Sequential PKC- and Cdc2-mediated phosphorylation events elicit zebrafish nuclear envelope disassembly

Molecular membrane (NEP55) and nuclear lamina (L68) were identified, partia lly 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 relate d to human LAP2 beta and lamin B, respectively, Exposure of zebrafish nucle i to meiotic cytosol elicits rapid phosphorylation of NEP55 and L68, and di sassembly of both proteins. L68 phosphorylation is completely inhibited by simultaneous inhibition of Cdc2 and PKC and only partially blocked by inhib ition of either kinase, NEP55 phosphorylation is completely prevented by in hibition 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 a nd L68 by both mammalian PKC and Cdc2, Inhibition of either kinase is suffi cient to abolish NE disassembly, Furthermore, novel two-step phosphorylatio n assays in cytosol and in vitro indicate that PKC-mediated phosphorylation of L68 prior to Cdc2-mediated phosphorylation of L68 and NEP55 is essentia l to elicit nuclear envelope breakdown. Phosphorylation elicited by Cdc2 pr ior to PKC prevents nuclear envelope disassembly even though NEP55 is phosp horylated. The results indicate that sequential phosphorylation events elic ited by PKC, followed by Cdc2, are required for zebrafish nuclear disassemb ly, They also argue that phosphorylation of inner nuclear membrane integral proteins is not sufficient to promote nuclear envelope breakdown, and sugg est a multiple-level regulation of disassembly of nuclear envelope componen ts during meiosis and at mitosis.