| Despite detailed knowledge of the components of the spindle-assembly checkpoint, a molecular explanation of how cells die after prolonged spindle checkpoint activation, and thus how microtubule inhibitors and other anti-mitotic drugs ultimately elicit their lethal effects, has yet to emerge. Mitotically arrested cells typically display extensive phosphorylation of two key anti-apoptotic proteins, Bcl-xL and Bcl-2, and evidence suggests that phosphorylation disables their anti-apoptotic activity. However, the responsible kinase has remained elusive. This dissertation presents evidence that Cdk1/cyclin B catalyzes Bcl-xL/Bcl-2 phosphorylation during mitosis and vinblastine-induced mitotic arrest. Preliminary experiments are presented first where two Bcl-xL kinase substrates, a full length protein (HisBclxL DeltaC) and a fusion peptide (GST-BL) were purified and tested as specific Bcl-xL kinase substrates. HisBclxL DeltaC was an ineffective and non-selective substrate because it was equally phosphorylated by size exclusion fractions, whole cell extracts, and cytosolic and mitochondrial fractions from both control and vinblastine treated KB-3 cells. In contrast, GST-BL, where the fused BL peptide contains amino acids 41-70 of Bcl-xL including the vinblastine induced phosphorylation site Ser62, was phosphorylated only in size exclusion fractions from vinblastine treated KB-3 cells. However, the GST-only negative control was also extensively phosphorylated indicating poor specificity of the substrate. Both HisBclxL DeltaC and GST-BL were therefore not suitable as Bcl-xL kinase substrates in partially-purified extracts or subcellular fractions, but the experiments did provide the foundation to develop a small peptide substrate, termed FL62, harboring only the vinblastine induced phosphorylation site, Ser62. Characterization of the kinase in vitro using FL62 as a substrate revealed it corresponded to Cdk1/cyclin B. Furthermore, Cdk inhibitors specifically inhibited Bcl-xL/Bcl-2 phosphorylation induced by vinblastine in cell culture. Furthermore, during normal mitosis, Bcl-xL and Bcl-2 were partially and transiently phosphorylated in a Cdk-dependent. The findings suggest a model whereby a switch in the duration of Cdk1 activation, from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-xL/Bcl-2 phosphorylation, resulting in inactivation of their anti-apoptotic function. Thus, phosphorylation of antiapoptotic Bcl-2 proteins acts as a sensor for Cdk1 signal duration and as a functional link coupling mitotic arrest to apoptosis. |
