Cyclin-dependent kinase 1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Cyclin-dependent kinase 1

Synonyms - cdc2, cdk1

Cytological map position - 31E1--31E7

Function - G2 phase cyclin dependent kinase

Keywords - cyclin dependent kinase, cell cycle, Function requires complex with either CycA, CycB or CycB3, phosphorylating hundreds of target proteins required for progression into and through mitotic and meiotic M phases

Symbol - Cdk1

FlyBase ID: FBgn0004106

Genetic map position - 2-[40]

Classification - cdc2 homolog

Cellular location - nuclear

NCBI link: Entrez Gene

Cdk1 orthologs: Biolitmine

Recent literature
Mirkovic, M., Hutter, L.H., Novák, B. and Oliveira, R.A. (2015). Premature sister chromatid separation is poorly detected by the spindle assembly checkpoint as a result of system-level feedback. Cell Rep [Epub ahead of print]. PubMed ID: 26456822
Sister chromatid cohesion, mediated by the cohesin complex, is essential for faithful mitosis. Nevertheless, evidence suggests that the surveillance mechanism that governs mitotic fidelity, the spindle assembly checkpoint (SAC), is not robust enough to halt cell division when cohesion loss occurs prematurely. The mechanism behind this poor response is not properly understood. Using developing Drosophila brains, this study shows that full sister chromatid separation elicits a weak checkpoint response resulting in abnormal mitotic exit after a short delay. Quantitative live-cell imaging approaches combined with mathematical modeling indicate that weak SAC activation upon cohesion loss is caused by weak signal generation. This is further attenuated by several feedback loops in the mitotic signaling network. The study proposes that multiple feedback loops involving cyclin-dependent kinase 1 (Cdk1) gradually impair error-correction efficiency and accelerate mitotic exit upon premature loss of cohesion. These findings explain how cohesion defects may escape SAC surveillance.

Wang, P., Larouche, M., Normandin, K., Kachaner, D., Mehsen, H., Emery, G. and Archambault, V. (2016). Spatial regulation of Greatwall by Cdk1 and PP2A-Tws in the cell cycle. Cell Cycle: [Epub ahead of print] PubMed ID: 26761639
Entry into mitosis requires the phosphorylation of multiple substrates by cyclin B-Cdk1, while exit from mitosis requires their dephosphorylation, which depends largely on the phosphatase PP2A in complex with its B55 regulatory subunit (Tws in Drosophila). At mitotic entry, cyclin B-Cdk1 activates the Greatwall kinase, which phosphorylates Endosulfine proteins, thereby activating their ability to inhibit PP2A-B55 competitively. The inhibition of PP2A-B55 at mitotic entry facilitates the accumulation of phosphorylated Cdk1 substrates. The coordination of these enzymes involves major changes in their localization. In interphase, Gwl is nuclear while PP2A-B55 is cytoplasmic. Gwl suddenly relocalizes from the nucleus to the cytoplasm in prophase, before nuclear envelope breakdown, and this controlled localization of Gwl is required for its function. Phosphorylation of Gwl by cyclin B-Cdk1 at multiple sites is required for its nuclear exclusion, but the precise mechanisms remained unclear. In addition, how Gwl returns to its nuclear localization was not explored. This study shows that cyclin B-Cdk1 directly inactivates a Nuclear Localization Signal in the central region of Gwl. This phosphorylation facilitates the cytoplasmic retention of Gwl, which is exported to the cytoplasm in a Crm1-dependent manner. In addition, this study shows that PP2A-Tws promotes the return of Gwl to its nuclear localization during cytokinesis. These results indicate that the cyclic changes in Gwl localization at mitotic entry and exit are directly regulated by the antagonistic cyclin B-Cdk1 and PP2A-Tws enzymes.

Varadarajan, R., Ayeni, J., Jin, Z., Homola, E. and Campbell, S. D. (2016). Myt1 inhibition of Cyclin A/Cdk1 is essential for fusome integrity and pre-meiotic centriole engagement in Drosophila spermatocytes. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27170181
Regulation of cell cycle arrest in pre-meiotic G2 phase coordinates germ cell maturation and meiotic cell division with hormonal and developmental signals by mechanisms that control Cyclin B synthesis and inhibitory phosphorylation of the M phase kinase, Cdk1. This study investigated how inhibitory phosphorylation of Cdk1 by Myt1 kinase regulates pre-meiotic G2 phase of Drosophila male meiosis. Immature spermatocytes lacking Myt1 activity exhibit two distinct defects: disrupted intercellular bridges (fusomes) and premature centriole disengagement. As a result the myt1 mutant spermatocytes enter meiosis with multipolar spindles. These myt1 defects could be suppressed by depletion of Cyclin A activity or ectopic expression of Wee1 (a partially redundant Cdk1 inhibitory kinase) and phenocopied by expression of a Cdk1F mutant defective for inhibitory phosphorylation. It is therefore concluded that Myt1 inhibition of Cyclin A/Cdk1 is essential for normal fusome behavior and centriole engagement during pre-meiotic G2 arrest of Drosophila male meiosis. These novel meiotic functions that were discovered for Myt1 kinase are spatially and temporally distinct from previously described functions of Myt1 as an inhibitor of Cyclin B/Cdk1 to regulate G2/MI timing.

Snee, M. J., Wilson, W. C., Zhu, Y., Chen, S. Y., Wilson, B. A., Kseib, C., O'Neal, J., Mahajan, N., Tomasson, M. H., Arur, S. and Skeath, J. B. (2016). Collaborative control of cell cycle progression by the RNA exonuclease Dis3 and Ras is conserved across species. Genetics 203: 749-762. PubMed ID: 27029730
Dis3 encodes a conserved RNase that degrades or processes all RNA species via an N-terminal PilT N terminus (PIN) domain and C-terminal RNB domain that harbor, respectively, endonuclease activity and 3'-5' exonuclease activity. In Schizosaccharomyces pombe, dis3 mutations cause chromosome missegregation and failure in mitosis, suggesting dis3 promotes cell division. In humans, apparently hypomorphic dis3 mutations are found recurrently in multiple myeloma, suggesting dis3 opposes cell division. Except for the observation that RNAi-mediated depletion of dis3 function drives larval arrest and reduces tissue growth in Drosophila, the role of dis3 has not been rigorously explored in higher eukaryotic systems. Using the Drosophila system and newly generated dis3 null alleles, this study found that absence of dis3 activity inhibits cell division. A conserved CDK1 phosphorylation site was found that when phosphorylated inhibits Dis3's exonuclease, but not endonuclease, activity. Leveraging this information, Dis3's exonuclease function was shown to be required for mitotic cell division: in its absence, cells are delayed in mitosis and exhibit aneuploidy and overcondensed chromosomes. In contrast, modest reduction dis3 function was found enhances cell proliferation in the presence of elevated Ras activity, apparently by accelerating cells through G2/M even though each insult by itself delays G2/M. Additionally, dis3 and ras were found to genetically interact in worms and that dis3 can enhance cell proliferation under growth stimulatory conditions in murine B cells. Thus, reduction, but not absence, of dis3 activity can enhance cell proliferation in higher organisms.
Deneke, V.E., Melbinger, A., Vergassola, M. and Di Talia, S. (2016). Waves of Cdk1 activity in S phase synchronize the cell cycle in Drosophila embryos. Dev Cell 38: 399-412. PubMed ID: 27554859
Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. Using biosensors of Cdk1 and Chk1 activities, this study dissected the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. Cdk1 waves were shown not to be controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. S phase Cdk1 waves were shown to be fundamentally distinct and propagate as active trigger waves in an excitable medium, while mitotic Cdk1 waves propagate as passive phase waves. These findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events. 

