The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase.

We have studied how the cell cycle-specific oscillations of mitotic B-type cyclins are generated in mouse fibroblasts. A reporter enzyme comprising the N-terminus of a B-type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked the oscillations in the level of the endogenous cyclin B2. These oscillations were largely conserved when the reporter was transcribed constitutively from the SV40 promoter. Pulse-chase experiments or addition of the proteasome inhibitors lactacystin and ALLN showed that cyclin synthesis continued after the end of mitosis. The destruction box-specific degradation of cyclins normally ceases at the onset of S phase, and is active in fibroblasts arrested in G0 and in differentiated C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells. Extracts of G1 cells degraded cyclin B1 whereas p27Kip1 was stable, in contrast, cyclin B1 remained stable and p27Kip1 was degraded in extracts of S phase cells.     

Sub-cellular localisation of GFP-tagged tobacco mitotic cyclins during the cell cycle and after spindle checkpoint activation

We have previously shown that the tobacco cyclin B1;1 protein accumulates during the G2 phase of the cell cycle and is subsequently destroyed during mitosis. Here, we investigated the sub-cellular localisation of two different B1-types and one A3-type cyclin during the cell cycle by using confocal imaging and differential interference contrast (DIC) microscopy. The cyclins were visualised as GFP-tagged fusion proteins in living tobacco cells. Both B1-type cyclins were found in the cytoplasm and in the nucleus during G2 but when cells entered into prophase, both cyclins became associated with condensing chromatin and remained on chromosomes until metaphase. As cells exited metaphase, the B1-type cyclins became degraded, as shown by time-lapse images. A stable variant of cyclin B1;1-GFP fusion protein, in which the destruction box had been mutated, maintained its association with the nuclear material at later phases of mitosis such as anaphase and telophase. Furthermore, we demonstrated that cyclin B1;1 protein is stabilised in metaphase-arrested cells after microtubule destabilising drug treatments. In contrast to the B1-type cyclins, the cyclin A3;1 was found exclusively in the nucleus in interphase cells and disappeared earlier than the cyclin B1 proteins during mitosis.     

The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.

The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.     

Mutagenic analysis of the destruction signal of mitotic cyclins and structural characterization of ubiquitinated intermediates.

Mitotic cyclins are abruptly degraded at the end of mitosis by a cell-cycle-regulated ubiquitin-dependent proteolytic system. To understand how cyclin is recognized for ubiquitin conjugation, we have performed a mutagenic analysis of the destruction signal of mitotic cyclins. We demonstrate that an N-terminal cyclin B segment as short as 27 residues, containing the 9-amino-acid destruction box, is sufficient to destabilize a heterologous protein in mitotic Xenopus extracts. Each of the three highly conserved residues of the cyclin B destruction box is essential for ubiquitination and subsequent degradation. Although an intact destruction box is essential for the degradation of both A- and B-type cyclins, we find that the Xenopus cyclin A1 destruction box cannot functionally substitute for its B-type counterpart, because it does not contain the highly conserved asparagine necessary for cyclin B proteolysis. Physical analysis of ubiquitinated cyclin B intermediates demonstrates that multiple lysine residues function as ubiquitin acceptor sites, and mutagenic studies indicate that no single lysine residue is essential for cyclin B degradation. This study defines the key residues of the destruction box that target cyclin for ubiquitination and suggests there are important differences in the way in which A- and B-type cyclins are recognized by the cyclin ubiquitination machinery.     

Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase

Mitosis is controlled by the specific and timely degradation of key regulatory proteins, notably the mitotic cyclins that bind and activate the cyclin-dependent kinases (Cdks). In animal cells, cyclin A is always degraded before cyclin B, but the exact timing and the mechanism underlying this are not known. Here we use live cell imaging to show that cyclin A begins to be degraded just after nuclear envelope breakdown. This degradation requires the 26S proteasome, but is not affected by the spindle checkpoint. Neither deletion of its destruction box nor disrupting Cdk binding prevents cyclin A proteolysis, but Cdk binding is necessary for degradation at the correct time. We also show that increasing the levels of cyclin A delays chromosome alignment and sister chromatid segregation. This delay depends on the proteolysis of cyclin A and is not caused by a lag in the bipolar attachment of chromosomes to the mitotic spindle, nor is it mediated via the spindle checkpoint. Thus, proteolysis that is not under the control of the spindle checkpoint is required for chromosome alignment and anaphase.     

