Role for cyclin A in the dependence of mitosis on completion of DNA replication.

THE cyclins were first identified by their cell-cycle-dependent synthesis and destruction and have a key role in the control of mitosis in Xenopus embryonic cell cycles. All higher eukaryotes have at least two types of cyclins, the A- and B-type, which can be distinguished by sequence motifs and the timing of their destruction in the cell cycle. The degradation of both cyclins is required for exit from mitosis, but the activation and destruction of cyclin A occur earlier in the cell cycle than with the B-type cyclins. This suggests that cyclin A has a distinct role in cell-cycle progression. We have used an antisense oligodeoxy-nucleotide directed against cyclin A to investigate this role. Ablation of cyclin A messenger RNA in cytostatic factor/metaphase-arrested extracts of Xenopus eggs, followed by in vitro progression into interphase, resulted in the premature appearance of cyclin B-cdc2-associated H1 kinase activity and premature entry into mitosis. Although cyclin A-ablated extracts could initiate DNA synthesis during interphase, S phase was not completed before entry into mitosis. The effects of cyclin A ablation were reversed by the addition of cyclin A mRNA or cyclin A protein to the extracts.     

Transient activation of calcineurin is essential to initiate embryonic development in Xenopus laevis

At fertilization, an increase of cytosolic calcium ions (Ca2+) triggers various activation responses in animal eggs. In vertebrates, these responses include exit from metaphase arrest in meiosis II (MII exit) and cortical remodelling initiated by cortical granule exocytosis. Although the essential requirement of Ca2+/calmodulin-dependent protein kinase II for inducing MII exit has been documented, a role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in egg activation has not been investigated. Here we show, using cell-free extracts from unfertilized eggs of Xenopus laevis, that calcineurin is transiently activated immediately after Ca2+ addition to a concentration that induces MII exit. When calcineurin activation is inhibited, cyclin-dependent kinase 1 (Cdk1) inactivation by means of cyclin B degradation is prevented and sperm chromatin incubated in the extracts remains condensed. Similarly, if calcineurin is inhibited in intact eggs, MII exit on egg activation is prevented. In addition, the activation contraction in the cortex is suppressed whereas cortical granule exocytosis occurs. We further demonstrate that, when a high level of calcineurin activity is maintained after activation, growth of sperm asters is prevented in egg extracts and, consistently, migration of male and female pronuclei towards each other is hindered in fertilized eggs. Thus, both activation and the subsequent inactivation of calcineurin in fertilized eggs are crucial for the commencement of vertebrate embryonic development.     

Release of mature starfish oocytes from interphase arrest by microinjection of human centrosomes

Mature oocytes (unfertilized eggs) are arrested at definite cell-cycle stages which vary from species to species. In frogs and mammals, the oocytes are arrested at the second metaphase of meiosis whereas in echinoderms they are blocked later, at the pronucleus stage. What causes the maturing oocytes to stop at some point in the cell cycle is not entirely clear. In frogs, the metaphase arrest seems to be maintained by a cytostatic factor. In echinoderms, which stop at interphase, no such a factor has so far been found. The fertilization process, beyond the introduction of paternal chromosomes, releases the oocyte from cell-cycle arrest and provides a functional centrosome to replace the endogenous centrosome which is apparently lost during oogenesis in most species. Several lines of evidence suggest that release from cell-cycle arrest is mediated by a Ca2+ burst which is associated with fertilization, and it is known that the functional centrosome provided by the sperm is necessary for mitotic spindle formation and cleavages. We report here that microinjection of purified human centrosomes into mature starfish oocytes is sufficient to release them from arrest at interphase and to support many cleavages leading to the occasional formation of normal embryos. In this species centrosome induced re-entry into the cell cycle does not require a transient calcium burst nor does it require intact microtubules.     

Triggering of cyclin degradation in interphase extracts of amphibian eggs by cdc2 kinase

The cell cycles of early Xenopus embryos consist of a rapid succession of alternating S and M phases. These cycles are controlled by the activity of a protein kinase complex (cdc2 kinase) which contains two subunits. One subunit is encoded by the frog homologue of the fission yeast cdc2+ gene, p34cdc2 and the other is a cyclin. The concentration of cyclins follows a sawtooth oscillation because they accumulate in interphase and are destroyed abruptly during mitosis. The association of cyclin and p34cdc2 is not sufficient for activation of cdc2 kinase, however; dephosphorylation of key tyrosine and threonine residues of p34cdc2 is necessary to turn on its kinase activity. The activity of cdc2 kinase is thus regulated by a combination of translational and post-translational mechanisms. The loss of cdc2 kinase activity at the end of mitosis depends on the destruction of the cyclin subunits. It has been suggested that this destruction is induced by cdc2 kinase itself, thereby providing a negative feedback loop to terminate mitosis. Here we report direct experimental evidence for this idea by showing that cyclin proteolysis can be triggered by adding cdc2 kinase to a cell-free extract of interphase Xenopus eggs.     