Ibar, C. and Glavic, A. (2017). Drosophila p115 is required for Cdk1 activation and G2/M cell cycle transition. Mech Dev [Epub ahead of print]. PubMed ID: 28396045
Golgi complex inheritance and its relationship with the cell cycle are central in cell biology. Golgi matrix proteins, known as golgins, are one of the components that underlie the shape and functionality of this organelle. In mammalian cells, golgins are phosphorylated during mitosis to allow fragmentation of the Golgi ribbon and they also participate in spindle dynamics; both processes are required for cell cycle progression. Little is known about the function of golgins during mitosis in metazoans in vivo. This is particularly significant in Drosophila, in which the Golgi architecture is distributed in numerous units scattered throughout the cytoplasm, in contrast with mammalian cells. This study examined the function of the ER/cis-Golgi golgin p115 during the proliferative phase of the Drosophila wing imaginal disc. Knockdown of p115 decreased tissue size. This phenotype was not caused by programmed cell death or cell size reductions, but by a reduction in the final cell number due to an accumulation of cells at the G2/M transition. This phenomenon frequently allows mitotic bypass and re-replication of DNA. These outcomes are similar to those observed following the partial loss of function of positive regulators of Cdk1 in Drosophila. In agreement with this, Cdk1 activation was reduced upon p115 knockdown. Interestingly, these phenotypes were fully rescued by Cdk1 overexpression and partially rescued by Myt1 depletion, but not by String (also known as Cdc25) overexpression. Additionally, the physical interaction between p115 and Cdk1 was confirmed, suggesting that the formation of a complex where both proteins are present is essential for the full activation of Cdk1 and thus the correct progression of mitosis in proliferating tissues.
Nag, R. N., Niggli, S., Sousa-Guimaraes, S., Vazquez-Pianzola, P. and Suter, B. (2018). Mms19 is a mitotic gene that permits Cdk7 to be fully active as a Cdk-activating kinase. Development 145(2). PubMed ID: 29361561
Mms19 encodes a cytosolic iron-sulphur assembly component. This study found that Drosophila Mms19 is also essential for mitotic divisions and for the proliferation of diploid cells. Reduced Mms19 activity causes severe mitotic defects in spindle dynamics and chromosome segregation, and loss of zygotic Mms19 prevents the formation of imaginal discs. The lack of mitotic tissue in Mms19(P/P) larvae can be rescued by overexpression of the Cdk-activating kinase (CAK) complex, an activator of mitotic Cdk1, suggesting that Mms19 functions in mitosis to allow CAK (Cdk7/Cyclin H/Mat1) to become fully active as a Cdk1-activating kinase. When bound to Xpd and TFIIH, the CAK subunit Cdk7 phosphorylates transcriptional targets and not cell cycle Cdks. In contrast, free CAK phosphorylates and activates Cdk1. Physical and genetic interaction studies between Mms19 and Xpd suggest that their interaction prevents Xpd from binding to the CAK complex. Xpd bound to Mms19 therefore frees CAK complexes, allowing them to phosphorylate Cdk1 and facilitating progression to metaphase. The structural basis for the competitive interaction with Xpd seems to be the binding of Mms19, core TFIIH and CAK to neighbouring or overlapping regions of Xpd.
Vergassola, M., Deneke, V. E. and Di Talia, S. (2018). Mitotic waves in the early embryogenesis of Drosophila: Bistability traded for speed. Proc Natl Acad Sci U S A 115(10): E2165-e2174. PubMed ID: 29449348
Early embryogenesis of most metazoans is characterized by rapid and synchronous cleavage divisions. Chemical waves of Cdk1 activity were previously shown to spread across Drosophila embryos, and the underlying molecular processes were dissected. This paper presents the theory of the physical mechanisms that control Cdk1 waves in Drosophila. The in vivo dynamics of Cdk1 are captured by a transiently bistable reaction-diffusion model, where time-dependent reaction terms account for the growing level of cyclins and Cdk1 activation across the cell cycle. Two distinct regimes were identified. The first one is observed in mutants of the mitotic switch. There, waves are triggered by the classical mechanism of a stable state invading a metastable one. Conversely, waves in wild type reflect a transient phase that preserves the Cdk1 spatial gradients while the overall level of Cdk1 activity is swept upward by the time-dependent reaction terms. This unique mechanism generates a wave-like spreading that differs from bistable waves for its dependence on dynamic parameters and its faster speed. Namely, the speed of "sweep" waves strikingly decreases as the strength of the reaction terms increases and scales as the powers 3/4, -1/2, and 7/12 of Cdk1 molecular diffusivity, noise amplitude, and rate of increase of Cdk1 activity in the cell-cycle S phase, respectively. Theoretical predictions are supported by numerical simulations and experiments that couple quantitative measurements of Cdk1 activity and genetic perturbations of the accumulation rate of cyclins. Finally, this analysis bears upon the inhibition required to suppress Cdk1 waves at the cell-cycle pause for the maternal-to-zygotic transition.
Deneke, V. E., Puliafito, A., Krueger, D., Narla, A. V., De Simone, A., Primo, L., Vergassola, M., De Renzis, S. and Di Talia, S. (2019). Self-organized nuclear positioning synchronizes the cell cycle in Drosophila embryos. Cell. PubMed ID: 30982601
The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm. Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. This study shows that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. This study demonstrates that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. It is concluded that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.
Gadre, P., Chatterjee, S., Varshney, B. and Ray, K. (2020). Cyclin E and Cdk1 regulate the termination of germline transit-amplification process in Drosophila testis. Cell Cycle 19(14): 1786-1803. PubMed ID: 32573329
An extension of the G1 is correlated with stem cell differentiation. The role of cell cycle regulation during the subsequent transit amplification (TA) divisions is, however, unclear. This paper reports that in the Drosophila male germline lineage, the transit amplification divisions accelerate after the second TA division. The cell cycle phases, marked by Cyclin E and Cyclin B, are progressively altered during the TA. Antagonistic functions of the bag-of-marbles and the Transforming-Growth-Factor-β signaling regulate the cell division rates after the second TA division and the extent of the Cyclin E phase during the fourth TA division. Furthermore, loss of Cyclin E during the fourth TA cycle retards the cell division and induces premature meiosis in some cases. A similar reduction of Cdk1 activity during this stage arrests the penultimate division and subsequent differentiation, whereas enhancement of the Cdk1 activity prolongs the TA by one extra round. Altogether, the results suggest that modification of the cell cycle structure and the rates of cell division after the second TA division determine the extent of amplification. Also, the regulation of the Cyclin E and CDK1 functions during the penultimate TA division determines the induction of meiosis and subsequent differentiation.
Falahati, H., Hur, W., Di Talia, S. and Wieschaus, E. (2021). Temperature-Induced uncoupling of cell cycle regulators. Dev Biol 470: 147-153. PubMed ID: 33278404
The early stages of development involve complex sequences of morphological changes that are both reproducible from embryo to embryo and often robust to environmental variability. To investigate the relationship between reproducibility and robustness this study examined cell cycle progression in early Drosophila embryos at different temperatures. The experiments show that while the subdivision of cell cycle steps is conserved across a wide range of temperatures (5-35 °C), the relative duration of individual steps varies with temperature. The transition into prometaphase is delayed at lower temperatures relative to other cell cycle events, arguing that it has a different mechanism of regulation. Using an in vivo biosensor, the ratio of activities were quantified of the major mitotic kinase, Cdk1 and one of the major mitotic phosphatases PP1. Comparing activation profile with cell cycle transition times at different temperatures indicates that in early fly embryos activation of Cdk1 drives entry into prometaphase but is not required for earlier cell cycle events. In fact, chromosome condensation can still occur when Cdk1 activity is inhibited pharmacologically. These results demonstrate that different kinases are rate-limiting for different steps of mitosis, arguing that robust inter-regulation may be needed for rapid and ordered mitosis.
Huang, J., Gujar, M. R., Deng, Q., S, Y. C., Li, S., Tan, P., Sung, W. K. and Wang, H. (2021). Histone lysine methyltransferase Pr-set7/SETD8 promotes neural stem cell reactivation. EMBO Rep: e50994. PubMed ID: 33565211
The ability of neural stem cells (NSCs) to switch between quiescence and proliferation is crucial for brain development and homeostasis. Increasing evidence suggests that variants of histone lysine methyltransferases including KMT5A are associated with neurodevelopmental disorders. However, the function of KMT5A/Pr-set7/SETD8 in the central nervous system is not well established. This study shows that Drosophila Pr-Set7 is a novel regulator of NSC reactivation. Loss of function of pr-set7 causes a delay in NSC reactivation and loss of H4K20 monomethylation in the brain. Through NSC-specific in vivo profiling, this study demonstrated that Pr-set7 binds to the promoter region of cyclin-dependent kinase 1 (cdk1) and Wnt pathway transcriptional co-activator earthbound1/jerky (ebd1). Further validation indicates that Pr-set7 is required for the expression of cdk1 and ebd1 in the brain. Similar to Pr-set7, Cdk1 and Ebd1 promote NSC reactivation. Finally, overexpression of Cdk1 and Ebd1 significantly suppressed NSC reactivation defects observed in pr-set7-depleted brains. Therefore, Pr-set7 promotes NSC reactivation by regulating Wnt signaling and cell cycle progression. These findings may contribute to the understanding of mammalian KMT5A/PR-SET7/SETD8 during brain development.

