Human cyclin F.

Cyclins are important regulators of cell cycle transitions through their ability to bind and activate cyclin-dependent protein kinases. In mammals several classes of cyclins exist which are thought to co-ordinate the timing of different events necessary for cell cycle progression. Here we describe the identification of a novel human cyclin, cyclin F, isolated as a suppressor of the G1/S deficiency of a Saccharomyces cerevisiae cdc4 mutant. Cyclin F is the largest cyclin, with a molecular weight of 87 kDa, and migrates as a 100-110 kDa protein. It contains an extensive PEST-rich C-terminus and a cyclin box region that is most closely related to cyclins A and B. Cyclin F mRNA is ubiquitiously expressed in human tissues. It fluctuates dramatically through the cell cycle, peaking in G2 like cyclin A and decreasing prior to decline of cyclin B mRNA. Cyclin F protein accumulates in interphase and is destroyed at mitosis at a time distinct from cyclin B. Cyclin F shows regulated subcellular localization, being localized in the nucleus in most cells, with a significant percentage of cells displaying only perinuclear staining. Overexpression of cyclin F, or a mutant lacking the PEST region, in human cells resulted in a significant increase in the G2 population, implicating cyclin F in the regulation of cell cycle transitions. The ubiquitous expression and phylogentic conservation of cyclin F suggests that it is likely to coordinate essential cell cycle events distinct from those regulated by other cyclins.     

Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.     

Cyclin I: A New Cyclin Encoded by a Gene Isolated from Human Brain

A new member of the cyclin family has been isolated from an equalized cDNA library derived from human forebrain cortex. This putative cyclin, designated cyclin I, contains a typical cyclin box near the N-terminus and a PEST sequence near the C-terminus. Cyclin I shows the highest sequence similarity in the cyclin box to cyclins G and E, while the similarity between cyclins I and G also extends toward the C-terminus from the cyclin box. Cyclin I mRNA was expressed at high levels in postmitotic tissues, including skeletal muscle, heart, and brain, and was expressed constantly during cell cycle progression. The expression of cyclin I mRNA does not correlate directly to the cell cycle, and therefore cyclin I may be a novel cyclin member that functions independently of the cell cycle control.     

Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the Golgi apparatus.

We have raised and characterized antibodies specific for human cyclin B2 and have compared the properties of cyclins B1 and B2 in human tissue culture cells. Cyclin B1 and B2 levels are very low in G1 phase, increase in S and G2 phases and peak at mitosis. Both B-type cyclins associate with p34cdc2; their associated kinase activities appear when cells enter mitosis and disappear as the cyclins are destroyed in anaphase. However, human cyclins B1 and B2 differ dramatically in their subcellular localization. Cyclin B1 co-localizes with microtubules, whereas cyclin B2 is primarily associated with the Golgi region. In contrast to cyclin B1, cyclin B2 does not relocate to the nucleus at prophase, but becomes uniformly distributed throughout the cell. The different subcellular locations of human cyclins B1 and B2 implicate them in the reorganization of different aspects of the cellular architecture at mitosis and indicate that different mitotic cyclin-cyclin-dependent kinase complexes may have distinct roles in the cell cycle.     

Cyclin A and B functions in the early Drosophila embryo.

In eukaryotes, mitotic cyclins localize differently in the cell and regulate different aspects of the cell cycle. We investigated the relationship between subcellular localization of cyclins A and B and their functions in syncytial preblastoderm Drosophila embryos. During early embryonic cycles, cyclin A was always concentrated in the nucleus and present at a low level in the cytoplasm. Cyclin B was predominantly cytoplasmic, and localized within nuclei only during late prophase. Also, cyclin B colocalized with metaphase but not anaphase spindle microtubules. We changed maternal gene doses of cyclins A and B to test their functions in preblastoderm embryos. We observed that increasing doses of cyclin B increased cyclin B-Cdk1 activity, which correlated with shorter microtubules and slower microtubule-dependent nuclear movements. This provides in vivo evidence that cyclin B-Cdk1 regulates microtubule dynamics. In addition, the overall duration of the early nuclear cycles was affected by cyclin A but not cyclin B levels. Taken together, our observations support the hypothesis that cyclin B regulates cytoskeletal changes while cyclin A regulates the nuclear cycles. Varying the relative levels of cyclins A and B uncoupled the cytoskeletal and nuclear events, so we speculate that a balance of cyclins is necessary for proper coordination during these embryonic cycles.     

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.     

