Human Cyclin E, a Nuclear Protein Essential for the G₁-to-S Phase Transition

Abstract

Cyclin E was first identified by screening human cDNA libraries for genes that would complement G 1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G 1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G 1 /S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G 1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G 1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G 1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G 0 . Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G 1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G 1 inhibited entry into S phase, whereas microinjection performed near the G 1 /S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G 1 /S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle.Cyclins and their catalytic subunits, the cyclin-dependent kinases (Cdks), play key roles in the regulation of eukaryotic cell cycle events (see references 10 and 82 for reviews). Cyclins are believed to determine the subcellular localization, substrate specificity, interaction with upstream regulatory proteins, and timing of activation of Cdks.Cell cycle control mechanisms have been highly conserved throughout evolution (see reference 59 for a review). Cyclins were originally discovered in marine invertebrates. In clam and sea urchin embryos, A-and B-type cyclins were found to accumulate to high levels during interphase before they were abruptly degraded during mitosis (17). Control of the cell cycle by these cyclins was demonstrated when injection of clam cyclin A mRNA was able to advance G 2 -arrested Xenopus oocytes into meiotic metaphase (89). Furthermore, after destruction of all mRNA in Xenopus egg extracts, readdition of sea urchin cyclin B message was sufficient to drive the extract into mitosis (56).In mammalian cells, several cyclins have been identified and classified according to their sequence simil...

K-ras modulates the cell cycle via both positive and negative regulatory pathways

“…Therefore, the e ect of activated K-ras on expression of both cyclin A and cyclin E (a G 1 cyclin; Ohtsubo et al, 1995), was studied. Western blotting analysis using a speci®c antibody against cyclin A (Figure 7a) indicated that induction of activated K-ras by Tet withdrawal in M2TK4 cells led to a signi®cant increase in cyclin A protein when cells were grown in media containing 10% serum (with or without Tet) (lanes 1 and 2).…”

BPV-4 E8 transforms NIH3T3 cells, up-regulates cyclin A and cyclin A-associated kinase activity and de-regulates expression of the cdk inhibitor p27Kip1

“…D-type cyclins are required for progression through early/mid G1 phase of the cell cycle and in the decision to embark on a new cell cycle or to enter a quiescent state (GO) after mitosis, linking cell exposure to external cues to entry into the cell cycle (Won et al, 1992;Baldin et al, 1993). Cyclin E is expressed in late G1 and is required for entry into Sphase in mammalian ®broblasts (Resnitzky et al, 1994;Ohtsubo et al, 1995), while Cyclin A is ®rst expressed at the G1/S transition (Henglein et al, 1994) and is required, in complex with cdk2, for the completion of S-phase and, in complex with cdc2, for passage through G2 and mitosis (Girard et al, 1991;Pagano et al, 1992).…”

Malignant transformation by cyclin E and Ha-Ras correlates with lower sensitivity towards induction of cell death but requires functional Myc and CDK4

“…Cyclin E is associated with CDK2 and this complex appears to be necessary for the onset of DNA replication. In this regard, cyclin E is thought to function downstream of cyclin D1 and is rate limiting for G1/S transition (Ko et al, 1992;Dulic et al, 1992;Pagano et al, 1993;Ohtsubo et al, 1995). A considerable number of experimental data suggests that the tumour suppressor protein Rb is one of the substrates of the cyclin D1/CDK4 and the cyclin E/ CDK2 kinase (Dowdy et al, 1993;Ewen et al, 1993;Hinds et al, 1994;Kato and Sherr, 1993;Matsushime et al, 1994;Meyerson and Harlow, 1994;Hu et al, 1992;Sherr, 1995).…”