One of the most striking and unique features of microtubule inhibitors such as vinca alkaloids and taxanes widely used in cancer chemotherapy is that they promote phosphorylation of two key anti-apoptotic proteins, Bcl-xL and Bcl-2. To identify the responsible kinase, a peptide termed FL62 (HLADSPAVN) harboring the in vivo Ser62 phosphorylation site in Bcl-xL was synthesized and tested as a substrate. Extracts from vinblastine-treated KB-3 cells exhibited 15-fold higher FL62 kinase activity than untreated cells. Characterization of the FL62 kinase indicated that it exhibited properties consistent with CDK1/cyclin B. To further investigate the role of CDK1 in Bcl-xL and Bcl-2 phosphorylation during mitosis and mitotic arrest, cells were synchronized by double thymidine block, treated with vehicle or vinblastine, and harvested at defined time-points thereafter. In the absence of vinblastine, both Bcl-xL and Bcl-2 were partially and transiently phosphorylated during normal mitotic progression, with kinetics that paralleled CDK1/cyclin B activation, and mitotic phosphorylation was sensitive to inhibition by the CDK inhibitors roscovitine, purvalanol A, or RO3306. When added to cultures of vinblastine-treated synchronized cells at the onset of mitotic arrest and under conditions which precluded mitotic slippage, the CDK inhibitors inhibited the extensive increase in Bcl-xL and Bcl-2 phosphorylation. Further, recombinant active CDK1 directly phosphorylated full-length Bcl-xL in vitro at the physiologically relevant site, Ser62. The results suggest a model whereby CDK1 acts normally during mitosis to induce transient phosphorylation of a subpopulation of Bcl-xL/Bcl-2 molecules. During sustained CDK1 activation after MTI treatment, when all Bcl-xL/Bcl-2 molecules become phosphorylated, it is hypothesized that a critical threshold is breached which inactivates their anti-apoptotic function thus priming the cells for apoptosis. The mechanism invoked by these studies, with sustained CDK1 activity disabling anti-apoptotic Bcl-2 proteins by direct phosphorylation, is attractive as a functional link between mitotic arrest and apoptosis. Supported by NIH CA109821.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3865.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO