A role for nuclear phosphatidylinositol-specific phospholipase C in the G₂/M phase transition

Protein kinase C (PKC) is activated at the nucleus during the G₂ phase of cell cycle, where it is required for mitosis. However, the mechanisms controlling cell cycle-dependent activation of nuclear PKC are not known. We now report that nuclear levels of the major physiologic PKC activator diacylglycerol (DAG) fluctuate during cell cycle. Specifically, nuclear DAG levels in G₂/M phase cells are 2.5-3-fold higher than in G₁ phase cells. In synchronized cells, nuclear DAG levels rise to a peak coincident with the G₂/M phase transition and return to basal levels in G₁ phase cells. This increase in DAG level is sufficient to stimulate β(II) PKC-mediated phosphorylation of its mitotic nuclear envelope substrate lamin B in vitro. Isolated nuclei from G₂ phase cells contain an active phospholipase activity capable of generating DAG in vitro. Nuclear phospholipase activity is inhibited by the selective phosphatidylinositol-specific phospholipase C (PI- PLC) inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and neomycin sulfate, but not by the phosphatidylcholine-PLC selective inhibitor D609 or inhibitors of phospholipase D-mediated DAG generation. Treatment of synchronized cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine leads to decreased nuclear PI-PLC activity and cell cycle blockade in the G₂ phase, suggesting a role for nuclear PI-PLC in the G₂/M phase transition. Our data are consistent with the hypothesis that nuclear PI-PLC generates DAG to activate nuclear β(II) PKC, whose activity is required for mitosis.