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 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
JD
Division of Basic Sciences, Howard Hughes Medical Institute, Seattle, Washington 98109 USA.
Gurian-West
M
M
Clurman
B
B
Roberts
J M
JM
D016475:3T3 Cells; D000818:Animals; D001755:Blastocyst / Q000166:cytology; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000502:physiology*; D003001:Cloning, Molecular; D044842:Cullin Proteins; D019927:Cyclin E / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000033:anatomy & histology; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008957:Models, Genetic; D000075687:NEDD8 Protein; D010641:Phenotype; D011485:Protein Binding; D016196:S Phase / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D015398:Signal Transduction; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism*
C503645:CUL3 protein, human; D018797:Cell Cycle Proteins; C503646:Cul3 protein, mouse; D044842:Cullin Proteins; D019927:Cyclin E; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C513172:Nedd8 protein, mouse; D014452:Ubiquitins