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p34cdc2 kinase-mediated release of lamins from nuclear ghosts is inhibited by cAMP-dependent protein kinase
1Institute for Cell and Tumour Biology, German Cancer Research Centre, Heidelberg.
Abstract:
During mitosis the lamins are found in a hyperphosphorylated and soluble state. p34cdc2 kinase (MPF), a protein kinase complex with a pivotal role during mitosis, has been found to phosphorylate the lamins and, in some cases, though not all, to cause depolymerization of the lamina in vitro. Due to the variety of protein interactions in the lamina, there is a probable requirement for multiple enzyme activities to effect its breakdown in mitosis. Using nuclear ghosts as substrate, we have fractionated a Xenopus mitotic extract into a lamin-releasing fraction (p34cdc2 kinase) and a fraction that inhibits p34cdc2 kinase-mediated lamin release if the nuclear ghosts are first preincubated in it. The lamin-release-inhibiting activity in the p34cdc2 kinase-depleted mitotic extract is, in turn, inhibited if PKI, a protein kinase inhibitor specific for PKA, is included in the preincubation reaction mixture. Furthermore, a similar degree of inhibition can be achieved by using purified PKA to preincubate the nuclear ghosts. This suggests that dephosphorylation of PKA substrate sites is necessary for lamin depolymerization.
Insights
Mitotic breakdown of the nuclear lamina involves p34cdc2 kinase and requires dephosphorylation. This process is regulated by protein kinase A (PKA) activity, suggesting a complex enzymatic control of lamin depolymerization during cell division.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- During mitosis, lamins transition to a hyperphosphorylated, soluble state.
- p34cdc2 kinase (MPF) phosphorylates lamins, potentially causing lamina depolymerization in vitro.
Purpose of the Study:
- To investigate the enzymatic activities involved in mitotic lamin breakdown.
- To identify factors regulating p34cdc2 kinase-mediated lamin release.
Main Methods:
- Fractionation of Xenopus mitotic extract using nuclear ghosts as substrate.
- Assays to measure lamin release and inhibition of p34cdc2 kinase activity.
- Use of protein kinase inhibitor PKI and purified PKA.
Main Results:
- A lamin-releasing fraction containing p34cdc2 kinase was identified.
- A fraction inhibiting p34cdc2 kinase-mediated lamin release was found.
- This inhibitory activity was dependent on PKA substrates and could be mimicked by purified PKA.
Conclusions:
- Mitotic lamin depolymerization likely requires multiple enzymatic activities beyond p34cdc2 kinase.
- Dephosphorylation of PKA substrate sites is a necessary step for lamin depolymerization.
- This suggests a regulatory role for PKA in controlling nuclear lamina disassembly during mitosis.