Analysis of the role of GSK3 in the mitotic checkpoint.

The mitotic checkpoint ensures proper chromosome segregation; defects in this checkpoint can lead to aneuploidy, a hallmark of cancer. The mitotic checkpoint blocks progression through mitosis as long as chromosomes remain unattached to spindle microtubules. Unattached kinetochores induce the formation of a mitotic checkpoint complex (MCC) composed of Mad2, BubR1, Bub1 and Bub3 which inhibits anaphase onset. Spindle toxins induce prolonged mitotic arrest by creating persistently unattached kinetochores which trigger MCC formation. We find that the multifunctional ser/thr kinase, glycogen synthase kinase 3 (GSK3) is required for a strong mitotic checkpoint. Spindle toxin-induced mitotic arrest is relieved by GSK3 inhibitors SB 415286 (SB), RO 318220 (RO) and lithium chloride. Similarly, targeting GSK3ß with knockout or RNAi reduced mitotic arrest in the presence of Taxol. GSK3 was required for optimal localization of Mad2, BubR1, and Bub1 at kinetochores and for optimal assembly of the MCC in spindle toxin-arrested cells. The WNT- and PI3K/Akt signaling pathways negatively regulate GSK3ß activity. Inhibition of WNT and PI3K/Akt signaling, in the presence of Taxol, induced a longer mitotic arrest compared to Taxol alone. Our observations provide novel insight into the regulation of the mitotic checkpoint and its connection to growth-signaling pathways.