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

Yi He Ling, Carmen Tornos, Roman Perez-Soler

Research output: Contribution to journalArticle

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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 antitubulin 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.

Original languageEnglish (US)
Pages (from-to)18984-18991
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number30
DOIs
StatePublished - Jul 24 1998
Externally publishedYes

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Phosphorylation
Cell Division
Cyclin B1
Paclitaxel
Apoptosis
Nocodazole
Phosphotransferases
HeLa Cells
Association reactions
Interphase
Mitosis
Proteins
Cell Count
Okadaic Acid
Staurosporine
Genistein
Protein-Serine-Threonine Kinases
Tubulin
Cell-Free System
Antigen-Antibody Complex

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphorylation of Bcl-2 is a marker of M phase events and not a determinant of apoptosis. / Ling, Yi He; Tornos, Carmen; Perez-Soler, Roman.

In: Journal of Biological Chemistry, Vol. 273, No. 30, 24.07.1998, p. 18984-18991.

Research output: Contribution to journalArticle

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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 antitubulin 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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