Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells

Cyclin E is an unstable protein that is degraded in a ubiquitin- and protea some- dependent pathway. Two factors stimulate cyclin E ubiquitination in v ivo: when it is free of its CDK partner, and when it is phosphorylated on t hreonine 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 res ulted 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 no t to cyclin E-Cdk2 complexes in mammalian cells, and that overexpression of Cul-3 increased ubiquitination of cyclin E but not other cyclins. Converse ly, deletion of the Cul-3 gene in mice caused increased accumulation of cyc lin E protein, and had cell-type-specific effects on S-phase regulation. In the extraembryonic ectoderm, in which cells undergo a standard mitotic cyc le, there was a greatly increased number of cells in S phase. In the trophe ctoderm, in which cells go through endocycles, there was a block to entry i nto S phase. The SCF pathway, which targets cyclins for ubiquitination on t he basis of their phosphorylation state, and the Cul-3 pathway, which selec ts cyclin E for ubiquitination on the basis of its assembly into CDK comple xes, may be complementary ways to control cyclin abundance.