Drosophila Cdc2 (cdc stands for cell division control) is the catalytic subunit (the cyclin dependent kinase or cdk) of the cyclin/cdk heterodimer responsible for the execution of the mitotic (M) phase of the cell cycle. While cyclin (the regulatory subunit in all cyclin/cdk heterodimers) alternates between synthesis and degradation during the cell cycle, steady levels of the cdk are maintained; cdk activity is regulated through phosphorylation. The cyclin dependent kinase Cdc2, also known as Cdk1, is maintained in an inactive hyperphosphorylated state during G1, S and G2 stages. The inhibition of Cdc2 activation during these stages involves phosphorylation at conserved residues threonine 14 and tyrosine 15, which overlap the ATP binding site of Cdc2.

The dephosphorylation of Cdc2 is highly regulated by what is termed checkpoint control. Checkpoint control ensures that M phase entry depends on the successful completion of S phase; entry into M phase is allowed only if DNA is fully replicated and is not damaged. Cdc2 is suddenly dephosphorylated by the phosphatase known as String at the boundary between G2 and M, thus driving the cell into mitosis. At this point Cdc2 fulfills a second biological role; the promotion of M phase. Cdc2 targets many cellular proteins involved in mitosis, activating them by catalytically adding phosphate residues to threonine and serine residues. In this second biological role, the Cdc2/Cyclin heterodimer is termed the mitosis promoting factor, or MPF. For more information about the G2 checkpoint, see Cyclin B.

Although the activity of Cdc2 is central to checkpoint control, inputs from developmentally regulated transcription factors, such as Escargot, are required. Escargot is involved in maintaining diploidy in imaginal cells, ensuring that these cells retain the ability to proliferate and differentiation upon metamorphosis. This requirement exemplifies the complex regulation that occurs at the G2/M boundary (Hayashi, 1996).

The mutant phenotypes of cdc2 are similar to those of escargot: many diploid cells in imaginal discs, salivary glands and the central nervous system enter an endocycle, characterized by DNA replication without a subsequent mitotic phase. Such endocycling cells are often polytene, possessing thick chromosomes with DNA replicated many times over. When escargot function is eliminated, diploid imaginal cells that were arrested in G2 lose Cyclin A, a regulatory subunit of G2/M cdk, and entered endocycle. escargot genetically interacts with cdc2, suggesting an intimate biological interaction. Since mitotically quiescent abdominal histoblasts still require cdc2 to remain diploid, the inhibitory activity of Cdc2 on DNA replication appears to be separable from its activity as the mitosis promoting factor. These results suggest that in G2, escargot is required to maintain a high level of G2/M cdk, which actively inhibits the entry into S phase. Expression of Cyclin A is lost in escargot mutants, suggesting that Cdc2 activity (dependent on its regulatory subunit Cyclin A), indirectly depends on escargot (Hayashi, 1996).

The notion of a correlation between cell fate and time of cell division remains a cornerstone of theories that explain the origin of patterns during development. For example, in the pupal wing disc, at pupariation, cells arrest in G2 before progressing through two programmed cell divisions, separated by an intervening S-phase. Cells in vein regions divide in a reproducible pattern before the cells in the adjacent intervein regions. In the early developing embryo, cells with a common developmental fate can be recognized as mitotic domains after cellularization because they divide almost synchronously at defined developmental stages. In the growing amphibian limb, treatment with colchicine inhibits cell divisions, reduces the size of the limb and also reduces the number of digets formed (references in Weigmenn, 1997).