Emi1 is required for cytostatic factor arrest in vertebrate eggs

Vertebrate eggs are arrested at metaphase of meiosis II with stable cyclin B and high cyclin B/Cdc2 kinase activity. The ability of the anaphase-promoting complex/cyclosome (APC), an E3 ubiquitin ligase, to trigger cyclin B destruction and metaphase exit is blocked in eggs by the activity of cytostatic factor (CSF) (reviewed in ref. 1). CSF was defined as an activity in mature oocytes that caused mitotic arrest when injected into dividing embryos. Fertilization causes a transient increase in cytoplasmic calcium concentration leading to CSF inactivation, APC activation, cyclin B destruction and mitotic exit. The APC activator Cdc20 is required for APC activation after fertilization. We show here that the APC(cdc20) inhibitor Emi1 (ref. 6) is necessary and sufficient to inhibit the APC and to prevent mitotic exit in CSF-arrested eggs. CSF extracts immunodepleted of Emi1 degrade cyclin B, and exit from mitosis prematurely in the absence of calcium. Addition of Emi1 to these Emi1-depleted extracts blocks premature inactivation of the CSF-arrested state. Emi1 is required to arrest unfertilized eggs at metaphase of meiosis II and seems to be the long-sought mediator of CSF activity.     

Phosphorylation of Erp1 by p90rsk is required for cytostatic factor arrest in Xenopus laevis eggs

Until fertilization, the meiotic cell cycle of vertebrate eggs is arrested at metaphase of meiosis II by a cytoplasmic activity termed cytostatic factor (CSF), which causes inhibition of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets mitotic cyclins-regulatory proteins of meiosis and mitosis-for degradation. Recent studies indicate that Erp1/Emi2, an inhibitor protein for the APC/C, has an essential role in establishing and maintaining CSF arrest, but its relationship to Mos, a mitogen-activated protein kinase (MAPK) kinase kinase that also has an essential role in establishing CSF arrest through activation of p90 ribosomal S6 kinase (p90rsk), is unclear. Here we report that in Xenopus eggs Erp1 is a substrate of p90rsk, and that Mos-dependent phosphorylation of Erp1 by p90rsk at Thr 336, Ser 342 and Ser 344 is crucial for both stabilizing Erp1 and establishing CSF arrest in meiosis II oocytes. Semi-quantitative analysis with CSF-arrested egg extracts reveals that the Mos-dependent phosphorylation of Erp1 enhances, but does not generate, the activity of Erp1 that maintains metaphase arrest. Our results also suggest that Erp1 inhibits cyclin B degradation by binding the APC/C at its carboxy-terminal destruction box, and this binding is also enhanced by the Mos-dependent phosphorylation. Thus, Mos and Erp1 collaboratively establish and maintain metaphase II arrest in Xenopus eggs. The link between Mos and Erp1 provides a molecular explanation for the integral mechanism of CSF arrest in unfertilized vertebrate eggs.     

Calcium triggers exit from meiosis II by targeting the APC/C inhibitor XErp1 for degradation

Vertebrate eggs awaiting fertilization are arrested at metaphase of meiosis II by a biochemical activity termed cytostatic factor (CSF). This activity inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that triggers anaphase onset and mitotic/meiotic exit by targeting securin and M-phase cyclins for destruction. On fertilization a transient rise in free intracellular calcium causes release from CSF arrest and thus APC/C activation. Although it has previously been shown that calcium induces the release of APC/C from CSF inhibition through calmodulin-dependent protein kinase II (CaMKII), the relevant substrates of this kinase have not been identified. Recently, we characterized XErp1 (Emi2), an inhibitor of the APC/C and key component of CSF activity in Xenopus egg extract. Here we show that calcium-activated CaMKII triggers exit from meiosis II by sensitizing the APC/C inhibitor XErp1 for polo-like kinase 1 (Plx1)-dependent degradation. Phosphorylation of XErp1 by CaMKII leads to the recruitment of Plx1 that in turn triggers the destruction of XErp1 by phosphorylating a site known to serve as a phosphorylation-dependent degradation signal. These results provide a molecular explanation for how the fertilization-induced calcium increase triggers exit from meiosis II.     