Cyclin B and Cyclin A Confer Different Substrate Recognition Properties on CDK2

The transitions of the cell cycle are regulated by the cyclin dependent protein kinases(CDKs). The cyclins activate their respective CDKs and confer substrate recognitionproperties. We report the structure of phospho-CDK2/cyclin B and show that cyclin Bconfers M phase-like properties on CDK2, the kinase that is usually associated with S phase.Cyclin B produces an almost identical activated conformation of CDK2 as that produced bycyclin A. There are differences between cyclin A and cyclin B at the recruitment site, whichin cyclin A is used to recruit substrates containing an RXL motif. Because of sequencedifferences this site in cyclin B binds RXL motifs more weakly than in cyclin A. Despitesimilarity in kinase structures, phospho-CDK2/cyclin B phosphorylates substrates, such asnuclear lamin and a model peptide derived from p107, at sequences SPXX that differ fromthe canonical CDK2/cyclin A substrate recognition motif, SPXK. CDK2/cyclin Bphosphorylation at these non-canonical sites is not dependent on the presence of a RXLrecruitment motif. The p107 peptide contained two SP motifs each followed by a noncanonicalsequence of which only one site (Ser640) is phosphorylated by pCDK2/cyclin Awhile two sites are phosphorylated by pCDK2/cyclin B. The second site is too close to theRXL motif to allow the cyclin A recruitment site to be effective, as previous work has shownthat there must be at least 16 residues between the catalytic site serine and the RXL motif.Thus the cyclins A and B in addition to their role in promoting the activatory conformationalswitch in CDK2, also provide differential substrate specificity.     

The A- and B-type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that define separable phases of the G2-M transition.

We show that the sequence of Drosophila cyclin B has greater identity with B-type cyclins from other animal phyla than with Drosophila cyclin A, suggesting that the two cyclins have distinct roles that have been maintained in evolution. Cyclin A is not detectable in unfertilized eggs and is present at low levels prior to cellularization of the syncytial embryo. In contrast, the levels of cyclin B remain uniformly high throughout these developmental stages. In cells within cellularized embryos and the larval brain, cyclin A accumulates to peak levels in prophase and is degraded throughout the period in which chromosomes are becoming aligned on the metaphase plate. The degradation of cyclin B, on the other hand, does not occur until the metaphase-anaphase transition. In cells arrested at c-metaphase by treating with microtubule destabilizing drugs to prevent spindle formation, cyclin A has been degraded in the arrested cells, whereas cyclin B is maintained at high levels. These observations suggest that cyclin A has a role in the G2-M transition that is independent of spindle formation, and that entry into anaphase is a key requirement for the degradation of cyclin B.     

A kinetic model of the cyclin E/Cdk2 developmental timer in Xenopus laevis embryos

Early cell cycles of Xenopus laevis embryos are characterized by rapid oscillations in the activity of two cyclin-dependent kinases. Cdk1 activity peaks at mitosis, driven by periodic degradation of cyclins A and B. In contrast, Cdk2 activity oscillates twice per cell cycle, despite a constant level of its partner, cyclin E. Cyclin E degrades at a fixed time after fertilization, normally corresponding to the midblastula transition. Based on published data and new experiments, we constructed a mathematical model in which: (1) oscillations in Cdk2 activity depend upon changes in phosphorylation, (2) Cdk2 participates in a negative feedback loop with the inhibitory kinase Wee1; (3) cyclin E is cooperatively removed from the oscillatory system; and (4) removed cyclin E is degraded by a pathway activated by cyclin E/Cdk2 itself. The model's predictions about embryos injected with Xic1, a stoichiometric inhibitor of cyclin E/Cdk2, were experimentally validated.     

Mitotic cyclin distribution during maize cell division: Implications for the sequence diversity and function of cyclins in plants

