Interactive Fly, Drosophila

cdc2 function in G1

Cyclin A (see Drosophila Cyclin A) is required at two points in the human cell cycle: (1) in association with E2F, it helps to terminate activation of transcription of genes involved in DNA replication. (2) Later, depletion of cyclin A arrests cells in G2, since it is required for progression from G2 to M. Cyclin A is associated with both cdk2 and cdc2. The cdc2 band associated with cyclin A in early S is hyperphosphorylated. This suggests that cyclin A binding is accompanied by tyrosine phosphorylation of cdc2, which keeps this kinase inactive until G2. The binding of cdk2 to cyclin A increases steadily during the cell cycle and occurs in both S and G2-enriched cell fractions (Pagano, 1992).

The p34cdc2 kinase is essential for progression past Start in the G1 phase of the fission yeast cell cycle, and also acts in G2 to promote mitotic entry. While very little is known about the G1 function of cdc2, the rum1 gene has recently been shown to encode an important regulator of Start in fission yeast; a model for rum1 function suggests that it inhibits p34cdc2 activity. rum1 maintains p34cdc2 in a pre-Start G1 form, inhibiting its activity until the cell achieves the critical mass required for Start. In the absence of rum1, p34cdc2 increasesStart activity in vivo. It is also known that either mutation of cdc2, or overexpression of rum1, can disrupt the dependency of S-phase upon mitosis, resulting in an extra round of S-phase in the absence of mitosis. cdc2 and rum1 interact in this process; dominant cdc2 mutants cause multiple rounds of S-phase in the absence of mitosis. It is suggested that the interaction of rum1 and cdc2 regulates Start, and that this interaction is important for the regulation of S-phase within the cell cycle (Labib, 1995).

The mitotic cyclin-dependent kinase affects DNA replication by inhibiting the accumulation and function of Cdc18, a critical regulator of S-phase entry in S. pombe. The ruml+ gene efficiently suppresses the lethality of a conditional cdc18 mutant. Conversely, deletion of ruml+ increases the severity of the cdc18 mutant phenotype, resulting in inappropriate cell division and a rapid loss of viability. Biochemical experiments indicate that Ruml potently inhibits Cdc2 phosphorylation of histone H1 by directly interacting with the Cdc2/cyclin B complex (See Drosophila Cyclin B). Overexpression of Ruml under conditions that promote re-replication of the genome induces a striking accumulation of Cdc18 protein by a largely post-transcriptional mechanism. Overexpression of SIC1, an unrelated cyclin-dependent kinase inhibitor from budding yeast, causes a similar accumulation of Cdc18 and also leads to re-replication. This work links a potent inhibitor of Cdc2 kinase to a key protein required for the initiation of DNA replication and strongly suggests that inhibition of Cdc18 by cyclin-dependent kinases has an important role in ensuring that the genome is duplicated precisely once each cell cycle (Jallepalli, 1996).

A variant form of p27 (see Drosophila Dacapo) was unexpectedly detected in a synchronized culture of NIH3T3 cells treated with serum. The expression levels of this form of p27 (which lacks its amino (NH2)-terminal region) reach their maximum during G2/M phase. Since the appearance of the NH2-terminal truncated form of p27 coincides with increased expression of Cdc2, it was hypothesized that p27 may play a role in regulating Cdc2 catalytic activity. To test this hypothesis, wild type p27, as well as the amino-terminal (Np27) and carboxyl-terminal (Cp27), were individually expressed, purified, and examined for the ability to regulate CDC2 kinase activity in vitro. Both p27 and Np27 inhibit CDC2 kinase activity. In marked contrast, Cp27 enhances the CDC2 kinase activity. In vitro kinase assays show that Cp27 and p27 are phosphorylated by CDC2, whereas Np27 is not. It was demonstrated that deletion of the putative CDC2 phosphorylation site in the carboxyl-terminal domain of Cp27 diminishes activation of CDC2 kinase activity otherwise stimulated by Cp27. A similar deletion does not have any effect on the inhibitory function of p27. Together these results suggest that the carboxyl-terminal domain of p27 may activate CDC2 kinase activity in vivo during G2/M and that this effect may be regulated by serine/threonine phosphorylation (Uren, 1997).

The fission yeast gene cdc18(+) is required for entry into S phase and for coupling mitosis to the successful completion of S phase. Cdc18 is a highly unstable protein that is expressed only once per cell cycle at the G1/S boundary. Overexpression of Cdc18 causes a mitotic delay and reinitiation of DNA replication, suggesting that the inactivation of Cdc18 plays a role in preventing re-replication within a given cell cycle. Cdc18 is associated with active cyclin-dependent kinase in vivo. Cdc18 was expressed as a glutathione S-transferase fusion in fission yeast: the fusion protein is functional in vivo. The Cdc18 fusion protein copurifies with a kinase activity capable of phosphorylating histone H1 and Cdc18. The activity was identified by a variety of methods as the cyclin-dependent kinase containing the product of the cdc2(+) gene. The amino terminus of Cdc18 is required for association with cyclin-dependent kinase, but the association does not require the consensus cyclin-dependent kinase phosphorylation sites in this region. Both G1/S and mitotic forms of cyclin-dependent kinase phosphorylate and interact with Cdc18. These interactions between Cdc18 and cyclin-dependent kinases suggest mechanisms by which cyclin-dependent kinases could activate the initiation of DNA replication and could prevent re-replication (Brown, 1997).

In the fission yeast Schizosaccharomyces pombe, the execution of Start requires the activity of the Cdc2 protein kinase and the Cdc10/Sct1 transcription complex. The loss of any of these genes leads to G1 arrest and activation of the mating pathway under appropriate conditions. A genetic and biochemical analysis of these genes and their protein products has been undertaken to elucidate the molecular mechanism that governs the regulation of Start. Serine-196 of Cdc10 is phosphorylated in vivo; phosphorylation of this residue is required for Cdc10 function. Substitution of serine-196 of Cdc10 with alanine (Cdc10 S196A) leads to inactivation of Cdc10. Cdc10 S196A is incapable of associating with Sct1 to form a heteromeric complex, whereas substitution of this serine with aspartic acid (S196D) restores DNA-binding activity by allowing Cdc10 to associate with Sct1. Cdc2 activity is required for the formation of the heteromeric Sct1/Cdc10 transcription complex; the Cdc10 S196D mutation alleviates this requirement. This is one mechanism by which the Cdc2 protein kinase may regulate Start in the fission yeast cell cycle (Connolly, 1997).

In fission yeast the mitotic B type cyclin Cdc13/Cdc2 kinase associates with replication origins in vivo. This association is dependent on the origin recognition complex (ORC) established as chromosomes are replicated, and is maintained during G2 and early mitosis. Cells expressing an orp2 (ORC2) allele that reduces binding of Cdc13 to replication origins are acutely prone to chromosomal reduplication. In synchronized endoreduplicating cells, following Cdc13 ablation, replication origins are coordinately licensed prior to each successive round of S phase with the same periodicity as in a normal cell cycle. Thus, ORC bound mitotic Cyclin B/Cdc2 kinase imposes the dependency of S phase on an intervening mitosis but not the temporal licensing of replication origins between each S phase (Wuarin, 2002).

These results suggest that the Cdc13/Cdc2 complex acts locally at replication origins to inhibit the formation of pre-RCs by phosphorylation of pre-RC components. These data support a simple model to explain why only G1 but not G2 nuclei have the potential to replicate. Namely, activation of the anaphase-promoting complex (APC), a specialized E3 ubiquitin ligase, during mitosis leads both to the dissolution of cohesion between sister chromatids and the removal of the mitotic Cyclin B/Cdc2 kinase from the origins of replicated nuclei. In this manner, licensing of replication origins and sister chromatid separation may be temporally coordinated. In addition, in endoreduplicating cells lacking Cdc13, replication origins are periodically and coordinately licensed prior to each successive S phase with the same timing as in a normal cell cycle. Thus, the mechanisms that determine the temporal licensing of replication origins can be experimentally distinguished from the controls that maintain the dependency of S phase on an intervening mitosis. Furthermore, since cells lacking Cdc13 undergo complete genome duplications, it was reasoned that the controls that maintain the correct order of origin firing within S phase and those that ensure each origin fires only once, each rereplicative S phase must remain intact in the absence of Cdc13. In this respect, the experimentally induced rereplication cycle in fission yeast described here closely resembles the naturally occurring endoreduplication cycles observed in plants, flies, and certain human tissues (Wuarin, 2002).

Phosphorylation of cdc2 early in the cell cycle

Activation of the cyclin-dependent kinases to promote cell cycle progression requires their association with cyclins as well as phosphorylation of a threonine residue. This phosphorylation is carried out by the Cdk-activating kinase (CAK) (see Drosophila Cyclin-dependent kinase 7). Purification of CAK from mammals, starfish, and Xenopus has identified it as a heterotrimeric complex composed of a catalytic subunit (p40MO15/cdk7), a regulatory subunit (cyclin H), and an assembly factor (MAT1). CAK phosphorylates not only c34cdc2 but also other Cdks, including p33cdk2 and cdk4, which function earlier in the cell cycle. The CAK subunits are components of TFIIH, a basal transcription factor involved in the initiation of transcription, phosphorylation of the C-terminal domain of the large subunit of RNA polymerase II and DNA repair. The cloning of the CAK from S. cerevisiae raises the possibility that the predominant CAK in vertebrate cell extracts may not function as a physiological CAK. S. cerevisiae CAK is active as a monomer and is not a component of the basal transcription factor (Kaldis, 1996 and references).

