Roughex mediates G1 arrest through a physical association with cyclin A

Differentiation in the developing Drosophila eye requires synchronization of cells in the G(1) phase of the cell cycle. The rougher gene product plays a key role in this synchronization by negatively regulating cyclin A protein levels in G(1). We show here that coexpressed Rougher and cyclin A physically interact in viva, Rougher 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 Rougher, cyclin A becomes strictly nuclear and is later degraded. Nuclear targeting of both Rougher and cyclin A under these conditions is dependent on a C-terminal nuclear localization signal in Rougher. Disruption of this signal results in cytoplasmic localization of both Rougher and cyclin it confirming a physical interaction between these molecules. Cyclin A interacts with both Cdc2 and Cdc2c, the Drosophila Cdk2 homolog, and Rougher 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 Rougher results from competition with the cyclin-dependent kinase subunit for binding to cyclin A. These findings suggest that Rougher can influence the intracellular distribution of cyclin A and define Rougher as a distinct and specialized cell cycle inhibitor for cyclin A-dependent kinase activity.