Sequential PKC- and Cdc2-mediated phosphorylation events elicit zebrafish nuclear envelope disassembly.
Sequential PKC- and Cdc2-mediated phosphorylation events elicit zebrafish nuclear envelope disassembly.
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作者：
P. Collas
斑马鱼内核膜（NEP55）和核纤层（L68）的分子标记物进行了鉴定，部分表征和用于证明，拆卸的斑马鱼核膜需要顺序磷酸化事件的第一个PKC，然后Cdc2激酶。NEP 55和L68分别与人LAP 2 β和核纤层蛋白B在免疫学和功能上相关。斑马鱼细胞核暴露于减数分裂的胞质溶胶中，可使NEP55和L68快速磷酸化，并使这两种蛋白解体。同时抑制Cdc2和PKC可完全抑制L68磷酸化，抑制任一激酶仅可部分阻断L68磷酸化。NEP55磷酸化完全防止抑制或免疫耗竭胞质Cdc2。cAMP依赖性激酶、MEK或CaM激酶II的抑制不影响NEP 55或L68磷酸化。在体外，核膜解体需要哺乳动物PKC和Cdc2磷酸化NEP55和L68。抑制任一激酶足以消除NE分解。此外，新的两步磷酸化试验在胞质溶胶和体外表明，PKC介导的磷酸化L68之前Cdc2介导的磷酸化L68和NEP55是必不可少的引起核膜破裂。在PKC之前由Cdc2引起的磷酸化阻止核膜解体，即使NEP 55被磷酸化。结果表明，顺序磷酸化事件引起的PKC，其次是Cdc2，是必需的斑马鱼核解体。他们还认为，内核膜整合蛋白的磷酸化不足以促进核膜分解，并建议在减数分裂和有丝分裂期间核膜组件的分解的多层次调节。
Molecular markers of the zebrafish inner nuclear membrane (NEP55) and nuclear lamina (L68) were identified, partially characterized and used to demonstrate that disassembly of the zebrafish nuclear envelope requires sequential phosphorylation events by first PKC, then Cdc2 kinase. NEP55 and L68 are immunologically and functionally related to human LAP2beta and lamin B, respectively. Exposure of zebrafish nuclei to meiotic cytosol elicits rapid phosphorylation of NEP55 and L68, and disassembly of both proteins. L68 phosphorylation is completely inhibited by simultaneous inhibition of Cdc2 and PKC and only partially blocked by inhibition of either kinase. NEP55 phosphorylation is completely prevented by inhibition or immunodepletion of cytosolic Cdc2. Inhibition of cAMP-dependent kinase, MEK or CaM kinase II does not affect NEP55 or L68 phosphorylation. In vitro, nuclear envelope disassembly requires phosphorylation of NEP55 and L68 by both mammalian PKC and Cdc2. Inhibition of either kinase is sufficient to abolish NE disassembly. Furthermore, novel two-step phosphorylation assays in cytosol and in vitro indicate that PKC-mediated phosphorylation of L68 prior to Cdc2-mediated phosphorylation of L68 and NEP55 is essential to elicit nuclear envelope breakdown. Phosphorylation elicited by Cdc2 prior to PKC prevents nuclear envelope disassembly even though NEP55 is phosphorylated. The results indicate that sequential phosphorylation events elicited by PKC, followed by Cdc2, are required for zebrafish nuclear disassembly. They also argue that phosphorylation of inner nuclear membrane integral proteins is not sufficient to promote nuclear envelope breakdown, and suggest a multiple-level regulation of disassembly of nuclear envelope components during meiosis and at mitosis.