Roughex mediates G(1) arrest through a physical association with cyclin A

Differentiation in the developing Drosophila eye requires synchronization o f 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 prote in levels in G(1). We show here that coexpressed Rougher and cyclin A physi cally interact in viva, Rougher is a nuclear protein, while cyclin A was pr eviously shown to be exclusively cytoplasmic during interphase in the embry o. 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 pre sence of ectopic Rougher, cyclin A becomes strictly nuclear and is later de graded. Nuclear targeting of both Rougher and cyclin A under these conditio ns is dependent on a C-terminal nuclear localization signal in Rougher. Dis ruption of this signal results in cytoplasmic localization of both Rougher and cyclin it confirming a physical interaction between these molecules. Cy clin A interacts with both Cdc2 and Cdc2c, the Drosophila Cdk2 homolog, and Rougher inhibits the histone H1 kinase activities of both cyclin A-Cdc2 an d cyclin A-Cdc2c complexes in whole-cell extracts. Two-hybrid experiments s uggested that the inhibition of kinase activity by Rougher results from com petition with the cyclin-dependent kinase subunit for binding to cyclin A. These findings suggest that Rougher can influence the intracellular distrib ution of cyclin A and define Rougher as a distinct and specialized cell cyc le inhibitor for cyclin A-dependent kinase activity.