A mammalian in vitro centriole duplication system: evidence for involvement of CDK2/cyclin E and nucleophosmin/B23 in centrosome duplication.
Centrosome duplication in mammalian cells is a highly regulated process, occurs in coordination of other cell cycle events. However, molecular exploration of this important cellular process had been difficult due to unavailability of a simple assay system. Here, using centrosomes loosely associated with nuclei isolated from cultured cells, we developed a cell-free centriole (duplication unit of the centrosome) duplication system: unduplicated centrosomes bound to the nuclei are able to undergo duplication in the presence of G1/S extracts. We show that the ability of G1/S extracts to induce centriole duplication in vitro depends on the presence of active CDK2/cyclin E. It has been shown that dissociation of centro-somal nucleophosmin (NPM)/B23 triggered by CDK2/cyclin E-mediated phosphorylation is required for initiation of centrosome duplication. We show that centriole duplication is blocked when nuclei were preincubated with the anti-NPM/B23 antibody that prevents phosphorylation of NPM/B23 by CDK2/cyclin E. These studies provide not only direct evidence for the requirement of CDK2/cyclin E and phosphorylation of NPM/B23 for centrosomes to initiate duplication, but a valuable experimental system for further exploration of the molecular regulation of centrosome duplication in somatic cells of higher animals.
Tarapore
Pheruza
P
Department of Cell Biology, University of Cincinnati College of Medicine, P.O. Box 670521, Ohio 45267-0521, USA.
Okuda
Masaru
M
Fukasawa
Kenji
K
D016475:3T3 Cells; D000818:Animals; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D002502:Centrioles / Q000378:metabolism / Q000502:physiology*; D018385:Centrosome / Q000378:metabolism / Q000502:physiology*; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016193:G1 Phase; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D000090243:Nucleophosmin; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016196:S Phase
D019927:Cyclin E; C000715570:Npm1 protein, mouse; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases