Caulerpenyne blocks MBP kinase activation controlling mitosis in sea urchin eggs

In a previous study, we demonstrated that caulerpenyne (Cyn), a natural ses quiterpene having an antiproliferative potency, blocked the mitotic cycle o f sea urchin embryos at metaphase and inhibited the phosphorylation of seve ral proteins, but did not affect histone H1 kinase activation (Pesando et a l, 1998, fur. J, Cell Biol, 77, 19-26), Here, we show that concentrations o f Cyn that blocked the first division of the sea urchin Paracentrotus livid us embryos in a metaphase-like stage (45 mu M) also inhibited the stimulati on of mitogen-activated protein kinase (MAPK) activity in vivo as measured in treated egg extracts using myelin basic protein (MBP) as a substrate (MB PK), However, Cyn had no effect on MBP phosphorylation when added in vitro to an untreated egg extract taken at the time of metaphase, suggesting that Cyn acts on an upstream activation process. PD 98059 (40 mu M), a previously characterized specific synthetic inhibitor of MAPK/extracellular signal-regulated kinase-1 (MEK1), also blocked sea u rchin eggs at metaphase in a way very similar to Cyn, Both molecules induce d similar inhibitory effects on MBP kinase activation in vivo, but had no d irect effect on MBP kinase activity in vitro, whereas they did not affect H 1 kinase activation neither in vivo nor in vitro. As a comparison, butyrola ctone 1 (100 mu M), a known inhibitor of H1 kinase activity, did inhibit H1 kinase of sea urchin eggs in vivo and in vitro, and blocked the sea urchin embryo mitotic cycle much before metaphase, Immunoblots of mitotic extract s, treated with anti-active MAP-kinase antibody, showed that both Cyn and P D 98059 reduced the phosphorylation of p42 MAP kinase (Erk2) in vivo. Our overall results suggest that Cyn blocks the sea urchin embryo mitotic c ycle at metaphase by inhibiting an upstream phosphorylation event in the MB PK activation pathway. They also show that H1 kinase and MBPK activation ca n be dissociated from each other in this model system.