Cyclin A


Regulation of Cyclin A transcription

A specific mutation in TAFII250, the largest subunit of the transcription factor TFIID, disrupts cell growth control in the temperature-sensitive mutant hamster cell line ts13. Transcription from the cyclin A and D1(but not the c-fos and myc promoters) is also dramatically reduced in ts13 cells at the nonpermissive temperature. These findings provide an intriguing link between TAF-mediated transcriptional regulation and cell cycle progression. An enhancer element in the cyclin A promoter (TSRE) has been mapped that responds to mutations in TAFII250. An analysis of chimeric promoter constructs reveals that the cyclin A TSRE can confer TAFII250 dependence to the core promoter of c-fos. Reciprocal hybrid promoter constructs suggest that TAFII250 also contributes to the transcriptional properties of the cyclin A core promoter. The cellular activators that specifically bind to the TSRE and mediate transcription in a TAFII250-dependent manner have been purified and identified. TSRE-binding proteins include members of the activating transcription factor (ATF) family (See Drosophila CrebB-17A). These results suggest that the ts13 mutation of TAFII250 has compromised the ability of TFIID to mediate activation of transcription by specific enhancer factors such as ATF, as well as its ability to perform certain core promoter functions. These defects in TAFII250 apparently result in the down-regulation of key molecules, such as cyclin A, which may be responsible for the ts13 cell cycle arrest phenotype (Wang, 1997).

Cyclin E is necessary and rate limiting for the passage of mammalian cells through the G1 phase of the cell cycle. Control of cell cycle progression by cyclin E involves cdk2 kinase, which requires cyclin E for catalytic activity. Expression of cyclin E/cdk2 leads to an activation of cyclin A gene expression, as monitored by reporter gene constructs derived from the human cyclin A promoter. Promoter activation by cyclin E/cdk2 requires an E2F binding site in the cyclin A promoter. Cyclin E/cdk2 kinase can directly bind to E2F/p107 complexes formed on the cyclin A promoter-derived E2F binding site, and this association is controlled by p27KIP1, most likely through direct protein-protein interaction. These observations suggest that cyclin E/cdk2 associates with E2F/p107 complexes in late G1 phase, once p27KIP1 has decreased below a critical threshold level. Since a kinase-negative mutant of cdk2 prevents promoter activation, it appears that transcriptional activation of the cyclin A gene requires an active cdk2 kinase tethered to its promoter region (Zerfass-Thome, 1997).

Expression of Cyclin A is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. This regulation is mediated by the transcription factor E2F that binds to a specific site in the Cyclin A promoter. The E2F binding site differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing Cyclin E and p107 but not pRb. Ectopic expression of Cyclin D1 triggers premature activation of the Cyclin A promoter by E2F. This effect is blocked by the tumor suppressor protein p16INK4 (Schulze, 1995).

E2F is a family of transcription factors that regulate both cellular proliferation and differentiation. To establish the role of E2F3 in vivo, an E2f3 mutant mouse strain was generated. E2F3-deficient mice arise at one-quarter of the expected frequency, demonstrating that E2F3 is important for normal development. To determine the molecular consequences of E2F3 deficiency, the properties of embryonic fibroblasts derived from E2f3 mutant mice were analyzed. Mutation of E2f3 dramatically impairs the mitogen-induced, transcriptional activation of numerous E2F-responsive genes. A number of genes, including B-myb, cyclin A, cdc2, cdc6, and DHFR, could be identified whose expression is dependent on the presence of E2F3 but not E2F1. A critical threshold level of one or more of the E2F3-regulated genes determines the timing of the G1/S transition, the rate of DNA synthesis, and thereby the rate of cellular proliferation. E2F3 is not required for cellular immortalization but is rate limiting for the proliferation of the resulting tumor cell lines. It is concluded that E2F3 is critical for the transcriptional activation of genes that control the rate of proliferation of both primary and tumor cells (Humbert, 2000).

The S/G2-specific transcription of the human cdc25C gene is due to the periodic occupation of a repressor element ('cell cycle-dependent element' or CDE) located in the region of the basal promoter. Protein binding to the major groove of the CDE in G0 and G1 results in a phase-specific repression of activated transcription. CDE-mediated repression is also the major principle underlying the periodic transcription of the human cyclin A and cdc2 genes. A single point mutation within the CDE results in a 10- to 20-fold deregulation in G0 and an almost complete loss of cell cycle regulation for all three genes. The cdc25C, cyclin A and cdc2 genes also share an identical 5 bp region ('cell cycle genes homology region' or CHR) starting at an identical position, six nucleotides 3' to the CDE. Strikingly, mutation of the CHR region in each of the three promoters produces the same phenotype as the mutation of the CDE, i.e. a dramatic deregulation in G0. In agreement with these results, in vivo DMS footprinting shows the periodic occupation of the cyclin A CDE in the major groove and of the CHR in the minor groove. All three genes bear conspicuous similarities in their upstream activating sequences (UAS). This applies in particular to the presence of NF-Y and Sp1 binding sites which, in the cdc25C gene, have been shown to be the targets of repression through the CDE (Zwicker, 1995).

The cdc25C, cdc2 and cyclin A promoters are controlled by transcriptional repression through two contiguous protein binding sites, termed the CDE and CHR. In the present study CDF-1 has been identified as the factor that interacts with the cdc25C CDE-CHR module. CDF-1 binds to the CDE in the major groove and to the CHR in the minor grove in a cooperative fashion in vitro, in a manner similar to that seen by genomic footprinting. In agreement with in vivo binding data and its putative function as a periodic repressor, DNA binding by CDF-1 in nuclear extracts is down-regulated during cell cycle progression. CDF-1 also binds avidly to the CDE-CHR modules of the cdc2 and cyclin A promoters, but not to the E2F site in the B-myb promoter. Conversely, E2F complexes do not recognize the cdc25C CDE-CHR and CDF-1 is immunologically unrelated to all known E2F and DP family members. This indicates that E2F- and CDF-mediated repression is controlled by different factors acting at different stages during the cell cycle. While E2F-mediated repression seems to be associated with genes such as B-myb that are up-regulated early (around mid G1), CDE-CHR-controlled genes (such as cdc25C, cdc2 and cyclin A), become derepressed later. The fractionation of native nuclear extracts on glycerol gradients leads to separation of CDF-1 from both E2F complexes and pocket proteins of the pRb family. This emphasizes the conclusion that CDF-1 is not an E2F family member and points to profound differences in the cell cycle regulation of CDF-1 and E2F (Liu, 1997).

Loss of adhesion leads to cell cycle arrest at the G1/S boundary in normal, adhesion-dependent, mesenchymal cells. This arrest is accompanied by the inability to produce Cyclin A. A CCAAT element mediates the adhesion-dependent transcriptional activation of Cyclin A in late G1 phase of the cell cycle. Specific binding of a novel 40/115-kDa heterodimeric protein complex, (CBP/cycA) to this CCAAT element is detectable in normal, growing rat kidney fibroblasts but not in G0-arrested or nonadherent fibroblasts. During G0, CBP/cycA appears to be present but sequestered by a retinoblastoma family member. These results suggest that expression of Cyclin A, which controls cell cycle progression by adhesion at the G1/S boundary, is regulated by CBP/cycA and by the phosphorylation status of the retinoblastoma protein or a retinoblastoma-related protein (Kramer, 1996).

Transforming growth factor (TGF)-beta1 prevents cell cycle progression by inhibiting several regulators, including cyclin A. The TGF-beta1-induced down-regulation of cyclin A promoter activity appears to be mediated via the activating transcription factor (ATF) site, because mutation of this site abolishes down-regulation. Surprisingly, although TGF-beta1 treatment for 24 h markedly decreases cyclin A promoter activity, it does not decrease the abundance of the ATF-binding proteins ATF-1 and cyclic AMP-responsive binding protein (CREB). However, a reduction in phosphorylated CREB and ATF-1 was observed in lung epithelial cells treated with TGF-beta1. TGF-beta1-induced down-regulation of cyclin A promoter activity is reversed by okadaic acid (a phosphatase inhibitor) and by cotransfection with plasmids expressing the cAMP-dependent protein kinase catalytic subunit or the simian virus small tumor antigen (Sm-t, an inhibitor of PP2A). These data indicate that TGF-beta1 may down-regulate cyclin A promoter activity by decreasing phosphorylation of CREB and ATF-1 (Yoshizumi, 1997).

