This work was supported in part by a grant from Mitotix Incorporated, and by grants from the National Institutes of Health to J.M.R. J.D.S. is supported by a fellowship from the Department of Defense, grant no. DADM17-98-1-8085. J.M.R. is an investigator of the Howard Hughes Medical Institute.
Genes & Development
Ubiquitin, Proteins -- Metabolism, Proteomics, Molecular biology, Mitosis
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.
Singer, J. D., Gurian-West, M., Clurman, B., & Roberts, J. M. (1999). Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells. Genes & development, 13(18), 2375-2387.