The Regulation of Human Cyclin B Protein Levels by the Ubiquitin Proteolytic Pathway

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 identity 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, where cells undergo a standard mitotic cycle, there was a greatly increased number of cells in S phase. In the trophectoderm, where cells go through endocycles, there was a block to entry into S phase.