Cyclin A plays an essential role in the G1 to S phase transition of the cell cycle. The expression of cyclin A is restrained during G0 and G1, but steeply induced at the G1/S boundary. Analysis of the rat cyclin A promoter elements with the 5' sequential deletion derivatives of the promoter fused to the luciferase cDNA indicate that the ATF/CRE motif primary determines the of inducibility at G1/S. Gel shift analysis of the complex formed at the ATF/CRE site indicates that the complex was not formed with the G0/G1 cell extract, but maximally formed with the late-G1 cell extract. The complex is supershifted by anti-JunD antibody; Western blot analysis of the immune complexes prepared with anti-JunD antibody reveals the presence of ATF2, suggesting heterodimerization of JunD with ATF2. The cyclin A promoter in a reporter plasmid is activated nearly 10-fold in quiescent rat 3Y1 cells by cotransfection with the expression of plasmids encoding ATF2 and Jun family members. In contrast, cotransfection with the ATF4 expression plasmid suppresses the promoter activation mediated by ATF2 and Jun family members. The expression of Jun family members during G1 to S progression is induced biphasically in early and late G1 and the level of JunD increases markedly at the G1/S, while that of ATF family members is gradually increased along with the G1 to S progression. These results indicate that the cyclin A promoter activity is regulated, at least in part, by relative amounts of the ATF and Jun family members (Shimizu, 1998).

Cyclin A1 is a recently cloned cyclin with high level expression in meiotic cells in the testis. However, it is also frequently expressed at high levels in acute myeloid leukemia. To elucidate the regulation of cyclin A1 gene expression, the genomic structure of cyclin A1 was cloned and analyzed. It consists of 9 exons within 13 kilobase pairs. The TATA-less promoter initiates transcription from several start sites, with the majority of transcripts beginning within a 4-base pair stretch. A construct containing a fragment from -190 to +145 shows the highest transcriptional activity. Transfection of cyclin A1 promoter constructs into S2 Drosophila cells demonstrates that Sp1 is essential for the activity of the promoter. Sp1, as well as Sp3, binds to four GC boxes between nucleotides -130 and -80 as observed by gel shift analysis. Mutations in two or more of the four GC boxes decreases promoter activity by >80%. The promoter is cell cycle-regulated with highest activities found in late S and G2/M phase. Further analyses suggests that cell cycle regulation is accomplished by periodic repression of the GC boxes in G1 phase. Taken together, these data show that cyclin A1 promoter activity critically depends on four GC boxes, and members of the Sp1 family appear to be involved in directing expression of cyclin A1 in both a tissue- and cell cycle-specific manner (Muller, 1999).

Rb forms a repressor complex containing histone deacetylase (HDAC) and the hSWI/SNF nucleosome remodeling complex, which inhibits transcription of genes for cyclins E and A and arrests cells in the G1 phase of the cell cycle. Phosphorylation of Rb by cyclin D/cdk4 disrupts association with HDAC, relieving repression of the cyclin E gene and G1 arrest. However, the Rb-hSWI/SNF complex persists and is sufficient to maintain repression of the cyclin A and cdc2 genes, inhibiting exit from S phase. HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF then appear to maintain the order of cyclin E and A expression during the cell cycle, which in turn regulates exit from G1 and from S phase, respectively (Zhang, 2000).

Degradation of Cyclin A

Progression through the cell cycle is regulated in part by the sequential activation and inactivation of cyclin-dependent kinases (CDKs). Many signals arrest the cell cycle through inhibition of CDKs by CDK inhibitors (CKIs). p27(Kip1) was first identified as a CKI that binds and inhibits cyclin A/CDK2 and cyclin E/CDK2 complexes in G1. p27 has an additional property, the ability to induce a proteolytic activity that cleaves cyclin A, yielding a truncated cyclin A lacking the mitotic destruction box. Other CKIs [p15(Ink4b), p16(Ink4a), p21(Cip1), and p57(Kip2)] do not induce cleavage of cyclin A; other cyclins (cyclin B, D1, and E) are not cleaved by the p27-induced protease activity. The C-terminal half of p27, which is dispensable for its kinase inhibitory activity, is required to induce cleavage. Mechanistically, p27 does not appear to cause cleavage through direct interaction with cyclin/CDK complexes. Instead, it activates a latent protease that, once activated, does not require the continuing presence of p27. Mutation of cyclin A at R70 or R71, residues at or very close to the cleavage site, and blocks cleavage. Noncleavable mutants are still recognized by the anaphase-promoting complex/cyclosome pathway responsible for ubiquitin-dependent proteolysis of mitotic cyclins, indicating that the p27-induced cleavage of cyclin A is part of a separate pathway. This protease is referred to as Tsap (pTwenty-seven-activated protease) (Bastians, 1998).

