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Background A previous screen of a human kinase and phosphatase shRNA library to select genes that mediate arsenite induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-bisphosphate (PIP2)-synthesizing enzyme. In this study, we explored how PIP4KIIγ regulates the assembly of mitotic spindles. Results PIP4KIIγ accumulates at the spindle pole before anaphase, and is required for the assembly of functional bipolar spindles. Depletion of PIP4KIIγ enhanced the spindle pole accumulation of mitotic centromere-associated kinesin (MCAK), a microtubule (MT)-depolymerizing kinesin, and resulted in a less stable spindle pole-associated MT. Depletion of MCAK can ameliorate PIP4KIIγ depletion-induced spindle abnormalities. In addition, PIP2 binds to polo-like kinase (PLK1) and reduces PLK1-mediated phosphorylation of MCAK. These results indicate that PIP4KIIγ and PIP2 may negatively regulate the MT depolymerization activity of MCAK by reducing PLK1-mediated phosphorylation of MCAK. Consequently, depletion of PLK1 has been shown to counteract the PIP4KIIγ depletion-induced instability of spindle pole-associated MT and cell resistance to arsenite. Conclusions Our current results imply that PIP4KIIγ may restrain MT depolymerization at the spindle pole through attenuating PLK1-mediated activation of MCAK before anaphase onset.

Arsenite induces centrosome amplification and spindle abnormalities, leading to chromosome missegregation and consequently resulting in mitotic cell apoptosis or aneuploidy. However, the underlying mechanism of how arsenite disrupts mitotic spindles is not known. In this study, we showed that CGL2 cells treated with arsenic trioxide (ATO) were arrested at mitotic stage in company with the formation of substantial spindle abnormalities and significant reduction of acetylated- and detyrosinated-tubulin, characteristics of decreased MT stability. Immunofluorescence staining of EB1, a plus-end microtubule-binding protein, revealed discrete EB1 spots distributing along microtubule (MT) nucleated from the two spindle poles in untreated mitotic cells. However, it showed a disorganized pattern in ATO-arrested mitotic cells with the spot-like EB1 clusters splashing all over the cells. In addition, immunoblotting and immunofluorescence staining revealed that the mitosis-specific inhibitory phospho-GSK3 was diminished in ATO-arrested mitotic cells. Pharmacological inhibition of GSK3 could prevent ATO induction of spindle abnormalities, mitotic arrest, and cell death. Since GSK3β has been reported to reduce MT stability by phosphorylating several microtubule-associated proteins, our current results indicated that GSK3β may mediate ATO induction of spindle abnormalities. Citation Format: Tz-Chi Lin, Ren-Meei Chen, Hsiao-Hui Kuo, Ling-Huei Yih. GSK3β mediated arsenite induction of spindle abnormalities. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1593. doi:10.1158/1538-7445.AM2014-1593