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Caulerpenyne blocks MBP kinase activation controlling mitosis in sea urchin eggs
D Pesando1, C Pesci-Bardon, P Huitorel
1Laboratoire de Physiologie et Toxicologie Environnementales, Université de Nice-Sophia Antipolis, Faculté des Sciences, Nice, France. pesando@unice.fr
Abstract:
In a previous study, we demonstrated that caulerpenyne (Cyn), a natural sesquiterpene having an antiproliferative potency, blocked the mitotic cycle of sea urchin embryos at metaphase and inhibited the phosphorylation of several proteins, but did not affect histone H1 kinase activation (Pesando et al, 1998, Eur. J. Cell Biol. 77, 19-26). Here, we show that concentrations of Cyn that blocked the first division of the sea urchin Paracentrotus lividus embryos in a metaphase-like stage (45 microM) also inhibited the stimulation of mitogen-activated protein kinase (MAPK) activity in vivo as measured in treated egg extracts using myelin basic protein (MBP) as a substrate (MBPK). 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 microM), a previously characterized specific synthetic inhibitor of MAPK/extracellular signal-regulated kinase-1 (MEK1), also blocked sea urchin eggs at metaphase in a way very similar to Cyn. Both molecules induced similar inhibitory effects on MBP kinase activation in vivo, but had no direct effect on MBP kinase activity in vitro, whereas they did not affect H1 kinase activation neither in vivo nor in vitro. As a comparison, butyrolactone 1 (100 microM), 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 extracts, treated with anti-active MAP-kinase antibody, showed that both Cyn and PD 98059 reduced the phosphorylation of p42 MAP kinase (Erk2) in vivo. Our overall results suggest that Cyn blocks the sea urchin embryo mitotic cycle at metaphase by inhibiting an upstream phosphorylation event in the MBPK activation pathway. They also show that H1 kinase and MBPK activation can be dissociated from each other in this model system.
Insights
Caulerpenyne (Cyn) inhibits sea urchin embryo cell division at metaphase by blocking mitogen-activated protein kinase (MAPK) activation, not by affecting histone H1 kinase. This suggests Cyn targets an upstream phosphorylation event in MAPK regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Marine Biology
Background:
- Caulerpenyne (Cyn), a natural sesquiterpene, previously showed antiproliferative effects by blocking sea urchin embryo mitosis at metaphase.
- Previous studies indicated Cyn inhibited protein phosphorylation but not histone H1 kinase activation.
Purpose of the Study:
- To investigate the mechanism by which caulerpenyne (Cyn) inhibits the sea urchin embryo mitotic cycle at metaphase.
- To determine if Cyn affects mitogen-activated protein kinase (MAPK) or histone H1 kinase activation.
- To compare the effects of Cyn with a specific MEK1 inhibitor, PD 98059.
Main Methods:
- Sea urchin (Paracentrotus lividus) embryos were treated with Cyn (45 microM) and PD 98059 (40 microM).
- Mitogen-activated protein kinase (MAPK) activity was measured in egg extracts using myelin basic protein (MBP) as a substrate (MBPK).
- Histone H1 kinase (H1K) activity was assessed in vivo and in vitro.
- Immunoblots using anti-active MAP-kinase antibody were performed on mitotic extracts.
Main Results:
- Cyn (45 microM) inhibited MAPK activation in vivo but not in vitro, suggesting an upstream target.
- PD 98059, a MEK1 inhibitor, mimicked Cyn's effects, blocking metaphase and inhibiting in vivo MAPK activation without affecting H1 kinase.
- Butyrolactone 1, an H1 kinase inhibitor, blocked the cell cycle earlier than metaphase and inhibited H1 kinase activity.
- Immunoblots showed Cyn and PD 98059 reduced p42 MAP kinase (Erk2) phosphorylation in vivo.
Conclusions:
- Caulerpenyne (Cyn) blocks the sea urchin embryo mitotic cycle at metaphase by inhibiting an upstream phosphorylation event in the MAPK activation pathway.
- Histone H1 kinase and MAPK activation are dissociable processes in this sea urchin model system.
- Cyn and PD 98059 target upstream MAPK activation, distinct from H1 kinase inhibition.