Using the N-terminus of cyclin A1 in a two-hybrid screen as a bait, a Xenopus protein, XDRP1, has been identified that contains a ubiquitin-like domain in its N-terminus and shows significant homology in its C-terminal 50 residues to Saccharomyces cerevisiae Dsk2 and Schizosaccharomyces pombe dph1. XDRP1 is a nuclear phosphoprotein in Xenopus cells, and its phosphorylation is mediated by cyclin A-dependent kinase. XDRP1 binds to both embryonic and somatic forms of cyclin A (A1 and A2) in Xenopus cells, but not to B-type cyclins. The N-terminal ubiquitin-like domain of XDRP1, but not the C-terminal Dsk2-like domain, is required for interaction with cyclin A. XDRP1 requires residues 130-160 of cyclin A1 for efficient binding, which do not include the destruction box of cyclin A. The addition of bacterially expressed XDRP1 protein to frog egg extract inhibits the Ca2+-induced degradation of cyclin A, but not that of cyclin B. The injection of XDRP1 protein into fertilized Xenopus eggs blocks embryonic cell division (Funakoshi, 1999).

XDRP1 binds to a conserved motif in the N-terminus of cyclin A in vitro and selectively inhibits cyclin A degradation in egg extracts. Because the N-terminus of mitotic cyclin including the destruction box is required for ubiquitin-dependent proteolysis, the binding of XDRP1 to the N-terminus of cyclin A may interfere with the interaction and/or the access of some protein(s) to cyclin A, which is required for proteolytic machinery. This would be the reason why XDRP1 selectively inhibits cyclin A degradation in Xenopus egg extracts and embryos. The state of XDRP1 phosphorylation is not directly related to the binding ability with cyclin A or cyclin A-CDK. Rather, the conformational change of cyclin A-CDK complex through XDRP1 binding to cyclin A would be an important step for cyclin A degradation, probably by protecting the cyclin substrate from the attack of the ubiquitin-dependent proteasome pathway. So far, no strong interaction between XDRP1 and cyclin A has been detected in vivo. However, it is suspected that XDRP1 might function through interaction with cyclin A in cells, because XDRP1 is specific for cyclin A as characterized by their interaction in cells and by a blockage of cyclin A degradation. A simple idea to explain these results is therefore that the selective binding of XDRP1 with cyclin A may be involved in the negative regulation of cyclin A degradation. The N-terminal ubiquitin-like domain is also shown to be essential for XDRP1 interaction with cyclin A. For example, Rad23, a protein containing the N-terminal ubiquitin-like domain, appears to interact with a subunit of XP-C to stabilize the repair complex XP-C. So, as shown previously for XP-C, XDRP1 containing a ubiquitin-like sequence in its N-terminus might function as a molecular chaperone of the cyclin-CDK complex to protect the cyclin A substrate. Several reports imply that mitotic cyclins must be protected from their degradation machinery under physiological conditions, even in a state of active proteasome in mitosis. However, it is not yet clear whether the protection of cyclin A from degradation is of any biological significance (Funakoshi, 1999).

The destruction box (D-box) consensus sequence has been defined as a motif mediating polyubiquitylation and proteolysis of B-type cyclins during mitosis. 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. Therefore, residual Cyclin A activity that survives the normal mitotic degradation process 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, 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 (Jacobs, 2001).

Therefore, the mitotic degradation of Drosophila Cyclin A does not depend on regions that fit the D-box consensus. Instead of a simple brief motif as in B-type cyclins, degradation of Drosophila Cyclin A is mediated by a complex degradation signal. Mitotic degradation of human cyclin A also involves a complex degradation signal. Complex degradation signals might thus be more common among A-type cyclins than initially suggested. It is pointed out that a number of cyclin A sequences from different species (i.e. mouse and hamster cyclin A2) do not contain regions matching even a very relaxed D-box consensus (RXXL) within the first hundred N-terminal amino acids (Jacobs, 2001).