During larval development, Drosophila imaginal discs increase in size about 1000-fold; cells are instructed to acquire distinct fates as a function of their position. The secreted signaling molecules Wingless and Decapentaplegic have been implicated as sources of positional information that globally control growth and patterning. Evidence has also been presented that local cell interactions play an important role in controlling cell proliferation in imaginal discs. A test was made of the role of cell division in pupal wing patterning in Drosophila by inactivating the mitotic kinase Cdc2 in developing imaginal discs. Clones of temperature sensitive cdc2 mutant cells were generated by mitotic recombination and the resulting patterns of tissue development were observed (Weigmenn, 1997).

Cell growth was seen to continue after inactivation of Cdc2, with little effect on overall patterning. The final size and shape of the pupal wing is not affected by inhibiting pupal cell division. Blocking cell division does not affect the spatial and temporal patterning of DNA replication in pupal wings. DNA replication is seen to continue at its normal pace in the absence of cell division, and larger than normal cells are generated, these cells attaining the architectural arrangement that smaller, normal cells achieve in the wild-type situation. Thus cell growth continues in the absence of cell division during larval stages and inhibition of cell division in mid-larval stages fails to block the patterning process. It is concluded that the mechanisms that regulate the size of the disc do not function by regulating cell division, but appear to act primarily by regulating size in terms of physical distance or tissue volume. In other words, overall size and shape of discs with mutant clones is unaffected and therefore disc size and structure is independent of cell number and pattern of cell division (Weigmann, 1997).

There are clear limitations to the universality of the above observations. If larvae are shifted to restrictive temperatures at early stages of development, wings fail to grow to normal size. In this case, where the cell number and overall dimensions of the disc are greatly reduced, pattern elements of gene expression do not resolve properly. This is consistent with the results of experiments with growing amphibian limbs (Weigmann, 1997).

There is already some precedence for these findings in the literature of Drosophila development. Simpson and Morata (1981) produced small clones of genetically marked, but otherwise wild-type cells in discs carrying large clones of Minute+ cells (which had been induced earlier). Mutant Minute cells divide more slowly than wild-type Mutant+ cells. Clones of wild-type cells located near the edge of the Minute+ clone grow larger than genetically equivalent clones far from the clone border. These observations suggest that cells within the Mutant+ territory but near the Minute mutant territory are stimulated to proliferate more strongly than cells surrounded by wild-type cells. It is concluded that the ability of cells to respond appropriately to long range patterning cues is relatively independent of cell number and that cell size can continue to increase if cell division is blocked, thus compensating for the presence of fewer cells (Weigmann, 1997). It might be that within a certain range, normal patterning mechanisms and compensatory mechanisms take presence over a deficiency in cell number to attain a genetically preprogrammed pattern.

Activating the DNA damage checkpoint in a developmental context

Studies in unicellular systems have established that DNA damage by irradiation invokes a checkpoint that acts to stall cell division. During metazoan development, the modulation of cell division by checkpoints must occur in the context of gastrulation, differential gene expression and changes in cell cycle regulation. To understand the effects of checkpoint activation in a developmental context, a study was performed of the effect of X-rays on post-blastoderm Drosophila embryos. In Drosophila, DNA damage delays anaphase chromosome separation during cleavage cycles that lack a G2 phase. In post-blastoderm cycles that include a G2 phase, irradiation delays the entry into mitosis. Gastrulation and the developmental program of string (Cdc25) gene expression, which normally regulates the timing of mitosis, occurs normally after irradiation. The radiation-induced delay of mitosis accompanies the exclusion of mitotic cyclins from the nucleus. Furthermore, a mutant form of the mitotic kinase Cdk1 that cannot be inhibited by phosphorylation drives a mitotic cyclin into the nucleus and overcomes the delay of mitosis induced by irradiation. It is concluded that developmental changes in the cell cycle, for example, the introduction of a G2 phase, dictate the response to checkpoint activation, for example, delaying mitosis instead of or in addition to delaying anaphase. This unprecedented finding suggests that different mechanisms are used at different points during metazoan development to stall cell division in response to checkpoint activation. The delay of mitosis in post-blastoderm embryos is due primarily to inhibitory phosphorylation of Cdk1, whereas nuclear exclusion of a cyclin-Cdk1 complex might play a secondary role. Delaying cell division has little effect on gastrulation and developmentally regulated string gene expression, supporting the view that development generally dictates cell proliferation and not vice versa (Su, 2000).

To examine the effect of DNA damage on the progression of the cell cycle during Drosophila embryogenesis, embryos 0-4.5 hours of age were exposed to 570 rads of X-rays. At this dose, 40%-60% of cellular embryos die and fail to hatch into larvae. This dose therefore corresponds to the half-maximal lethal dose (LD50). When syncytial embryos are exposed to X-rays: nuclei enter mitosis normally but chromosome segregation is delayed. The delay is transient such that nuclei enter the next interphase without completely separating sister chromosomes, resulting in polyploid nuclei (Su, 2000).

In cellularized embryos, changes in cell cycle indicators that are consistent with a delay in the entry into mitosis are observed. In untreated embryos at these stages, cells divide in stereotypical clusters termed 'mitotic domains'. Both the location of a mitotic domain within the embryo and the time at which it goes through mitosis are invariant from embryo to embryo. The timing of morphogenetic movements that comprise gastrulation is likewise invariant from embryo to embryo. Thus, the wild-type pattern of mitotic cells at any specific time during this period, as indicated by the extent of gastrulation, is easily recognizable. In irradiated samples, embryos were found in which expected mitotic domains were not in mitosis, as judged by the absence of condensed chromosomes and mitotic figures. Antibody staining for a mitotic-specific phospho-epitope on histone H3 (PH3), and staining with wheatgerm agglutinin (WGA) to detect nuclear envelope breakdown, has confirmed the absence of mitoses in these embryos. It is inferred that irradiation delays the entry into mitosis in cellularized embryos, whereas under identical conditions, chromosome segregation is delayed in syncytial embryos. Treatment of cellularized embryos with a DNA-damaging agent, methyl methane sulfonate, results in a similar delay of mitosis. Therefore, the observed effect of irradiation on mitosis is probably due to the DNA-damaging activity of X-rays (Su, 2000).

It is an unprecedented finding that irradiation leads to two different cell cycle responses in a single organism: either the delay of anaphase chromosome segregation or the delay of mitosis. Mitotic chromosome segregation and the initiation of mitosis are regulated by different mechanisms. The former requires the proteolysis of proteins, such as PDS1 in budding yeast and cyclin A in Drosophila, whereas the latter requires the activation of mitotic cyclin-Cdk complexes. It is suggested that checkpoint activation by the same dose of radiation under identical conditions must have used different downstream mechanisms in order to delay chromosome segregation in the syncytium and mitosis in the cellularized embryos. Although mechanisms that operate in the syncytium remain elusive, the mechanisms used by cellularized embryos were addressed in this study (Su, 2000).

