Abstract
Entry into mitosis requires the coordinated action of multiple mitotic protein kinases. In this report, we investigate the involvement of protein kinase C in the control of mitosis in human cells. Treatment of synchronized HL60 cells with the highly selective protein kinase C (PKC) inhibitor chelerythrine chloride leads to profound cell cycle arrest in G2 phase. The cellular effects of chelerythrine are not due to either direct or indirect inhibition of the known mitotic regulator p34(cdc2)/cyclin B kinase. Rather, several lines of evidence demonstrate that chelerythrine-mediated G2 phase arrest results from selective inhibition and degradation of betaII protein kinase C. First, chelerythrine causes dose-dependent inhibition of betaII PKC in vitro with an IC50 identical to that for G2 phase blockade in whole cells. Second, chelerythrine specifically inhibits betaII PKC-mediated lamin B phosphorylation and mitotic nuclear lamina disassembly. Third, chelerythrine leads to selective loss of betaII PKC during G2 phase in synchronized cells. Fourth, chelerythrine mediates activation-dependent degradation of PKC, indicating that betaII PKC is selectively activated during G2 phase of cell cycle. Taken together, these data demonstrate that betaII PKC activation at G2 phase is required for mitotic nuclear lamina disassembly and entry into mitosis and that betaII PKC-mediated phosphorylation of nuclear lamin B is important in these events.
Highlights
Increases in cytosolic free calcium have been implicated in the regulation of mitosis in a number of cell systems (8 –12)
We show that protein kinase C (PKC) activation is required for phosphorylation of key mitotic sites on nuclear lamin B previously implicated in mitotic nuclear lamina disassembly [17, 18]
In order to assess the effects of chelerythrine on cell cycle progression, HL60 cells were synchronized in G1/S phase with aphidicolin and allowed to progress through cell cycle
Summary
The regulation of mitosis in a number of cell systems (8 –12). Intracellular calcium release triggers nuclear envelope breakdown in sea urchin embryos, and calcium transients are required for nuclear envelope breakdown in mammalian fibroblasts (8 –12). PKC has been implicated in the regulation of mitosis in a number of systems [4, 13,14,15,16,17,18,19]. Staurosporine, a potent but relatively nonselective protein kinase C inhibitor, arrests cells at two cell cycle phases, G1 and G2/M phase, depending on the concentration used [15]. Several staurosporine analogues and other structurally distinct PKC inhibitors lead to inhibition of cell cycle at G2/M phase [16, 19]. We utilize the highly selective PKC inhibitor chelerythrine to demonstrate that PKC activity is required for entry of human cells into mitosis. We find that II PKC is a critical PKC isotype involved in the G2/M phase transition
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