The levels of Cyclin A1, B1, B2, and E1, as well as Cdc2 and Cdk2 kinase activity, and Cdc25C phosphorylation states, were all monitored during early Xenopus embryonic cell cycles. Cyclin B1 and B2 protein levels are high in the unfertilized egg, decline upon fertilization, and reaccumulate to the same level during the first cell cycle, a pattern repeated during each of the following 11 divisions. Cyclin A1 shows a similar pattern, except that its level is lower in the egg than in the cell cycles after fertilization. Cyclin B1/Cdc2 kinase activity oscillates, peaking before each cleavage, and Cdc25C alternates between a highly phosphorylated and a less phosphorylated form that correlates with high and low cyclin B1/Cdc2 kinase activity, respectively. Unlike the mitotic cyclins, the level of cyclin E1 does not oscillate during embryogenesis, although its associated Cdk2 kinase activity cycles twice for each oscillation of cyclin B1/Cdc2 activity, consistent with a role for cyclin E1 in both S-phase and mitosis. Although the length of the first embryonic cycle is regulated by both the level of cyclin B and the phosphorylation state of Cdc2, cyclin accumulation alone is rate-limiting for later cycles, since overexpression of a mitotic cyclin after the first cycle causes cell cycle acceleration. The activity of Cdc2 closely parallels the accumulation of cyclin B2, but cell cycle acceleration, caused by cyclin B overexpression, is not associated with elevation of Cdc2 activity to higher than metaphase levels. Tyrosine phosphorylation of Cdc2, absent during cycles 2-12, reappears at the midblastula transition coincident with the disappearance of cyclin E1. Cyclin A1 disappears later, at the beginning of gastrulation. These results suggest that the timing of the cell cycle in the Xenopus embryo evolves from regulation by accumulation of mitotic cyclins to mechanisms involving periodic G1 cyclin expression and inhibitory tyrosine phosphorylation of Cdc2 (Hartley, 1996).

In higher eukaryotes, Cdk2 kinase plays an essential role in regulating the G1-S transition. Cycling Xenopus egg extracts are used to examine the requirement for Cdk2 kinase upon progression into mitosis. When Cdk2 kinase activity is inhibited by the Cdk-specific inhibitor, p21Cip1, a block to mitosis occurs, and inactive Cdc2-cyclin B accumulates. This block occurs in the absence of nuclei and is not due to direct inhibition of Cdc2 by Cip. Importantly, this block to mitosis is reversible by restoring Cdk2-cyclin E kinase activity to a Cip-treated cycling extract. Immunodepletion of Cdk2 from interphase extracts prevents activation of Cdc2 upon the addition of exogenous cyclin B. Thus, Cdk2 kinase is a positive regulator of Cdc2-cyclin B complexes and a link is established between Cdk2 kinase and cell cycle progression into mitosis (Guadagno, 1996).

Cln-Cdc28 is the G1 phase cyclin-cyclin dependent kinase dimer in budding yeast Stability of the mitotic B-type cyclin Clb2 is tightly cell cycle-regulated. B-type cyclin proteolysis is initiated during anaphase and persists throughout the G1 phase. Cln-Cdc28 kinase activity at START is required to repress B-type cyclin-specific proteolysis. Clb-dependent kinases, when expressed during G1, are also capable of repressing the B-type cyclin proteolysis machinery. Furthermore, inactivation of Cln- and Clb-Cdc28 kinases is found to be sufficient to trigger Clb2 proteolysis and sister-chromatid separation in G2/M phase-arrested cells, where the B-type cyclin-specific proteolysis machinery is normally inactive. These results suggest that Cln- and Clb-dependent kinases are both capable of repressing B-type cyclin-specific proteolysis and that these kinases are required to maintain the proteolysis machinery in an inactive state in S and G2/M phase-arrested cells. It is proposed that in yeast, as cells pass through START, Cln-Cdc28-dependent kinases inactivate B-type cyclin proteolysis. As Cln-Cdc28-dependent kinases decline during G2, Clb-Cdc28-dependent kinases take over this role, ensuring that B-type cyclin proteolysis is not activated during S phase and early mitosis (Amon, 1997).