Despite the finding that irradiation does not interfere with String expression, it might have antagonized String activity. Cdc25Stg activates Cdk1 by removing the inhibitory phosphates on Thr14 and Tyr15. A Cdk1 mutant in which these residues have been mutated (Cdk1AF) bypasses the requirement for String. If the mechanism by which radiation delays mitosis solely involves inhibitory phosphorylation of Cdk1, Cdk1AF should bypass the radiation-induced delay. To test this hypothesis, Cdk1 or Cdk1AF, in conjunction with a mitotic cyclin, was expressed from a heat-inducible (hs) promoter during interphase 14. It was then asked whether irradiation could delay the onset of mitosis 14 in embryos expressing these transgenes. It was found that many cells of heat-shocked embryos that carried hs-Cdk1AF and hs-mitotic cyclin transgenes fail to delay mitosis after irradiation. This effect was seen with mitotic cyclins A, B or Bs -- a truncated version of cyclin B that is resistant to proteolysis. In contrast, embryos carrying hs-Cdk1, in combination with the same cyclins, behave like wild-type embryos and delay mitosis. It is concluded that Cdk1AF, and not Cdk1, can overcome the radiation-induced delay in mitosis. It is inferred that inhibitory phosphorylation on Cdk1 is required to delay mitosis in response to DNA damage, in agreement with previous results from fission yeast and vertebrates (Su, 2000).

Interestingly, the ability of Cdk1AF and cyclins to overcome the delay of mitosis in Drosophila was seen only in certain cells of the embryos, and these cells represent mitotic domains, for example, domain 4. Cells of mitotic domains are distinguished from their neighbors by their accumulation of String protein. Although further experiments are required to demonstrate the importance of String, the perfect coincidence of clusters of irradiated cells that entered mitosis in the presence of Cdk1AF as well as accumulated String, has led to the following suggestion: although cyclin-Cdk1AF activity is not present in sufficient quantities to promote mitosis by itself under these experimental conditions, this activity can induce endogenous String to activate endogenous Cdk1 and induce mitosis. A similar feedback mechanism has been proposed for human Cdk1 and Cdc25. It follows then that endogenous String and Cdk1 might be inhibited by irradiation, but that this inhibition can be overcome by a small amount of Cdk1AF activity (Su, 2000).

The same amount of Cdk1AF activity overcomes another consequence of irradiation, namely, the nuclear exclusion of a mitotic cyclin. Nuclear cyclin/Cdk1 activity is a prerequisite to mitosis and the exclusion of cyclin B1 from the nucleus appears to contribute to the delay in mitosis after irradiation in human cells. In cellular-stage Drosophila embryos, cyclins A and B remain enriched in the cytoplasm in interphase. Cyclin A accumulates in the nucleus of cells that initiate mitosis, as does cyclin B. In irradiated embryos, both cyclins A and B are excluded from nuclei although their levels remain unchanged. In cells that express Cdk1AF (with a mitotic cyclin) that enter mitosis even after irradiation, nuclear accumulation of cyclin A is evident. Thus, a low level of Cdk1 activity, provided by Cdk1AF in these experiments, leads to both the nuclear accumulation of a cyclin and the entry into mitosis (Su, 2000).

Given these two observations -- that Cdk1AF drives the nuclear accumulation of cyclin A and that nuclear accumulation of mitotic cyclins coincides with the entry into mitosis in unperturbed cell cycles -- it has been proposed that Cdk1 activity normally drives the nuclear accumulation of cyclin-Cdk1 complexes. In support of this idea, Cyclin A remains excluded from nuclei in string mutants. In accordance with this, Cdk1AF, in conjunction with endogenous String, overcomes the radiation-induced delay of mitosis because Cdk1AF can start the feedback loop that activates endogenous Cdk1 by endogenous String and Cdk1 activity can drive the nuclear accumulation of cyclin-Cdk1. These ideas help explain previous observations in human cells. In the latter, although the exclusion of cyclin B1 from nuclei appears to be of some importance to regulating mitotic entry, Cdk1AF can overcome the checkpoint-induced delay of mitosis, regardless of whether cyclin B1 or NLS-cyclin B1, which is constitutively localized to the nucleus, is co-expressed. Thus, Cdk1AF in human cells, as in Drosophila, might also drive the nuclear accumulation of cyclin-Cdk complexes and the entry into mitosis by initiating a positive feedback loop for the activation of endogenous Cdk1. Whether a similar feedback loop of Cdk1, String and cyclin-localization operates to control mitosis in other tissues, such as larval imaginal discs, remains to be seen (Su, 2000).

Drosophila myt1 is the major cdk1 inhibitory kinase for wing imaginal disc development

Mitosis is triggered by activation of Cdk1, a cyclin-dependent kinase. Conserved checkpoint mechanisms normally inhibit Cdk1 by inhibitory phosphorylation during interphase, ensuring that DNA replication and repair is completed before cells begin mitosis. In metazoans, this regulatory mechanism is also used to coordinate cell division with critical developmental processes, such as cell invagination. Two types of Cdk1 inhibitory kinases have been found in metazoans. They differ in subcellular localization and Cdk1 target-site specificity: one (Wee1) being nuclear and the other (Myt1), membrane-associated and cytoplasmic. Drosophila has one representative of each: dMyt1 and dWee1. Although dWee1 and dMyt1 are not essential for zygotic viability, loss of both resulted in synthetic lethality, indicating that they are partially functionally redundant. Bristle defects in myt1 mutant adult flies prompted a phenotypic analysis that revealed cell-cycle defects, ectopic apoptosis, and abnormal responses to ionizing radiation in the myt1 mutant imaginal wing discs that give rise to these mechanosensory organs. Cdk1 inhibitory phosphorylation was also aberrant in these myt1 mutant imaginal wing discs, indicating that dMyt1 serves Cdk1 regulatory functions that are important both for normal cell-cycle progression and for coordinating mitosis with critical developmental processes (Jin, 2008).

Multicellular organisms regulate Cdk1 by inhibitory phosphorylation to prevent mitosis when DNA is being replicated or repaired and to ensure that mitosis does not interfere with critical developmental processes that require remodeling of the cytoskeleton. Previous studies of Drosophila Wee1 and Myt1 revealed that these conserved Cdk1 inhibitory kinases were required during early embryogenesis and gametogenesis, respectively. This study has characterized imaginal and adult developmental defects caused by loss of dMyt1 activity (and to a much lesser extent, dWee1), that confirm the importance of Cdk1 inhibitory phosphorylation for coordinating cell-cycle events with critical developmental processes (Jin, 2008).