In addition to the D-box, the KEN box has recently been shown to mediate APC/C-dependent degradation of proteins like human p55CDC, Nek2, Cdc6 and mouse B99. The N-terminus of Drosophila Cyclin A also includes KEN box-like sequences, but these sequences are clearly not sufficient for normal mitotic Cyclin A degradation. It is not excluded that KEN-box 2 contributes to the degradation signal, perhaps by cooperating with one of the D-box-like motifs, dbs2, which alone is also not sufficient for normal degradation. The combined function of a KEN and a D-box mediate degradation of human Cdc6 during G1. However, the same signal combination is unlikely to operate in the case of Drosophila Cyclin A because KEN-boxes are thought to mediate ubiquitylation exclusively via FZR/Cdh1, which does not appear to be expressed during the early embryonic mitoses, during which Cyclin A, nevertheless, is efficiently degraded (Jacobs, 2001).

The finding that the degradation of A- and B-type cyclins does not rely on identical signals might be a first step towards understanding why Cyclin A but not Cyclin B is degraded in cells arrested by the spindle assembly and chromosome attachment checkpoint. It is conceivable, for instance, that FZY/Cdc20 might have different binding sites for A- and B-type degradation signals. At unattached kinetochores, the spindle checkpoint protein Mad2 protein is thought to be converted into a form that binds and inhibits FZY/Cdc20. If Mad2 were to block only the binding site for B-type signals, Cyclin A could still be degraded in arrested cells in a FZY/Cdc20-dependent manner (Jacobs, 2001).

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 (homologous to Drosophila Fizzy) arrests cells at metaphase and stabilizes both cyclins A and B1. Cyclin A is efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A is not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 is 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 delays the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrests cells in anaphase (Geley, 2001).

Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism of how cyclin-CDKs interact with APC subunits remains unresolved. This study substantiates the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating the Cdk2 periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, a conserved cyclin-binding motif has been identified within the Cdh1 WD-40 domain; its disruption abolishes the Cdh1-cyclin A-Cdk2 interaction, eliminates Cdh1-associated histone H1 kinase activity, and impairs Cdh1 phosphorylation by cyclin A-Cdk2 in vitro and in vivo. Overexpression of cyclin binding-deficient Cdh1 stabilizes the APC-Cdh1 interaction and induces prolonged cell cycle arrest at the G(1)/S transition. Conversely, cyclin binding-deficient Cdh1 loses its capability to support APC-dependent proteolysis of cyclin A but not that of other APC substrates such as cyclin B and securin Pds1. Collectively, these data provide a mechanistic explanation for the mutual functional interplay between cyclin A-Cdk2 and APC-Cdh1 and the first evidence that Cdh1 may activate the APC by binding specific substrates (Sorensen, 2001).

The anaphase-promoting complex (APC) coordinates mitosis and G1 by sequentially promoting the degradation of key cell-cycle regulators. Following the degradation of its substrates in G1, the APC catalyzes the autoubiquitination of its E2 UbcH10. This stabilizes cyclin A and allows it to inactivate APC(Cdh1). How the APC establishes this complex temporal sequence of ubiquitinations, referred to as substrate ordering, is not understood. This study shows that substrate ordering depends on the relative processivity of substrate multiubiquitination by the APC. Processive substrates obtain ubiquitin chains in a single APC binding event. The multiubiquitination of distributive substrates requires multiple rounds of APC binding, which render it sensitive to lower APC concentrations, competition by processive substrates, and deubiquitination. Consequently, more processive substrates are preferentially multiubiquitinated in vitro and degraded earlier in vivo. The processivity of multiubiquitination is strongly influenced by the D box within the substrate, suggesting that substrate ordering is established by a mechanism intrinsic to APC and its substrates and similar to kinetic proofreading (Rape, 2006).

Mutation of Cyclin A

Cyclins are regulatory subunits of cyclin-dependent kinases. Cyclin A, the first cyclin ever cloned, is thought to be an essential component of the cell-cycle engine. Mammalian cells encode two A-type cyclins, testis-specific cyclin A1 and ubiquitously expressed cyclin A2. This study tested the requirement for cyclin A function using conditional knockout mice lacking both A-type cyclins. Acute ablation of cyclin A in fibroblasts did not affect cell proliferation, but led to prolonged expression of another cyclin, cyclin E, across the cell cycle. However, combined ablation of all A- and E-type cyclins extinguished cell division. In contrast, cyclin A function was essential for cell-cycle progression of hematopoietic and embryonic stem cells. Expression of cyclin A is particularly high in these compartments, which might render stem cells dependent on cyclin A, whereas in fibroblasts cyclins A and E play redundant roles in cell proliferation (Kalaszczynska, 2009).

