Roughex mediates G(1) 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 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.
Avedisov
S N
SN
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Krasnoselskaya
I
I
Mortin
M
M
Thomas
B J
BJ
D000818:Animals; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D017124:Conserved Sequence; D019925:Cyclin A / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D016193:G1 Phase; D017353:Gene Deletion; D007150:Immunohistochemistry; D008156:Luciferases / Q000378:metabolism; D018613:Microscopy, Confocal; D010766:Phosphorylation; D017956:Photoreceptor Cells, Invertebrate / Q000196:embryology; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques
D019925:Cyclin A; D029721:Drosophila Proteins; D005136:Eye Proteins; C088084:rux protein, Drosophila; D008156:Luciferases; D011494:Protein Kinases; C102333:histone H1 kinase; D016203:CDC2 Protein Kinase