In Drosophila and other organisms, G2/M delays can be induced by overexpression of Myt1 kinases, suggesting a specific role for Myt1 in regulating this stage of the cell cycle. Further evidence of a role for Myt1 in G2/M regulation comes from studies of oocyte maturation in frogs, starfish, and nematodes. Not all data indicate that Myt1 is required for G2 phase arrest, however, and there is no evidence that dMyt1 regulates oocyte maturation in Drosophila. Nor is there evidence that dMyt1 activity is responsible for the timing of the G2/M meiotic transition that follows a prolonged 4-day-long G2 phase arrest, in Drosophila primary spermatocytes. Moreover, a recent study showed that functional depletion of human Myt1 by siRNA did not affect the proportion of cells in G2 phase, but instead affected membrane dynamics during mitotic exit (Nakajima, 2008). More needs to be learned about Myt1 mediated regulatory mechanisms before these apparent discrepancies in Myt1 functions are resolved (Jin, 2008).

Previous work showed that Cdk1 inhibitory phosphorylation is required for proper development of thoracic mechanosensory organs. This study has now identified dMyt1 as the primary Cdk1 inhibitory kinase for this developmental program. Several molecular mechanisms could explain the role of dMyt1 in mechanosensory bristle development. One obvious possibility is that myt1 mutant sensory organ precursor (SOP) cells and their descendants might divide prematurely due to a defect in G2/M regulation, resulting in aberrant segregation of cell fate determinants. If there was a relatively narrow window for coordinating specific developmental events with the G2/M transition, disrupting this regulatory mechanism could account for the observed loss and duplication of bristles and socket cells in myt1 mutants. Live analysis of mechanosensory organ development could test this possibility (Jin, 2008).

Alternatively, myt1 mutant phenotypes could reflect defects in Myt1-mediated regulatory mechanisms that are important for the control of intracellular membrane dynamics during mitosis, particularly the Golgi apparatus and endoplasmic reticulum. The Drosophila Golgi apparatus undergoes significant morphological changes that have been linked to specific developmental states and so the observed myt1 mutant developmental defects might reflect problems in the structure or function of this organelle. Further support for this idea comes from a recent study showing that asymmetrical segregation of mouse Numb (a conserved cell fate determinant) requires the Golgi apparatus, leading to the suggestion that Golgi fragmentation and reconstitution could represent a mechanism for coupling cell-fate specification and cell-cycle progression (Jin, 2008).

Another possible explanation for myt1 mutant defects concerns the large quantities of actin that are synthesized and packaged to form the large mechanosensory bristle shafts. This process involves extensive reorganization of the endoplasmic reticulum and Golgi apparatus to accommodate increased membrane trafficking. Defects in the structure or function of the Golgi apparatus and ER caused by loss of dMyt1 activity could therefore account for defects or diminution in these bristles. Resolving which of these potential mechanisms best explain the role of dMyt1 during mechanosensory organ development will be a major challenge of future research (Jin, 2008).

Intriguing cell-cycle defects (higher mitotic index, aberrant chromatin condensation, and ectopic apoptosis), as well as defects in responses to ionizing radiation in proliferating cells, were observed in myt1 mutant imaginal wing discs. These observations suggest an important role for dMyt1 in conserved cell-cycle checkpoint responses that target Cdk1 by inhibitory phosphorylation. It was not anticipated that dMyt1 would serve such functions, since Wee1 kinases are generally assumed to be responsible for checkpoint responses that protect the nucleus from premature Cdk1 activity. It was not clear that myt1 mutants were deficient in conventional premitotic checkpoint responses, however. Indeed, the partial decline in myt1 mutant PH3-labeled cells observed immediately after exposure to ionizing radiation could reflect activation of an otherwise dispensable Wee1-regulated premitotic checkpoint mechanism. The remaining PH3-positive cells that persisted long after irradiation in myt1 mutant discs could be arrested in mitosis by an alternative regulatory mechanism that was responsive to DNA damage. Further studies will be needed to clarify the respective roles of dMyt1 and dWee1 in cellular responses to DNA damage (Jin, 2008).

This study also observed profound defects in Cdk1 inhibitory phosphorylation in myt1 mutant imaginal discs. Phosphorylation of the T14 residue of Cdk1 was eliminated, demonstrating that dMyt1 is solely responsible for this regulatory modification, like Myt1 homologs described in other organisms. It was also observed that phosphorylation of the Y15 residue of Cdk1 was markedly reduced in myt1 mutant extracts, demonstrating for the first time that dMyt1 functions as a dual specificity Cdk1 inhibitory kinase, in vivo. Why dWee1 activity is insufficient for maintaining normal levels of phosphorylation of the Y15 residue is not clear, since Cdk1 complexes are thought to shuttle between the nucleus and cytoplasm. One possible explanation is that the doubly phosphorylated Cdk1 isoform may be more refractory to dephosphorylation by Cdc25 phosphatases, and hence more stably inhibited, than Cdk1 phosphorylated on a single residue. Another possibility is that the kinase-independent Myt1 mechanism proposed to tether phospho-inhibited Cdk1 complexes in the cytoplasm until cells are ready for mitosis might also protect them from dephosphorylation. Loss of either of these regulatory mechanisms could therefore underlie the cell-cycle defects observed in myt1 mutants. Testing these hypotheses promises to yield interesting new insights into cell-cycle regulation and the diverse developmental roles of dMyt1 and similar regulatory kinases in other organisms (Jin, 2008).

Control of PNG kinase, a key regulator of mRNA translation, is coupled to meiosis completion at egg activation

The oocyte-to-embryo transition involves extensive changes in mRNA translation, regulated in Drosophila by the PNG kinase complex whose activity is shown in this study to be under precise developmental control. Despite presence of the catalytic PNG subunit and the PLU and GNU activating subunits in the mature oocyte, GNU is phosphorylated at Cyclin B/CDK1 sites and unable to bind PNG and PLU. In vitro phosphorylation of GNU by CyclinB/CDK1 blocks activation of PNG. Meiotic completion promotes GNU dephosphorylation and PNG kinase activation to regulate translation. The critical regulatory effect of phosphorylation is shown by replacement in the oocyte with a phosphorylation-resistant form of GNU, which promotes PNG-GNU complex formation, elevation of Cyclin B, and meiotic defects consistent with premature PNG activation. After PNG activation GNU is destabilized, thus inactivating PNG. This short-lived burst in kinase activity links development with maternal mRNA translation and ensures irreversibility of the oocyte-to-embryo transition (Hara, 2017).

The massive changes in mRNA translation accompanying egg activation occur in a matter of minutes and must be linked to completion of meiosis in the oocyte. This study found that in Drosophila the solution to this developmental challenge is the regulation of PNG kinase activity. The results show that GNU is phosphorylated at CycB/CDK1 sites in mature oocytes, and in vitro CycB/CDK1 can directly phosphorylate GNU and thereby inhibit its ability to activate PNG kinase via inhibition of formation of the complex. In mature oocytes that are arrested at metaphase I, GNU is phosphorylated at CDK1 consensus sites and prevented from interaction with the PNG-PLU sub-complex. Following egg activation, as CycB protein and H1 kinase activity decline, GNU is dephosphorylated. This corresponds to the completion of meiosis and activation of the PNG kinase complex. Consistent with dephosphorylation of GNU being the crucial event for activation of PNG, substitution of a phosphorylation-resistant form of GNU into the oocyte results in premature elevation of CycB protein, implying PNG activation. Thus it is proposed that control of PNG kinase activity via GNU phosphorylation by CycB/CDK1 links meiotic completion and translational control of maternal mRNA to coordinate their timing precisely during egg activation. Active PNG leads to decreased GNU protein levels. This makes a negative feedback to shut down PNG kinase activity, thereby ensuring PNG kinase activity is constrained to the short developmental window of the oocyte-to-embryo transition (Hara, 2017).