The APC/C inhibitor, Emi1, is essential for prevention of rereplication

Emi1 (early mitotic inhibitor) inhibits APC/C (anaphase-promoting complex/cyclosome) activity during S and G2 phases, and is believed to be required for proper mitotic entry. Emi1 plays an essential function in cell proliferation by preventing rereplication. Rereplication seen after Emi1 depletion is due to premature activation of APC/C that results in destabilization of geminin and cyclin A, two proteins shown in this study to play redundant roles in preventing rereplication in mammalian cells. Geminin is known to inhibit the replication initiation factor Cdt1. The rereplication block by cyclin A is mediated through its association with S and G2/M cyclin-dependent kinases (Cdks), Cdk2 and Cdk1, suggesting that phosphorylation of proteins by cyclin A-Cdk is responsible for the block. Rereplication upon Emi1 depletion activates the DNA damage checkpoint pathways. These data suggest that Emi1 plays a critical role in preserving genome integrity by blocking rereplication, revealing a previously unrecognized function of this inhibitor of APC/C (Machida, 2007).

DNA replication initiates from the specific regions of chromosomal DNA called origins. A critical question in the field of genomic stability is how cells manage to restrict the firing of origins to once and only once per cell cycle. Not only is this regulation achieved despite the fact that thousands of origins fire asynchronously all over the genome, but the regulation is selectively breached during certain stages of development as in endoreduplicating trophoblasts. Most of this regulation appears to be executed at the level of prereplicative complex (pre-RC) formation or origin licensing. The origin recognition complex (ORC) recruits Cdc6 and Cdt1 to origins in G1, which in turn load the putative replicative helicase, Mcm2-7, to form pre-RCs. After replication initiation in the subsequent S phase, Mcm2-7 are depleted from origins, but reformation of pre-RCs is prevented until chromosomes are segregated in M phase. Higher eukaryotes, including human cells, express geminin a protein that binds to Cdt1 to prevent loading of Mcm2-7 on post-initiation origins. Knockdown of geminin by RNA interference (RNAi) is sufficient to induce rereplication in many human cell lines, and geminin-null mice show enhanced endoreduplication in trophoblasts of early embryos. These results suggest that geminin is a major inhibitor of rereplication in mammalian cells. Yeast, however, do not encode geminin, and rereplication is prevented solely by the high levels cyclin-dependent kinase (Cdk) activity seen during S and G2 phases of the cell cycle. A role of high Cdk activity in rereplication block has also been suggested in human cells, although cancer cells that show rereplication after geminin knockdown do so without any obvious mechanism to concurrently inhibit Cdk2 activity. It is therefore currently unclear whether geminin and Cdk play redundant roles in rereplication block, or whether these mechanisms function in different parts of the cell cycle or in different tissues (Machida, 2007 and references therein).

The regulated degradation of proteins by proteasomes through a carefully orchestrated polyubiquitination program is a critical component of cell cycle regulation. For example, Cdks are important for progression through S, G2, and M, but their activity is regulated by the periodic accumulation and destruction of different types of cyclins. Levels of cyclins in the cell cycle are regulated by two ubiquitin ligases, SCF and anaphase-promoting complex/cyclosome (APC/C). SCF complex uses an F-box protein as a substrate recognition subunit. For example, SCFFBXW7 polyubiquitinates the G1 cyclin, cyclin E, for degradation in S phase. There are >60 F-box proteins in the human genome but the cellular functions of most of them are not yet known. APC/C, on the other hand, uses substrate recognition subunits Cdc20 and Cdh1 to polyubiquitinate substrates like cyclins A and B (and geminin) in mitosis and the subsequent G1. Cdc20 activates APC/C in mitosis while Cdh1 activates APC/C in late M and G1 phases. Both these subunits target substrates by recognizing a destruction motif called D-box, while Cdh1, in addition, recognizes a KEN-box (Machida, 2007 and references therein).

Since cyclin A/Cdk kinase activity is essential for DNA replication, the inactivation of APC/C at the G1/S transition is critical for the accumulation of cyclin A for S-phase progression. Emi1 (early mitotic inhibitor) is a cellular inhibitor of APC/C that is not present in yeasts, and is induced by the E2F transcription factor to inactivate APC/C at the G1/S transition. Emi1-mediated reduction in APC/C activity allows cells to accumulate cyclin A, and then the accumulated cyclin A-Cdk complex in S phase further suppresses APC/C activity by phosphorylating Cdh1. Because of its role in suppression of APC/C and because the latter is critical for mitosis, the primary role of Emi1 is believed to ensure proper mitotic entry. At the onset of mitosis, Emi1 is degraded following phosphorylation by Plk1 and ubiquitination by SCF-TrCP, and this allows the activation of APC/C that is critical for progression through M phase and the subsequent G1 phase (Machida, 2007 and references therein).