Summary Cyclin proteins are components of the regulatory system that controls the orderly progression of the events of cell division. Their sub-cellular location, as well as their fluctuating abundance and their affinities for the cyclin-dependent kinases (CDKs) to which they bind, determine their successive roles during the cell cycle. Here we employ species-specific antibodies to monitor changes in quantity and location of four maize cyclins and maize Cdc2-kinase in dividing maize root tip cells. Maize cyclin Ia occurs in the nuclear matrix and is released when the nuclear envelope breaks down. In contrast, cyclin Ib is cytoplasmic until prophase; it associates transiently with the nuclear envelope and preprophase band (PPB) just before these structures break down and then associates with the condensed chromosomes and spindle region before declining at anaphase. Cyclin II and Cdc2 also occur in the PPB. Occurrence of cyclin Ib and Cdc2 at the PPB concurrent with initiation of breakdown is consistent with previous studies in which microinjection of cyclin-dependent protein kinase indicated that removal of the PPB at the time of nuclear-envelope breakdown is catalysed by a CDK. While cyclins Ia and III are predominantly nuclear prior to mitosis, cyclins Ib and II are predominantly cytoplasmic until prophase then become nuclear. The initial cytoplasmic retention of cyclins Ib and II correlates with their possession of a sequence similar to the cytoplasmic-retention signal of animal cyclin B1. Cyclin II binds to all microtubule arrays during the cell cycle, becoming markedly concentrated in the phragmoplast, and cyclin III associates with the spindle and then the phragmoplast. Cdc2 also occurs in the phragmoplast. Persistence of mitotic cyclins and CDK after mitosis into the cytokinetic stage, as seen in maize, is not paralleled in animal cells, where the cytokinetic mid-body is not so labelled, presumably reflecting the key role of the phragmoplast apparatus in plant cell division.Abbreviations CDK cyclin-dependent kinase - CRS cytoplasmicretention signal - NE nuclear envelope - NEB nuclear-envelope breakdown - NLS nuclear-location signal - PPB preprophase band - FITC fluorescein isothiocyanate - TRITC tetramethylrhodamine isothiocyanate     

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.     

Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.

M-phase-promoting factor (MPF), a complex of cdc2 and a B-type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. We show here that cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)-dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES-dependent transport, resulted in nuclear accumulation of cyclin B1 in G2 phase. Disruption of an NES which has been identified in cyclin B1 here abolished the nuclear export of this protein, and consequently the NES-disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Moreover, we show that expression of the NES-disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage-induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage-induced G2 checkpoint.     

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Cyclin B is a regulatory subunit of CDK1 within MPF complex. Degradation of cyclin B via ubiquitin-proteasome pathway seemed to be absolutely required for the M-phase exit. However, inhibition of the proteasome proteolytic activity upon the exit from the meiotic metaphase II-arrest in Xenopus cell-free extract revealed that the proteasome-dependent dissociation of cyclin B from CDK1 is sufficient to inactivate MPF without cyclin B degradation. In this study we analyse whether the same mechanism operates during the exit from mitotic M-phase. We show in Xenopus cell-free extract undergoing the first or the second embryonic mitosis that CDK1 oscillations are not affected by proteasome inhibition with MG132 or ALLN despite effective inhibition of cyclins B degradation. The majority of cyclins B1 and B2 surviving CDK1 inactivation is CDK-free and cyclin B2 becomes resistant to phosphatase ? dephosphorylation. The pool of cyclins B remaining after CDK1 inactivation in the presence of MG132 is mitotically inert, while exogenous or newly synthesised cyclin B activates CDK1. This suggests that cyclins B remain sequestered within the proteasome upon MPF inactivation in the presence of MG132. Comparison of the dynamics of the decline of total and CDK-bound pools of cyclins B1, B2 and B4 upon mitotic exit in absence of protein synthesis reveals that CDK-bound cyclins B diminish clearly faster. Our results thus show that cyclin B dissociation from CDK1 precedes cyclins B degradation upon CDK1 inactivation in mitotic embryo extracts and that proteasome proteolytic activity is dispensable for both activation and inactivation of CDK1 in such extracts.     

Structural basis for the ORC1‐Cyclin A association

Progression of cell cycle is regulated by sequential expression of cyclins, which associate with distinct cyclin kinases to drive the transition between different cell cycle phases. The complex of Cyclin A with cyclin‐dependent kinase 2 (CDK2) controls the DNA replication activity through phosphorylation of a set of chromatin factors, which critically influences the S phase transition. It has been shown that the direct interaction between the Cyclin A‐CDK2 complex and origin recognition complex subunit 1 (ORC1) mediates the localization of ORC1 to centrosomes, where ORC1 inhibits cyclin E‐mediated centrosome reduplication. However, the molecular basis underlying the specific recognition between ORC1 and cyclins remains elusive. Here we report the crystal structure of Cyclin A‐CDK2 complex bound to a peptide derived from ORC1 at 2.54 å resolution. The structure revealed that the ORC1 peptide interacts with a hydrophobic groove, termed cyclin binding groove (CBG), of Cyclin A via a KXL motif. Distinct from other identified CBG‐binding sequences, an arginine residue flanking the KXL motif of ORC1 inserts into a neighboring acidic pocket, contributing to the strong ORC1‐Cyclin A association. Furthermore, structural and sequence analysis of cyclins reveals divergence on the ORC1‐binding sites, which may underpin their differential ORC1‐binding activities. This study provides a structural basis of the specific ORC1‐cyclins recognition, with implication in development of novel inhibitors against the cyclin/CDK complexes.     

