Analysis of the role of GSK3 in the mitotic checkpoint

www.nature.com/scientificreports OPEN Analysis of the role of GSK3 in the mitotic checkpoint M. S. Rashid, T. Mazur, W. Ji, S. T. Liu & W. R. Taylor Received: 25 April 2018 Accepted: 6 September 2018 Published: xx xx xxxx 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 GSK3β 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 GSK3β 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. The mitotic checkpoint monitors attachment of chromosomes to spindle microtubules and blocks anaphase onset until all the chromosomes attain bi-orientation1–3. A defining feature of cancer cells is having fewer or more than 2 copies of each chromosome and/or chromosomal segment, typically called aneuploidy. Imbalances in chromosome number may contribute to overexpression of oncogenes or loss of tumor suppressor loci. Additionally, chromosome mis-segregation produces cytosolic DNA which triggers the GAS-STING inflammatory pathway which drives metastasis4,5. Increased rates of chromosome mis-segregation, called chromosome instability (CIN), has been proposed to occur in part from mutations in mitotic checkpoint genes during cancer progression6–8. The origins and consequences of aneuploidy and CIN in cancer are not completely understood9. The mitotic checkpoint is composed of a group of evolutionarily conserved proteins including Mad1, Mad2, BubR1, Bub1, Bub3 and Mps1. These proteins localize to unattached kinetochores, where they generate the mitotic checkpoint complex (MCC) consisting of Mad2, BubR1-Bub3 and Cdc203,10. Aberrant attachments such as syntelic, where microtubules from both poles bind to the same kinetochore, or monotelic attachments, where microtubules from only one pole bind to one kinetochore, result in unattached kinetochores and can cause chromosome mis-segregation if allowed to proceed10. In response, the mitotic checkpoint generates MCC which inhibits the multisubunit E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C) to prevent mitotic progression3,11. The APC/C contains a “destruction box” via which it targets Securin and Cyclin B for degradation by the proteasome12. Degradation of Securin allows sister-chromatid cohesion to dissolve, and degradation of Cyclin B inactivates CDK1 to promote mitotic exit. Thus, an active APC/C promotes chromosome disjunction and mitotic exit. Cdc20 is an APC/C co-activator and contains WD-40 domains which are bound by APC/C substrates. When the MCC binds the APC/CCdc20, it shifts the position of the APC/C-bound Cdc20 preventing substrate recognition. In this manner, the MCC inhibits APC/C in response to mis-aligned chromosomes and prevents mitotic progression13–17. The chromosomal passenger complex (CPC) composed of INCENP, Survivin, Borealin and Aurora B kinase provides an additional layer to monitor proper chromosome attachment to the spindle18. In this case, CPC destabilizes inappropriate attachments of chromosomes to the spindle (for example, both kinetochores attached to microtubules from the same pole). This destabilization creates unattached kinetochores that activate the mitotic checkpoint18. CPC malfunction can lead to cytokinesis defects, chromosome congression and segregation defects, spindle checkpoint malfunction and improper spindle pole formation19. Department of Biological Sciences, University of Toledo, 2801 W. Bancroft Street, MS601, Toledo, OH, 43606, USA. Correspondence and requests for materials should be addressed to W.R.T. (email: ) Scientific ReporTS | (2018) 8:14259 | DOI:10.1038/s41598-018-32435-w 1 www.nature.com/scientificreports/ Checkpoint activation involves the hierarchal recruitment of the mitotic checkpoint proteins to kinetochores to generate a catalytic platform. First, Mad1 and Bub1 are recruited to the kinetochores by Mps1 phosphorylation of Knl1 MELT repeats20–22. At the kinetochore Mad1 binds to open-Mad2 (o-Mad2) and catalyzes its refolding to an alternative tertiary conformation: closed-Mad2 (c-Mad2)23,24. Simultaneously, Bub1 recruits and stabilizes BubR1 at unattached kinetochores, where BubR1 binds Cdc20 in complex with c-Mad225. Importantly, only c-Mad2 is incorporated into MCC. c-Mad2 and BubR1-Bub3 co-operatively inhibit Cdc20 substrate recognition by the APC/C to inhibit anaphase onset11,24,26. Mammalian cells may exit mitosis in the presence of spindle toxins by several mechanisms. In mitotic slippage, a basal level of APC/C activity degrades Cyclin B below a threshold level whereupon the cells exit mitosis without satisfying the mitotic checkpoint27,28. Alternatively, mitotic exit may be due to a gradual decrease in MCC abundance during prolonged arrest. A “weakened” mitotic checkpoint may be caused by mutations in or reduced expression of the mitotic checkpoint proteins and this can translate to an accelerated rate of mitotic exit29. Mad2 localization at kinetochores correlates with mitotic checkpoint strength; decreased Mad2 levels at the kinetochores resulted in shorter mitotic duration times in the presence of spindle toxins13,30,31. Additionally, levels of Mad2-Cdc20 complex determines the rate of Cyclin B degradation and mitotic exit, with decreasing levels of Mad2-Cdc20 interactions translating to increased Cyclin B degradation32. Understanding how mitotic checkpoint strength is modulated, and how key players such as Mad2 are regulated in this modulation, has implications for the clinical effectiveness of spindle toxins. GSK3 contributes to a plethora of biological processes functioning downstream of the WNT pathway and responsive to Akt-dependent signaling pathways33. Both WNT-signaling and Akt signaling activity peak during mitosis, and WNT plays a clear role in spindle dynamics as well as modulating mitotic protein levels34,35. GSK3 regulates the G1/S transition by affect (...truncated)