Both cyclin A delta 60 and B delta 97 are stable and arrest cells in M-phase, but only cyclin B delta 97 turns on cyclin destruction.

Previous work has established that destruction of cyclin B is necessary for exit from mitosis and entry into the next interphase. Sea urchin cyclin B lacking an N-terminal domain is stable, permanently activates cdc2 kinase, resulting in mitotic arrest, and permanently activates the destruction pathway acting on full length cyclin B. Here we have compared the properties of clam cyclins A and B lacking related N-terminal domains. Both cyclin A delta 60 and B delta 97 bind to cdc2 kinase, keep it hyperactivated and block the completion of mitosis. By adding purified delta cyclin proteins to a cell-free system at different cell cycle times, we find that when the cell-free system reaches the cyclin destruction point in the presence of either A delta 60 or B delta 97, the cyclin destruction pathway acting on full length cyclins fails to be turned off. However, the two cyclins differ dramatically in their ability to turn on cyclin destruction. When added to emetine-arrested interphase lysates devoid of endogenous cyclins, only cyclin B delta 97 activates the cyclin destruction system; cyclin A delta 60 does not. This functional difference between the two cyclin types, the first to be described, provides strong support for the idea that the two cyclins have different roles in the cell cycle and suggests that one specialized role of the cyclin B-cdc2 complex is to activate the cyclin destruction pathway and drive cells into interphase of the next cell cycle.     

Cell cycle-regulated degradation of Xenopus cyclin B2 requires binding to p34cdc2.

The protein kinase activity of the cell cycle regulator p34cdc2 is inactivated when the mitotic cyclin to which it is bound is degraded. The amino (N)-terminus of mitotic cyclins includes a conserved "destruction box" sequence that is essential for degradation. Although the N-terminus of sea urchin cyclin B confer cell cycle-regulated degradation to a fusion protein, a truncated protein containing only the N-terminus of Xenopus cyclin B2, including the destruction box, is stable under conditions where full length molecules are degraded. In an attempt to identify regions of cyclin B2, other than the destruction box, involved in degradation, the stability of proteins encoded by C-terminal deletion mutants of cyclin B2 was examined in Xenopus egg extracts. Truncated cyclin with only the first 90 amino acids was stable, but other C-terminal deletions lacking between 14 and 187 amino acids were unstable and were degraded by a mechanism that was neither cell cycle regulated nor dependent upon the destruction box. None of the C-terminal deletion mutants bound p34cdc2. To investigate whether the binding of p34cdc2 is required for cell cycle-regulated degradation, the behavior of proteins encoded by a series of full length Xenopus cyclin B2 cDNA with point mutations in conserved amino acids in the p34cdc2-binding domain was examined. All of the point mutants failed to form stable complexes with p34cdc, and their degradation was markedly reduced compared to wild-type cyclin. Similar results were obtained when the mutant cyclins were synthesized in reticulocyte lysates and when cyclin mRNA was translated directly in a Xenopus egg extract. These results indicate that mutations that interfere with p34cdc2 binding also interfere with cyclin destruction, suggesting that p34cdc2 binding is required for the cell cycle-regulated destruction of Xenopus cyclin B2.     

The KEN box regulates Clb2 proteolysis in G1 and at the metaphase-to-anaphase transition.

Clb2 mitotic cyclin inhibits cell cycle progression by preventing mitotic exit and DNA synthesis. To allow cell cycle progression, Clb2 proteolysis is triggered by Cdc20 during the metaphase-to-anaphase (M-A) transition and by Hct1 during mitotic exit and G1 [1-6]. A cis element called the destruction box is required for this proteolysis [7-11]. Recently, an additional cis element called the "KEN box" was also shown to be required for proteolysis of human CDC20 and Securin [3,12]. Using a novel color assay, we show that a Clb2 KEN box is required to target a fusion protein containing the first 124 amino acids of Clb2 for proteolysis. We further show that full-length Clb2 bearing mutations in the KEN box is degraded efficiently during the M-A transition, but poorly during G1. If the destruction box of Clb2 is mutated in combination with mutation of the KEN box, then this form of Clb2 is more stable than Clb2 bearing either mutation by itself during both M-A and G1. Our results show that the KEN box and the destruction box act together during both M-A and G1 to regulate Clb2 proteolysis.     