Cyclin gene expression and growth control in normal and neoplastic human breast epithelium

Recent advances in defining the molecular mechanisms of cell cycle control in eukaryotes provide a basis for beter understanding the hormonal control of cell proliferation in normal and neoplastic breast epithelium. It is now clear that a number of critical steps in cell cycle progression are controlled by families of serine/threonine kinases, the cdks. These kinases are activated by interactions with various cyclin gene products which form the regulatory subunits of the kinase complexes. Several families of cyclins control cell cycle progression in G₁ phase, cyclins C, D and E, or in S, G₂ and mitosis, cyclins A and B. Recent studies have defined the expression and regulation of cyclin genes in normal breast epithelial cells and in breast cancer cell lines. Following growth arrest of T-47D breast cancer cells by serum deprivation restimulation with insulin results in sequential induction of cyclin genes. Cyclin D1 mRNA increases within 1 h of mitogenic stimulation and is followed by increased expression of cyclins D3 and E in G₁ phase, cyclin A in late G₁/early S phase and cyclin B1 in G₂. Similar results were observed following epidermal growth factor stimulation of normal breast epithelial cells. Other hormones—oestrogens and progestins—and growth factors—insulin-like investigated for their effects on G₁ cyclin gene expression. In all cases there was an excellent correlation between the induction of cyclin D1 mRNA and subsequent entry into S phase. Furthermore, growth inhibition by antioestrogens and concurrent G₁ arrest were preceded by an acute decrease in cyclin D1 gene expression. These observations suggest a likely role for cyclin D1 in mediating many of the known hormonal effects on cell proliferation in breast epithelial cells.     

The Kinetics of G2 and M Transitions Regulated by B Cyclins

B cyclins regulate G2-M transition. Because human somatic cells continue to cycle after reduction of cyclin B1 (cycB1) or cyclin B2 (cycB2) by RNA interference (RNAi), and because cycB2 knockout mice are viable, the existence of two genes should be an optimization. To explore this idea, we generated HeLa BD™ Tet-Off cell lines with inducible cyclin B1- or B2-EGFP that were RNAi resistant. Cultures were treated with RNAi and/or doxycycline (Dox) and bromodeoxyuridine. We measured G2 and M transit times and 4C cell accumulation. In the absence of ectopic B cyclin expression, knockdown (kd) of either cyclin increased G2 transit. M transit was increased by cycB1 kd but decreased by cycB2 depletion. This novel difference was further supported by time-lapse microscopy. This suggests that cycB2 tunes mitotic timing, and we speculate that this is through regulation of a Golgi checkpoint. In the presence of endogenous cyclins, expression of active B cyclin-EGFPs did not affect G2 or M phase times. As previously shown, B cyclin co-depletion induced G2 arrest. Expression of either B cyclin-EGFP completely rescued knockdown of the respective endogenous cyclin in single kd experiments, and either cyclin-EGFP completely rescued endogenous cyclin co-depletion. Most of the rescue occurred at relatively low levels of exogenous cyclin expression. Therefore, cycB1 and cycB2 are interchangeable for ability to promote G2 and M transition in this experimental setting. Cyclin B1 is thought to be required for the mammalian somatic cell cycle, while cyclin B2 is thought to be dispensable. However, residual levels of cyclin B1 or cyclin B2 in double knockdown experiments are not sufficient to promote successful mitosis, yet residual levels are sufficient to promote mitosis in the presence of the dispensible cyclin B2. We discuss a simple model that would explain most data if cyclin B1 is necessary.     

The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog.

WHI1-1 is a dominant mutation that reduces cell volume by allowing cells to commit to division at abnormally small sizes, shortening the G1 phase of the cell cycle. The gene was cloned, and dosage studies indicated that the normal gene activated commitment to division in a dose-dependent manner, and that the mutant gene had a hyperactive but qualitatively similar function. Mild over-expression of the mutant gene eliminated G1 phase, apparently entirely relaxing the normal G1 size control, but revealing hitherto cryptic controls. Sequence analysis showed that the hyperactivity of the mutant was caused by the loss of the C-terminal third of the wild-type protein. This portion of the protein contained PEST regions, which may be signals for protein degradation. The WHI1 protein had sequence similarity to clam cyclin A, to sea urchin cyclin and to Schizosaccharomyces pombe cdc13, a cyclin homolog. Since cyclins are inducers of mitosis, WHI1 may be a direct regulator of commitment to division. A probable accessory function of the WHI1 activator is to assist recovery from alpha factor arrest; WHI1-1 mutant cells could not be permanently arrested by pheromone, consistent with a hyperactivation of division.