PMID:11027291

Citation	Avedisov, SN, Krasnoselskaya, I, Mortin, M and Thomas, BJ (2000) Roughex mediates G(1) arrest through a physical association with cyclin A. Mol. Cell. Biol. 20:8220-9
Abstract	Differentiation in the developing Drosophila eye requires synchronization of cells in the G(1) phase of the cell cycle. The roughex gene product plays a key role in this synchronization by negatively regulating cyclin A protein levels in G(1). We show here that coexpressed Roughex and cyclin A physically interact in vivo. Roughex is a nuclear protein, while cyclin A was previously shown to be exclusively cytoplasmic during interphase in the embryo. In contrast, we demonstrate that in interphase cells in the eye imaginal disk cyclin A is present in both the nucleus and the cytoplasm. In the presence of ectopic Roughex, cyclin A becomes strictly nuclear and is later degraded. Nuclear targeting of both Roughex and cyclin A under these conditions is dependent on a C-terminal nuclear localization signal in Roughex. Disruption of this signal results in cytoplasmic localization of both Roughex and cyclin A, confirming a physical interaction between these molecules. Cyclin A interacts with both Cdc2 and Cdc2c, the Drosophila Cdk2 homolog, and Roughex inhibits the histone H1 kinase activities of both cyclin A-Cdc2 and cyclin A-Cdc2c complexes in whole-cell extracts. Two-hybrid experiments suggested that the inhibition of kinase activity by Roughex results from competition with the cyclin-dependent kinase subunit for binding to cyclin A. These findings suggest that Roughex can influence the intracellular distribution of cyclin A and define Roughex as a distinct and specialized cell cycle inhibitor for cyclin A-dependent kinase activity.
Links	PubMed PMC86431
Keywords	Animals; Blotting, Western; CDC2 Protein Kinase/metabolism; Cell Nucleus/metabolism; Cells, Cultured; Conserved Sequence; Cyclin A/metabolism; Cytoplasm/metabolism; Dose-Response Relationship, Drug; Drosophila/genetics; Drosophila/metabolism; Drosophila Proteins; Eye Proteins/genetics; Eye Proteins/metabolism; Eye Proteins/physiology; G1 Phase; Gene Deletion; Immunohistochemistry; Luciferases/metabolism; Microscopy, Confocal; Phosphorylation; Photoreceptor Cells, Invertebrate/embryology; Plasmids/metabolism; Point Mutation; Precipitin Tests; Protein Binding; Protein Kinases/metabolism; Transfection; Two-Hybrid System Techniques
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