A role for glycogen synthase kinase-3 in mitotic spindle dynamics and chromosome alignment.

Glycogen synthase kinase-3 (GSK-3) is a conserved, multifunctional kinase that is constitutively active in resting cells, and inactivated through phosphorylation by protein kinase B (PKB). We have investigated the temporal and spatial control of GSK-3 phosphorylation during the cell cycle in mammalian cells. We show that GSK-3 is present along the length of spindle microtubules and that a fraction of GSK-3 is phosphorylated during mitosis. Phospho-GSK-3 is abundant at the centrosomes and spindle poles but absent from other areas of the spindle. GSK-3 phosphorylation occurs concomitantly with the appearance of phosphorylated and active PKB at the centrosome, which suggests that PKB is the kinase responsible for phosphorylating and inactivating GSK-3 at the centrosome during mitosis. We demonstrate that lithium and two structurally distinct inhibitors of GSK-3 promote defects in microtubule length and chromosomal alignment during prometaphase. Treated cells contain mono-oriented chromosomes concentrated at the plus ends of astral microtubules, which are longer than in untreated cells. Live microscopy of cells expressing Histone-2B-GFP confirms that the inhibition of GSK-3 suppresses mitotic chromosome movement and leads to a prometaphase-like arrest. We propose that GSK-3 is regulated in a temporal and spatial manner during mitosis and, through controlling microtubule dynamics, plays an important role in chromosomal alignment on the metaphase plate.