These findings highlight the linchpin role the GNU subunit plays in the developmental control of PNG kinase activity. This regulation is exerted both at the levels of GNU protein and via its phosphorylation state. Although the presence of all three PNG kinase complex proteins is limited to late oogenesis through the early embryo, GNU is present over a narrower time window. GNU protein is undetectable in stage 10 oocytes and is rapidly accumulated during oocyte maturation. A previous genome-wide study showed that GNU protein accumulation during oocytes maturation relies on translational activation of its mRNA. Although the regulatory mechanisms for gnu translational activation remain to be defined, it is clearly dependent on CDK1, but not MOS, activity. Thus CDK1 promotes the appearance of GNU, permitting all PNG subunits to be present in the mature oocyte and poised for activation, while it simultaneously prevents activation by phosphorylation of GNU (Hara, 2017).

Several previous experimental observations now have clear significance in support of the role of CDK1 phosphorylation of GNU in inhibiting PNG complex formation and kinase activation. Ectopic GNU protein expression in early stage oocytes, which are in prophase I, causes png-dependent premature CycB protein expression and actin disorganization. Importantly, these defects result from expression of GNU at a developmental stage when although PNG and PLU are present at low levels, CycB/CDK1 is not active, and thus would not be able to block formation of the PNG complex. Analysis of the phosphorylation state of GNU in mutants for the calcipressin Sarah (sra) or the meiosis-specific APC/C activator Cortex (cort) showed that GNU remains hyperphosphorylated in eggs laid from both types of mutant mothers. Interestingly, both of these mutants fail to complete meiosis, with sra mutant eggs arrested in anaphase I, and cort mutants arrested in metaphase II. The failure of GNU to be dephosphorylated in both of these mutants agrees with the demonstration that GNU becomes hypophosphorylated after the completion of meiosis (Hara, 2017).

Although PNG and PLU levels in embryos are gradually decreased 2-4 hr after laying GNU seems to disappear after the completion of meiosis but before the initiation of embryogenesis. This is evidenced by GNU protein levels being decreased in unfertilized in vivo activated eggs, which complete meiosis II but do not enter into the embryonic cycles. This rapid GNU disappearance requires PNG kinase activity, revealing the existence of a negative feedback to shut off PNG activity shortly after its activation. It remains to be determined how GNU protein levels decline. PNG phosphorylates GNU on sites other than the CDK1 sites, and this phosphorylation may target GNU for degradation mediated by an E3 ubiquitin ligase of the SCF or the APC/C families. It is noted that GNU levels fail to decline in cort mutants, which lack one form of the APC/C. This may be an indirect effect of failure of dephosphorylation of the CDK1 sites in GNU and thus absence of PNG activation, but it is also possible that GNU is targeted directly to the APC/Ccort after completion of meiosis (Hara, 2017).

A key question raised by the demonstration of the regulatory role of GNU phosphorylation is whether developmental control of phosphatase activity is required. One possibility is that the phosphatase responsible for dephosphorylation of the CDK1 sites in GNU is constitutively active. CycB/CDK1 activity is high in the metaphase I-arrested mature oocyte, but egg activation triggers meiotic resumption and CDK1 inactivation. Reduction of CycB/CDK1 activity in the presence of active phosphatase may be sufficient for hypophosphorylated GNU. Alternatively, egg activation could lead to activation of a phosphatase. The identity of the GNU phosphatase for the CDK1 sites awaits elucidation. Although it has been shown that PP1 is capable of dephosphorylating GNU in mature oocytes, PP2A also is a possible GNU phosphatase during the process, particularly given it is the major phosphatase that removes phosphates on CDK1 substrates in mitotic exit (Hara, 2017).

The partial activity of the GNU 9A protein form provides insights into the mechanism by which GNU activates the PNG kinase. This mutant protein binds to the PNG-PLU sub-complex even in high CDK activity, however, it does not activate PNG kinase to the full extent that wild-type GNU does. This implies that there is an additional step to PNG kinase activation after GNU and the PNG-PLU sub-complex interaction. It is likely that the kinase activation following complex formation involves the N-terminus of GNU. A point mutation in the GNU N-terminal region (changing Pro 17 to Leu) retains ability to bind the PNG-PLU sub-complex but not does not activate PNG kinase activity in vitro (Hara, 2017).

The PNG kinase is significant for the understanding of how a kinase can rapidly control translation of hundreds of mRNAs. In addition to these insights into mRNA translation, identifying and defining the role of regulators involved in triggering the profound changes accompanying the oocyte-to-embryo transition is crucial for understanding of the onset of development, with implications for human fertility. This study has shown two forms of regulation of PNG kinase activity: one being regulation of protein expression of PNG kinase complex components and another being regulation of its activity. Strikingly, a cell cycle regulator, CDK1, controls both. This implies that CDK1 precisely regulates PNG kinase activity, a translational regulator, thus coordinating cell cycle progression and the translational landscape change during the oocyte-to-embryo transition. There are interesting parallels between the current findings and those in C. elegans. In C. elegans another kinase, the MBK-2 member of the conserved DYRK family of dual specificity tyrosine kinases, like PNG is crucial for the oocyte-to-embryo transition. MBK-2 activation is linked to the meiotic cell cycle by being downstream of the APC/C. MBK-2 controls proteolysis of oocyte proteins through the SCF E3 ubiquitin ligase, and it also affects RNA granule dynamics and thus likely impacts translation. Although PNG and MBK-2 are distinct kinases, these distantly related invertebrates utilize parallel approaches of coupling to cell cycle regulators to limit kinase activity to the oocyte-to-embryo transition. In both organisms, this links meiotic progression to gene expression changes after egg activation. This conservation suggests that this strategy may be employed for the onset of mammalian development (Hara, 2017).

Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk

According to the prevailing 'clock' model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the 'ruler' model. This study found that Cdk1 remains active during anaphase due to ongoing APC/C(Cdc20)- and APC/C(Cdh1)-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular 'rulers' and 'clocks' licenses mitotic exit only after proper chromosome separation (Afonso, 2019).

