Nuclear phospholipase C-β 1b activation during G 2/M and late G 1 phase in nocodazole-synchronized HL-60 cells

Abstract

In this study, the activity of nuclear phosphatidylinositol-specific phosholipase C (PI-PLC) was investigated in HL-60 cells blocked at G 2/M phase by the addition of nocodazole, and released into medium as synchronously progressing cells. Two peaks of an increase in the nuclear PI-PLC activities were detected; an early peak reached a maximum at 1 h after release from the nocodazole block, and a second increase was detected at 8.5 h after the release. Immunoprecipitation studies indicated that the increase in the activity was due to the activation of the nuclear PI-PLC-β 1. Western blot analysis demonstrated no changes in the level of both a and b splicing variants of PI-PLC-β 1 in the nuclei of cells isolated at either 1 h or 8.5 h after the block. However, an increase in the serine-phosphorylation of PI-PLC-β 1b was detected in the nuclei of HL-60 cells isolated at 1 and 8.5 h after the block, and the presence of MEK-inhibitor PD98059 completely inhibited both the serine phosphorylation and the increase in the PI-PLC activities in vitro. The presence of PI-PLC inhibitor prevented the progression of HL-60 cells through the G 1 into S phase of the cell cycle. These results demonstrate that two peaks of nuclear PI-PLC activities, which are due to a PD98059-sensitive phosphorylation of nuclear PLC-β 1b on serine, occur at the G 2/M and late G 1 phase and are necessary for the progression of the cells through the cell cycle.