Cyclin A-Cys41 does not undergo cell cycle-dependent degradation in Xenopus extracts.

Truncated cyclin A and cyclin B lacking the N-terminal domain comprising the 'destruction box' escape from proteolysis and arrest cells at metaphase. Mutation of a conserved arginine residue of the destruction domain makes cyclin B resistant to proteolysis. Here we show that mutation of the same residue also makes cyclin A resistant to proteolysis, in either of two situations in which the cyclin degradation pathway is turned on: (i) in Xenopus extracts of activated eggs where the degradation pathway has been permanently turned on by adding a recombinant undegradable cyclin B in which the arginine residue of the destruction box has been substituted by alanine; (ii) in extracts of metaphase II-arrested oocytes after Ca(2+)-dependent inactivation of the cytostatic factor (CSF).     

Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3.

While entry into mitosis is triggered by activation of cdc2 kinase, exit from mitosis requires inactivation of this kinase. Inactivation results from proteolytic degradation of the regulatory cyclin subunits during mitosis. At least three different cyclin types, cyclins A, B and B3, associate with cdc2 kinase in higher eukaryotes and are sequentially degraded in mitosis. We show here that mutations in the Drosophila gene fizzy (fzy) block the mitotic degradation of these cyclins. Moreover, expression of mutant cyclins (delta cyclins) lacking the destruction box motif required for mitotic degradation affects mitotic progression at distinct stages. Deltacyclin A results in a delay in metaphase, deltacyclin B in an early anaphase arrest and deltacyclin B3 in a late anaphase arrest, suggesting that mitotic progression beyond metaphase is ordered by the sequential degradation of these different cyclins. Coexpression of deltacyclins A, B and B3 allows a delayed separation of sister chromosomes, but interferes wit chromosome segregation to the poles. Mutations in fzy block both sister chromosome separation and segregation, indicating that fzy plays a crucial role in the metaphase/anaphase transition.     

Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1

Progress through mitosis is controlled by the sequential destruction of key regulators including the mitotic cyclins and securin, an inhibitor of anaphase whose destruction is required for sister chromatid separation. Here we have used live cell imaging to determine the exact time when human securin is degraded in mitosis. We show that the timing of securin destruction is set by the spindle checkpoint; securin destruction begins at metaphase once the checkpoint is satisfied. Furthermore, reimposing the checkpoint rapidly inactivates securin destruction. Thus, securin and cyclin B1 destruction have very similar properties. Moreover, we find that both cyclin B1 and securin have to be degraded before sister chromatids can separate. A mutant form of securin that lacks its destruction box (D-box) is still degraded in mitosis, but now this is in anaphase. This destruction requires a KEN box in the NH2 terminus of securin and may indicate the time in mitosis when ubiquitination switches from APCCdc20 to APCCdh1. Lastly, a D-box mutant of securin that cannot be degraded in metaphase inhibits sister chromatid separation, generating a cut phenotype where one cell can inherit both copies of the genome. Thus, defects in securin destruction alter chromosome segregation and may be relevant to the development of aneuploidy in cancer.     

A complex degradation signal in Cyclin A required for G1 arrest, and a C-terminal region for mitosis

The destruction box (D-box) consensus sequence has been defined as a motif mediating polyubiquitylation and proteolysis of B-type cyclins during mitosis. We show here that the regions with similarity to D-boxes are not required for mitotic degradation of Drosophila Cyclin A. Instead of a simple D-box, a complex N-terminal degradation signal is present in this cyclin. Mutations that impair or abolish mitotic Cyclin A destruction delay progression through metaphase, but only when overexpressed. Moreover, these mutations prevent epidermal cells from entering the first G1 phase of embryogenesis and lead to a complete extra division cycle instead of a timely cell proliferation arrest. Residual Cyclin A activity after mitosis, therefore, has S phase-promoting activity. In principle, an S phase defect could also explain why epidermal cells fail to enter mitosis 16 in mutants lacking zygotic Cyclin A function. However, we demonstrate that this failure of mitosis is not caused simply by DNA replication or damage checkpoints. Entry into mitosis requires a function of Cyclin A that does not depend on the presence of the N-terminal region.     