Since SCF is involved in many aspects of cell cycle regulation, a search was initiated for F-box proteins that are required for cell proliferation by RNAi screening. This screen identified Emi1, an F-box protein known as FBXO5, to be essential for cell proliferation. Surprisingly, the block to cell proliferation was accompanied by extensive rereplication after Emi1 depletion. This rereplication is due to premature activation of APC/C in S and G2 cells. Among many APC/C substrates, cyclin A and geminin are critical for prevention of rereplication, and the two proteins act simultaneously in S and G2 cells as redundant barriers to rereplication. These data suggest that an essential function of Emi1 in S and G2 cells is to prevent rereplication via stabilization of inhibitors of rereplication such as cyclin A and geminin (Machida, 2007).

Cyclin A is a component of a replication competent complex

A replication competent (RC) complex has been isolated from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. The RC complex has now been isolated from nuclear extracts of synchronized HeLa cells; the complex contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. There is a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associates with the complex in S phase, and cyclin B1 and Cdk1 are present exclusively in G2/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes (Frouin, 2002).

These results suggest the presence of two complexes: (1) one bound to the chromatin that contains replication proteins, cyclin A and no Cdks; and (2) a soluble complex in the nucleus containing the same replicative proteins as the chromatin bound complexes, except PCNA, which is absent. This DNA-unbound complex is associated with Cdk2/cyclin A in S phase and Cdk1/cyclin A and B1 in G2 phase. This complex displays a kinase activity that is due to Cdks. Cdk/cyclins are known to phosphorylate several DNA replication proteins, such as SV40 T antigen, RP-A, pol alpha, pol delta and PCNA. Accordingly, Cdk-dependent phosphorylation of different proteins has been detected within the complex. Cdk-dependent phosphorylation of DNA replication proteins appears to have a regulatory role. For example, cyclin A/Cdk2 has been shown to inhibit the replication activity of human pol alpha primase in an SV40 initiation assay, whereas the activities of pol alpha and the tightly associated primase were not impaired in simpler in vitro assays. In addition to the role in modulating the activity of DNA replication enzymes, the results seem to suggest a role for cyclin/Cdk complexes in regulating the association of replication complexes to chromatin during the cell cycle. It could be that a stable association of cyclin A to replication complexes during S phase has the role of recruiting Cdk2, which in turn can regulate the dynamic association of the replication proteins to the chromatin. This might represent an example of intra-phase regulation, perhaps correlated to a different timing of origin firing. Cyclin A could have an 'informational' role, independent of its association to a Cdk. The appearance of Cdk1/cyclin B1 associated with replication complexes in G2/M, concomitantly with the disappearance of Cdk2, could reflect an interphase regulatory mechanism, which prevents re-binding of replication complexes to chromatin during G2/M phase. This hypothesis fits well with the so-called Cdk-driven 'replication switch' model, which predicts that Cdk activity serves both to activate initiation complexes and to inhibit further initiation complex assembly (Frouin, 2002).

Initiation of DNA replication is regulated by cyclin-dependent protein kinase 2 (Cdk2) in association with two different regulatory subunits, cyclin A and cyclin E. But why two different cyclins are required and why their order of activation is tightly regulated are unknown. Using a cell-free system for initiation of DNA replication that is based on G1 nuclei, G1 cytosol and recombinant proteins, it is found that cyclins E and A have specialized roles during the transition from G0 to S phase. Cyclin E stimulates replication complex assembly by cooperating with Cdc6, to make G1 nuclei competent to replicate in vitro. Cyclin A has two separable functions: it activates DNA synthesis by replication complexes that are already assembled, and it inhibits the assembly of new complexes. Thus, cyclin E opens a 'window of opportunity' for replication complex assembly that is closed by cyclin A. The dual functions of cyclin A ensure that the assembly phase (G1) ends before DNA synthesis (S) begins, thereby preventing re-initiation until the next cell cycle (Coverley, 2002).

Cyclin A/cdk targets

Evolutionary homologs continued part 3/3 | back to part 1/3

Cyclin A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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