Taken together, this work reveals that degradation of B-type Cyclins specifically during anaphase is rate-limiting for mitotic exit among animals that diverged more than 900 million years ago. Most importantly, Cdk1 activity during anaphase was shown to be a function of chromosome separation and is spatially regulated by Aurora B localization and activity at the spindle midzone. In concert with previous work, the findings unveil an unexpected crosstalk between molecular 'rulers' (Aurora B) and 'clocks' (B-type Cyclins-Cdk1) that ensures that cells only exit mitosis after proper chromosome separation during anaphase, consistent with the previously proposed chromosome separation checkpoint hypothesis. An Aurora B-dependent spatial control mechanism regulating normal NER in human cells has been recently confirmed. However, nuclear envelope defects associated with incomplete chromosome separation during anaphase (namely, anaphase lagging chromosomes due to mitotic errors) were proposed as an inevitable pathological condition. The present work provides yet additional evidence for a molecular network operating during anaphase that promotes chromosome segregation fidelity by controlling mitotic exit in space and time. According to this model, APC/CCdc20 mediates the initial degradation of Cyclin B1 during metaphase under SAC control. The consequent decrease in Cdk1 activity as cells enter anaphase targets Aurora B to the spindle midzone (via Subito/Mklp2/kinesin-6); Aurora B at the spindle midzone (counteracted by PP1/PP2A phosphatases on chromatin establishes a phosphorylation gradient that locally delays APC/CCdc20- and APC/CCdh1-mediated degradation of residual Cyclin B1 (and possibly B3) at the spindle midzone, at least in Drosophila cells. Localization experiments in human cells suggest that Cdk1 itself might be enriched at the spindle midzone. Consequently, as chromosomes separate and move away from the spindle midzone, Cdk1 activity decreases, allowing the PP1/PP2A-mediated dephosphorylation of Cdk1 and Aurora B substrates (e.g., Lamin B and Condensin I) necessary for mitotic exit. This model is consistent with the recent demonstration that Cdk1 inactivation promotes the recruitment of PP1 phosphatase to chromosomes to locally oppose Aurora B phosphorylation and recent findings in budding yeast demonstrating equivalent phosphorylation and dephosphorylation events during mitotic exit. It is also consistent with a premature Greatwall inactivation and PP2A:B55 reactivation that would be predicted after acute Cdk1 inactivation during anaphase. Most important, this model provides an explanation for the coordinated action of two unrelated protein kinases that likely regulate multiple substrates required for mitotic exit (Afonso, 2019).

Previous landmark work has carefully monitored the kinetics of Cyclin B1 degradation in living human HeLa and rat kangaroo Ptk1 cells during mitosis, and concluded that Cyclin B1 was degraded by the end of metaphase, becoming essentially undetectable as cells entered anaphase. However, it was noticed that, consistent with the current findings, a small pool of Cyclin B1 continued to be degraded during anaphase in Ptk1 cells. Subsequent work investigating cellular response to anti-mitotic drugs has also shown that human DLD-1 cells undergoing normal mitosis entered anaphase with as much as 32% of Cyclin B1 compared to metaphase levels, suggesting that human cells enter anaphase with significant Cdk1 activity. Indeed, quantitative analysis with a FRET biosensor in human HeLa cells also revealed residual Cdk1 activity during anaphase. However, the significance of persistent Cdk1 activity for the control of anaphase duration and mitotic exit was not investigated in these original studies. Previous works also clearly demonstrated that forcing Cdk1 activity during anaphase through expression of non-degradable Cyclin B1 (and Cyclin B3 in Drosophila) prevents chromosome decondensation and NER. However, while these works suggested the existence of different Cyclin B1 thresholds that regulate distinct mitotic transitions, expression of non-degradable Cyclin B1 could be interpreted as an artificial gain of function that preserves Cdk1 activity during anaphase. For example, it was shown that expression of non-degradable Cyclin B1 during anaphase 'reactivates' the SAC, inhibiting APC/CCdc20. The current work demonstrates in five different experimental systems, from flies to humans, including primary tissues, that Cdk1 activity persists during anaphase and is rate-limiting for the control of mitotic exit. Failure to degrade B-type Cyclins during anaphase blocked cells in an anaphase-like state with separated sister chromatids that remained condensed for several hours, whereas complete Cdk1 inactivation in anaphase triggered chromosome decondensation and NER. Importantly, if a positive feedback loop imposed by phosphatases was sufficient to drive mitotic exit simply by reverting the effect of Cdk1 phosphorylation prior to anaphase, cells would exit mitosis regardless of the remaining pool of B-type Cyclins that sustains Cdk1 activity during anaphase. The main conceptual implication of these findings is that, contrary to what was previously assumed, mitotic exit is determined during anaphase and not at the metaphase-anaphase transition under SAC control (Afonso, 2019).

This model also implies that persistent Cyclin B1-Cdk1 in anaphase is spatially regulated by a midzone Aurora B gradient. Indeed, a residual pool of Cyclin B1-Cdk1 was identified enriched at the spindle midzone and midbody, this localization was dependent on Aurora B activity and localization at the spindle midzone. Interestingly, human Cyclin B2 (which contains a recognizable KEN box), as well as Cdk1, were identified at the midbody and Cdk1 inactivation during late mitosis was required for the timely completion of cytokinesis in human cells. Thus, it is possible that in human cells, Cdk1 activity during anaphase is regulated not only by Cyclin B1, but also by Cyclin B2. Importantly, this model predicted the existence of a midzone-centered Cdk1 activity gradient during anaphase, which was confirmed experimentally by targeting a FRET reporter of Cdk1 activity to chromosomes (Afonso, 2019).

Finally, the experiments indicate that Aurora B activity regulates Cyclin B1 homeostasis and consequently anaphase duration in the presence of incompletely separated chromosomes. One possibility is that direct Cyclin B1 phosphorylation by Aurora B spatially regulates Cyclin B1 degradation during anaphase, mediated by both APC/CCdc20 and APC/CCdh1. Another non-mutually exclusive possibility is that Aurora B indirectly controls Cyclin B1 during anaphase by regulating APC/CCdc20 and/or APC/CCdh1 activity, as recently shown for Cdk1. Future work will be necessary to test these hypotheses (Afonso, 2019).

In conclusion, this study has uncovered an unexpected level of regulation at the end of mitosis in metazoans and reconciled what were thought to be antagonistic models of mitotic exit relying either on molecular 'clocks' or on 'rulers'. These findings have profound implications to fundamental understanding of how tissue homeostasis is regulated, perturbation of which is a hallmark of human cancers (Afonso, 2019).


cDNA clone length - 1058

Bases in 5' UTR - 45

Bases in 3' UTR - 99


Amino Acids - 297

Structural Domains

The two Drosophila Cdc2 proteins, Cdc2 and Cdc2c exhibit a 56% sequence identity. Cdc2 is more closely related to the identified Cdc2 sequences of other species than is Cdc2c. This suggests that the evolutionary separation of Cdc2 and Cdc2c is ancient. The major differences between the Drosophila homologs are confined to regions that are not conserved among known p34cdc2 homologs (Lehner, 1990). Cdc2c functioning during G1 and S phases, partnering Cyclin E, is required for progression through S phase of the mitotic cycle (Knoblich, 1994).

cdc2: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 10 July 2021 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.