Cyclin-dependent kinases (Cdks) are fully active only when phosphorylated by a Cdk-activating kinase (CAK). Metazoan CAK is itself a Cdk (Cdk7), whereas the CAK of Saccharomyces cerevisiae is a distinct enzyme unrelated to Cdks. The Mcs6-Mcs2 complex of Schizosaccharomyces pombe is a putative CAK related to the metazoan enzyme. Although the loss of Mcs6 is lethal, it results in a phenotype that is inconsistent with a failure to activate Cdc2, the major Cdk in S. pombe. A test was made of the ability of Csk1, a putative regulator of Mcs6, to activate Cdk-cyclin complexes in vitro. Csk1 activates both the monomeric and the Mcs2-bound forms of Mcs6. Surprisingly, Csk1 also activates Cdc2 in complexes with either Cdc13 or Cig2 cyclins. When a double mutant carrying a csk1 deletion and a temperature-sensitive mcs6 allele is incubated at the restrictive temperature, Cdc2 is not activated and the cells undergo a cell division arrest prior to mitosis. Cdc2-cyclin complexes isolated from the arrested cells can be activated in vitro by recombinant CAK, whereas complexes from wild-type cells or either of the single mutants are refractory to activation. Thus, fission yeast contains two partially redundant CAKs: the Mcs6-Mcs2 complex and Csk1. Inactivation of both CAKs is necessary and sufficient to prevent Cdc2 activation and cause a cell-cycle arrest. Mcs6, which is essential, may therefore have required functions other than Cdk activation (Lee, 1999).

In the early embryonic cell cycle, exit from M phase is immediately followed by entry into S phase without an intervening gap phase. To understand the regulatory mechanisms for the cell cycle transition from M to S phase, dependence on Cdc2 inactivation of cell-cycle events occurring during the M-S transition period was examined using Xenopus egg extracts in which the extent of Cdc2 inactivation at M phase exit was quantitatively controlled. The results demonstrate that the occurance of MCM binding and the initiation of DNA replication of nuclear chromatin depends on the decrease of Cdc2 activity to critical levels. Similarly, it was found that Cdc2 inhibitory phosphorylation and cyclin B degradation are turned on and off, respectively, depending on the decrease in Cdc2 activity. However, sensitivity of these processes to Cdc2 activity was different, with the turning-on of Cdc2 inhibitory phosphorylation occurring at higher Cdc2 activity levels than the turning-off of cyclin B degradation. This means that, when cyclin B degradation ceases at M phase exit, Cdc2 inhibitory phosphorylation is necessarily activated. In the presence of constitutive synthesis of cyclin B, this condition favors the occurrence of the Cdc2 inactivation period after M phase exit, thereby ensuring progression through S phase. Thus, M phase exit and S phase entry are coordinately regulated by the Cdc2 activity level in the early embryonic cell cycle (Iwabuchi, 2002).

ErbB2 overexpression confers resistance to taxol-induced apoptosis by inhibiting p34Cdc2 activation. One mechanism is via ErbB2-mediated upregulation of p21Cip1, which inhibits Cdc2. The inhibitory phosphorylation on Cdc2 tyrosine (Y)15 (Cdc2-Y15-p) is elevated in ErbB2-overexpressing breast cancer cells and primary tumors. ErbB2 binds to and colocalizes with cyclin B-Cdc2 complexes and phosphorylates Cdc2-Y15. The ErbB2 kinase domain is sufficient to directly phosphorylate Cdc2-Y15. Increased Cdc2-Y15-p in ErbB2-overexpressing cells corresponds with delayed M phase entry. Expressing a nonphosphorylatable mutant of Cdc2 renders cells more sensitive to taxol-induced apoptosis. Thus, ErbB2 membrane RTK can confer resistance to taxol-induced apoptosis by directly phosphorylating Cdc2 (Tan, 2002).

Cdc2 interaction with a homolog of Cdc6

Fission yeast Cdc18, a homolog of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase. These findings suggest that illegitimate activation of origins may be prevented through the inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, it is reported that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle (Lopez-Girona, 1998).

Cdk1 association with 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).

ORC is involved in formation of prereplicative complexes (pre-RCs) on replication origins in the G1 phase. At the G1/S transition, elevated cyclin E-CDK2 activity triggers 1DNA replication to enter S phase. The CDK cycle works as an engine that drives progression of cell cycle events by successive activation of different types of cyclin-CDK. However, how the CDK cycle is coordinated with replication initiation remains elusive. This study report that acute depletion of ORC2 by RNA interference (RNAi) arrests cells with low cyclin E-CDK2 activity. This result suggests that loss of a replication initiation protein prevents progression of the CDK cycle in G1. p27 and p21 proteins accumulate following ORC2 RNAi and are required for the CDK2 inhibition. Restoration of CDK activity by co-depletion of p27 and p21 allows many ORC2-depleted cells to enter S phase and go on to mitosis. However, in some cells the release of the CDK2 block causes catastrophic events like apoptosis. Therefore, the CDK2 inhibition observed following ORC2 RNAi seems to protect cells from premature S phase entry and crisis in DNA replication. These results demonstrate an unexpected role of ORC2 in CDK2 activation, a linkage that could be important for maintaining genomic stability (Machida, 2005).

Table of contents

cdc2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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