A process independent of the anaphase-promoting complex contributes to instability of the yeast S phase cyclin Clb5

Proteolytic destruction of many cyclins is induced by a multi-subunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C). In the budding yeast Saccharomyces cerevisiae, the S phase cyclin Clb5 and the mitotic cyclins Clb1-4 are known as substrates of this complex. The relevance of APC/C in proteolysis of Clb5 is still under debate. Importantly, a deletion of the Clb5 destruction box has little influence on cell cycle progression. To understand Clb5 degradation in more detail, we applied in vivo pulse labeling to determine the half-life of Clb5 at different cell cycle stages and in the presence or absence of APC/C activity. Clb5 is significantly unstable, with a half-life of approximately 8-10 min, at cell cycle periods when APC/C is inactive and in mutants impaired in APC/C function. A Clb5 version lacking its cyclin destruction box is similarly unstable. The half-life of Clb5 is further decreased in a destruction box-dependent manner to 3-5 min in mitotic or G(1) cells with active APC/C. Clb5 instability is highly dependent on the function of the proteasome. We conclude that Clb5 proteolysis involves two different modes for targeting of Clb5 to the proteasome, an APC/C-dependent and an APC/C-independent mechanism. These different modes apparently have overlapping functions in restricting Clb5 levels in a normal cell cycle, but APC/C function is essential in the presence of abnormally high Clb5 levels.     

Destruction of Xenopus cyclins A and B2, but not B1, requires binding to p34cdc2.

The specific and rapid destruction of cyclins A and B during mitosis is their most remarkable property. A short peptide motif of approximately 10 amino acids near the N-terminus, known as the destruction box, is absolutely required for programmed proteolysis. In this paper we show that although the destruction box is necessary for the degradation of cyclin A, it is not sufficient. Mutant versions of cyclin A that cannot form complexes with p34cdc2 are stable, which we interpret to mean that this cyclin must bind to p34cdc2 in order to undergo programmed proteolysis. Thus, N-terminal fragments of cyclin A containing little more than the destruction box and its surroundings are indestructible. p34cdc2 binding also appears to be required for the destruction of cyclin B2. In contrast, cyclin B1 does not require p34cdc2 binding for specific proteolysis. The systems for the proteolysis of cyclins A, B1 and B2 thus appear to show important differences in the way they recognize their substrates.     

Fission yeast Cut2 required for anaphase has two destruction boxes.

The fission yeast Schizosaccharomyces pombe cut2(+) gene is essential for sister chromatid separation. Cut2 protein, which locates in the interphase nucleus and along the metaphase spindle, disappears in anaphase with the same timing as mitotic cyclin destruction. This proteolysis depends on the APC (Anaphase-Promoting Complex)-cyclosome which contains ubiquitin ligase activity. The N-terminus of Cut2 contains two stretches similar to the mitotic cyclin destruction box. We show that both sequences (33RAPLGSTKQ and 52RTVLGGKST) serve as destruction boxes and are required for in vitro polyubiquitination and proteolysis. Cut2 with doubly mutated destruction boxes inhibits anaphase, whereas Cut2 with singly mutated boxes can suppress cut2 mutations. Strong expression of the N-terminal 73 residues containing the destruction boxes leads to the accumulation of endogenous cyclin and Cut2, and arrests cells in metaphase, whereas the same fragment with the mutated boxes does not. Cut2 proteolysis occurs in vitro using Xenopus mitotic extracts in the presence of functional destruction boxes. Furthermore, Cut2 is polyubiquitinated in an in vitro system using HeLa extracts, and this polyubiquitination requires the destruction boxes.     

Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis

CENP-E is a kinesin-like protein that binds to kinetochores through the early stages of mitosis, but after initiation of anaphase, it relocalizes to the overlapping microtubules in the midzone, ultimately concentration in the developing midbody. By immunoblotting of cells separated at various positions in the cell cycle using centrifugal elutriation, we show that CENP-E levels increase progressively across the cycle peaking at approximately 22,000 molecules/cell early in mitosis, followed by an abrupt (> 10 fold) loss at the end of mitosis. Pulse-labeling with [35S]methionine reveals that beyond a twofold increase in synthesis between G1 and G2, interphase accumulation results primarily from stabilization of CENP-E during S and G2. Despite localizing in the midbody during normal cell division, CENP-E loss at the end of mitosis is independent of cytokinesis, since complete blockage of division with cytochalasin has no affect on CENP-E loss at the M/G1 transition. Thus, like mitotic cyclins, CENP-E accumulation peaks before cell division, and it is specifically degraded at the end of mitosis. However, CENP-E degradation kinetically follows proteolysis of cyclin B in anaphase. Combined with cyclin A destruction before the end of metaphase, degradation of as yet unidentified components at the metaphase/anaphase transition, and cyclin B degradation at or after the anaphase transition, CENP-E destruction defines a fourth point in a mitotic cascade of timed proteolysis.     

The destruction box of the cyclin Clb2 binds the anaphase-promoting complex/cyclosome subunit Cdc23

Properly regulated cyclin proteolysis is critical for normal cell cycle progression. A nine-amino acid peptide motif called the destruction box (D box) is present at the N terminus of the yeast mitotic cyclins. This short sequence is required for cyclin ubiquitination and subsequent proteolysis. The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 required for cyclin ubiquitination. We have tested the D box of five mitotic cyclins for interaction with six APC/C subunits. The APC/C subunit Cdc23, but not five other subunits tested, interacted by two-hybrid analysis with the N terminus of wild-type Clb2. None of these subunits interacted with the N termini of the cyclins Clb1, Clb3, or Clb5. Mutations in the D box sequences of Clb2 inhibited interaction with Cdc23 both in vivo and in vitro. Our results provide the first evidence for a direct interaction between an APC/C substrate (Clb2) and an APC/C subunit (Cdc23).     

Cyclin F is degraded during G2-M by mechanisms fundamentally different from other cyclins

Cyclin F, a cyclin that can form SCF complexes and bind to cyclin B, oscillates in the cell cycle with a pattern similar to cyclin A and cyclin B. Ectopic expression of cyclin F arrests the cell cycle in G(2)/M. How the level of cyclin F is regulated during the cell cycle is completely obscure. Here we show that, similar to cyclin A, cyclin F is degraded when the spindle assembly checkpoint is activated and accumulates when the DNA damage checkpoint is activated. Cyclin F is a very unstable protein throughout much of the cell cycle. Unlike other cyclins, degradation of cyclin F is independent of ubiquitination and proteasome-mediated pathways. Interestingly, proteolysis of cyclin F is likely to involve metalloproteases. Rapid destruction of cyclin F does not require the N-terminal F-box motif but requires the COOH-terminal PEST sequences. The PEST region alone is sufficient to interfere with the degradation of cyclin F and confer instability when fused to cyclin A. These data show that although cyclin F is degraded at similar time as the mitotic cyclins, the underlying mechanisms are entirely distinct.     

The requirements for protein synthesis and degradation, and the control of destruction of cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo

Fertilization of clam oocytes initiates a series of cell divisions, of which the first three--meiosis I, meiosis II, and the first mitotic division--are highly synchronous. After fertilization, protein synthesis is required for the successful completion of every division except meiosis I. When protein synthesis is inhibited, entry into meiosis I and the maintenance of M phase for the normal duration of meiosis occur normally, but the chromosomes fail to interact correctly with the spindle in meiosis II metaphase. By contrast, inhibition of protein synthesis immediately after completion of meiosis or mitosis stops cells entering the next mitosis. We describe the behavior of cyclins A and B in relation to these "points of no return." The cyclins are synthesized continuously and are rapidly destroyed shortly before the metaphase-anaphase transition of the mitotic cell cycles, with cyclin A being degraded in advance of cyclin B. Cyclin destruction normally occurs during a 5-min window in mitosis, but in the monopolar mitosis that occurs after parthenogenetic activation of clam oocytes, or when colchicine is added to fertilized eggs about to enter first mitosis, the destruction of cyclin B is strongly delayed, whereas proteolysis of cyclin A is maintained in an activated state for the duration of metaphase arrest. Under either of these abnormal conditions, inhibition of protein synthesis causes a premature return to interphase that correlates with the time when cyclin B disappears.