Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells (original) (raw)
- Jeffrey D. Singer,
- Mark Gurian-West,
- Bruce Clurman, and
- James M. Roberts
- Division of Basic Sciences, Howard Hughes Medical Institute, and Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 USA
Abstract
Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome- dependent pathway. Two factors stimulate cyclin E ubiquitination in vivo: when it is free of its CDK partner, and when it is phosphorylated on threonine 380. We pursued the first of these pathways by using a two-hybrid screen to identify proteins that could bind only to free cyclin E. This resulted in the isolation of human Cul-3, a member of the cullin family of E3 ubiquitin–protein ligases. We found that Cul-3 was bound to cyclin E but not to cyclin E-Cdk2 complexes in mammalian cells, and that overexpression of Cul-3 increased ubiquitination of cyclin E but not other cyclins. Conversely, deletion of the Cul-3 gene in mice caused increased accumulation of cyclin E protein, and had cell-type-specific effects on S-phase regulation. In the extraembryonic ectoderm, in which cells undergo a standard mitotic cycle, there was a greatly increased number of cells in S phase. In the trophectoderm, in which cells go through endocycles, there was a block to entry into S phase. The SCF pathway, which targets cyclins for ubiquitination on the basis of their phosphorylation state, and the Cul-3 pathway, which selects cyclin E for ubiquitination on the basis of its assembly into CDK complexes, may be complementary ways to control cyclin abundance.
Footnotes
↵Corresponding author.
E-MAIL jroberts{at}fred.fhcrc.org; FAX (206) 667-6877.
- Received July 8, 1999.
- Accepted July 30, 1999.
Cold Spring Harbor Laboratory Press