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Multifunctional Ca2+/calmodulin-dependent protein kinase is necessary for nuclear envelope breakdown
C Baitinger1, J Alderton, M Poenie
1Department of Pharmacology, Stanford University School of Medicine, California 94305-5332.
Abstract:
The role of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in nuclear envelope breakdown (NEB) was investigated in sea urchin eggs. The eggs contain a 56-kD polypeptide which appears to be a homologue of neuronal CaM kinase. For example, it undergoes Ca2+/calmodulin-dependent autophosphorylation that converts it to a Ca2(+)-independent species, a hallmark of multifunctional CaM kinase. It is homologous to the alpha subunit of rat brain CaM kinase. Autophosphorylation and substrate phosphorylation by the sea urchin egg kinase are inhibited in vitro by CaMK(273-302), a synthetic peptide corresponding to the autoinhibitory domain of the neuronal CaM kinase. This peptide inhibited NEB when microinjected into sea urchin eggs. Only one mAb to the neuronal enzyme immunoprecipitated the 56-kD polypeptide. Only this antibody blocked or significantly delayed NEB when microinjected into sea urchin eggs. These results suggest that sea urchin eggs contain multifunctional CaM kinase, and that this enzyme is involved in the control of NEB during mitotic division.
Insights
Sea urchin eggs possess a multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) homologous to neuronal CaM kinase. This enzyme plays a crucial role in controlling nuclear envelope breakdown during cell division.
Area of Science:
- Cell Biology
- Molecular Biology
- Developmental Biology
Background:
- Nuclear envelope breakdown (NEB) is a critical event in cell division.
- Ca2+/calmodulin-dependent protein kinase (CaM kinase) is known for its roles in neuronal function.
- The specific role of CaM kinase in the cell cycle of non-neuronal cells, like sea urchin eggs, is less understood.
Purpose of the Study:
- To investigate the presence and function of multifunctional CaM kinase in sea urchin eggs.
- To determine if this enzyme is involved in the process of nuclear envelope breakdown (NEB).
Main Methods:
- Biochemical characterization of a 56-kD polypeptide in sea urchin eggs, including autophosphorylation assays.
- Comparison of the sea urchin egg polypeptide with neuronal CaM kinase using homology and antibody cross-reactivity.
- Microinjection of a synthetic peptide inhibitor (CaMK(273-302)) and a specific monoclonal antibody (mAb) into sea urchin eggs to assess their effect on NEB.
Main Results:
- A 56-kD polypeptide in sea urchin eggs was identified, exhibiting Ca2+/calmodulin-dependent autophosphorylation characteristic of multifunctional CaM kinase and showing homology to rat brain CaM kinase alpha subunit.
- A synthetic peptide corresponding to the autoinhibitory domain of neuronal CaM kinase inhibited both autophosphorylation and substrate phosphorylation in vitro, and also inhibited NEB when microinjected into eggs.
- A specific mAb recognizing neuronal CaM kinase immunoprecipitated the 56-kD sea urchin egg polypeptide and also inhibited NEB upon microinjection.
Conclusions:
- Sea urchin eggs contain a multifunctional CaM kinase homologous to its neuronal counterpart.
- This CaM kinase is implicated in the regulation of nuclear envelope breakdown during mitotic division in sea urchin eggs.