Abstract
BackgroundA 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.ResultsPIP4KIIγ 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.ConclusionsOur current results imply that PIP4KIIγ may restrain MT depolymerization at the spindle pole through attenuating PLK1-mediated activation of MCAK before anaphase onset.
Highlights
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
PIP4KIIγ accumulates at the spindle pole and associates with α‐tubulin Cellular localization of PIP4KIIγ was examined in CGL2 cells by immunofluorescence staining with two commercial antibodies
PIP4KIIγ has not been experimentally verified to localize at the centrosome, our immunofluorescence staining and immunoprecipitation findings indicate that PIP4KIIγ accumulates at the spindle pole during mitosis
Summary
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. The spindle microtubule (MT) exhibits highly regulated dynamic instability, with frequent polymerization and depolymerization occurring at both the plus and minus ends. Phosphatidylinositol 4,5-bisphosphate (PI4,5P2, hereafter referred to as PIP2) either binds to intracellular proteins and directly modulates their subcellular localization and activity or acts as a precursor for the generation of other second messengers. In this way, PIP2 regulates numerous diverse cellular activities, including modulation of the actin cytoskeleton, vesicle trafficking, focal adhesion formation, and several important nuclear events, such as cell cycle progression, apoptosis, chromatin remodeling, transcriptional regulation, and mRNA processing [5,6,7]. Because the members of the PIP5KI and PIP4KII families exhibit specific and distinct subcellular localization patterns, it has been postulated that local increases in PIP2 concentration by a specific kinase member may underlie its diverse signaling functions [5, 8]
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