng/ml paclitaxel resulted in mitotic arrest with a symmetrical bell-shaped curve over time. The number of mitotic cells was highest at 24 h (82%), then declined as arrested cells progressed into apoptosis, and barely no mitotic cells were present at 48-60 h. The time curves of paclitaxel-induced cyclin B1 accumulation and stimulation of Cdc2/cyclin B1 kinase activity were identical and superimposable to that of M phase arrest. In contrast, apoptosis was first detected at 12 h and steadily increased thereafter until the termination of the experiments at 48-60 h, when about 80-96% of cells were apoptotic. Bcl-2 phosphorylation was closely associated in time with M phase arrest, accumulation of cyclin B1, and activation of Cdc2/cyclin B1 kinase, but not with apoptosis. At 24 h, when about 82% of the cells were 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, indicating that the association of paclitaxel-induced M phase arrest and Bcl-2 phosphorylation is not restricted to HeLa cells. We used short exposure to nocodazole and double thymidine to synchronize HeLa cells and investigate the association of Bcl-2 phosphorylation with mitosis. These studies demonstrated that Bcl-2 phosphorylation occurs in tight association with the number of mitotic cells in experimental conditions that do not lead to apoptosis. However, a continuous exposure to nocodazole resulted in a pattern of Bcl-2 phosphorylation, M phase arrest, and apoptosis similar to that observed with paclitaxel. The phosphatase inhibitor okadaic acid was found to inhibit the dephosphorylation of phosphorylated Bcl-2 and to delay the progression of nocodazole M phase-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 demonstrated that Bcl-2 protein can be a substrate of Cdc2/cyclin B1 kinase isolated from paclitaxel-treated cells arrested in M phase. Taken together, these studies suggest that Bcl-2 phosphorylation is tightly associated with mitotic arrest and fail to demonstrate that it is a determinant of progression into apoptosis after mitotic arrest induced by anti-tubulin agents.

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