Collas P
Department of Biochemistry, Norwegian College of Veterinary Medicine, and Institute of Medical Biochemistry, University of Oslo, PO Box 1112, Blindern, Norway.
J Cell Sci. 1999 Mar;112 ( Pt 6):977-87. doi: 10.1242/jcs.112.6.977.
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.
斑马鱼内核膜（NEP55）和核纤层（L68）的分子标记物已被鉴定、部分表征，并用于证明斑马鱼核膜的解体需要先由蛋白激酶C（PKC），然后由细胞周期蛋白依赖性激酶2（Cdc2激酶）进行顺序磷酸化事件。NEP55和L68分别与人LAP2β和核纤层蛋白B在免疫和功能上相关。将斑马鱼细胞核暴露于减数分裂胞质溶胶中会引发NEP55和L68的快速磷酸化以及这两种蛋白质的解体。同时抑制Cdc2和PKC可完全抑制L68的磷酸化，而仅抑制其中一种激酶时则部分受阻。抑制或免疫耗尽胞质溶胶中的Cdc2可完全阻止NEP55的磷酸化。抑制环磷酸腺苷依赖性激酶、丝裂原活化蛋白激酶（MEK）或钙调蛋白激酶II不会影响NEP55或L68的磷酸化。在体外，核膜解体需要哺乳动物PKC和Cdc2对NEP55和L68进行磷酸化。抑制其中任何一种激酶都足以消除核膜解体。此外，在胞质溶胶和体外进行的新型两步磷酸化分析表明，在Cdc2介导的L68和NEP55磷酸化之前，PKC介导的L68磷酸化对于引发核膜破裂至关重要。即使NEP55已被磷酸化，Cdc2在PKC之前引发的磷酸化也会阻止核膜解体。结果表明，斑马鱼核解体需要PKC引发的顺序磷酸化事件，随后是Cdc2。它们还表明，内核膜整合蛋白的磷酸化不足以促进核膜破裂，并提示在减数分裂和有丝分裂期间核膜成分解体的多层次调节。