Phosphorylation of two small GTP-binding proteins of the Rab family by p34cdc2

Entry of a cell into mitosis induces a series of structural and functional changes including arrest of intracellular transport. Knowledge of how the mitotic cycle is driven progressed substantially with the identification of the p34cdc2 protein kinase as a subunit of maturation-promoting factor, the universal regulating component of the mitotic cycle. Activation of the kinase at the onset of mitosis is thought to trigger the important mitotic events by phosphorylating key proteins. Small guanine nucleotide-binding proteins have been implicated in regulating transport pathways. For instance, two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, control distinct stages of the secretory pathway in budding yeast. The GTP-binding proteins of the Rab family in rats and humans display strong homologies with Sec4p and Ypt1p, and might therefore also be involved in regulating intracellular transport. Indeed, distinct Rab proteins are located in the exocytotic and endocytotic compartments. Interruption of vesicular transport during mitosis might involve modification of these proteins. We now present biochemical evidence for a mitosis-specific p34cdc2 phosphorylation of Rab1Ap and Rab4p. By contrast, Rab2p and Rab6p are not phosphorylated. We also show that the distribution of Rab1Ap and Rab4p between cytosolic and membrane-bound forms is different in interphase and mitotic cells. This may provide a clue to the mechanism by which phosphorylation could affect membrane traffic during mitosis.     

Cyclin synthesis drives the early embryonic cell cycle

We have produced extracts of frog eggs that can perform multiple cell cycles in vitro. Destruction of the endogenous messenger RNA arrests the extracts in interphase. The addition of exogenous cyclin mRNA is sufficient to produce multiple cell cycles. The newly synthesized cyclin protein accumulates during each interphase and is degraded at the end of each mitosis.     

A direct link of the Mos-MAPK pathway to Erp1/Emi2 in meiotic arrest of Xenopus laevis eggs

In vertebrates, unfertilized eggs (or mature oocytes) are arrested at metaphase of meiosis II by a cytoplasmic activity called cytostatic factor (CSF). The classical Mos-MAPK pathway has long been implicated in CSF arrest of vertebrate eggs, but exactly how it exerts CSF activity remains unclear. Recently, Erp1 (also called Emi2), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C) required for degradation of the mitotic regulator cyclin B (ref. 5), has also been shown to be a component of CSF in both Xenopus and mice. Erp1 is destroyed on fertilization or egg activation, like Mos. However, despite these similarities the Mos-MAPK (mitogen-activated protein kinase) pathway and Erp1 are thought to act rather independently in CSF arrest. Here, we show that p90rsk, the kinase immediately downstream from Mos-MAPK, directly targets Erp1 for CSF arrest in Xenopus oocytes. Erp1 is synthesized immediately after meiosis I, and the Mos-MAPK pathway or p90rsk is essential for CSF arrest by Erp1. p90rsk can directly phosphorylate Erp1 on Ser 335/Thr 336 both in vivo and in vitro, and upregulates both Erp1 stability and activity. Erp1 is also present in early embryos, but has little CSF activity owing, at least in part, to the absence of p90rsk activity. These results clarify the direct link of the classical Mos-MAPK pathway to Erp1 in meiotic arrest of vertebrate oocytes.     

Independent inactivation of MPF and cytostatic factor (Mos) upon fertilization of Xenopus eggs.

In vertebrates, mature eggs are arrested at the second meiotic metaphase by the cytostatic factor (CSF), now known to be the c-mos proto-oncogene product (Mos). Fertilization or egg activation triggers a transient increase in the cytoplasmic free calcium and releases the meiotic arrest by inactivating maturation/mitosis-promoting factor (MPF). CSF or Mos, which is also inactivated by the calcium transient, seems to stabilize MPF in mature eggs and CSF-injected embryos. Thus, it was assumed that CSF inactivation is the primary cause of MPF inactivation on meiotic release. We have directly compared the degradation kinetics of CSF (Mos) and MPF during meiotic release, using the same batch of Xenopus eggs. We report here that, at the molecular level, cyclin subunits of MPF are degraded before Mos is degraded and, at the physiological level, that MPF activity is inactivated before CSF activity during activation of Xenopus eggs. These results, in conjunction with circumstantial evidence, support the novel view that a calcium transient on fertilization induces a CSF-independent pathway for MPF inactivation, whereas CSF inactivation during meiotic release serves only to allow the fertilized egg to enter mitosis.     

Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2

Membrane transport between the endoplasmic reticulum and the plasma membrane, which involves the budding and fusion of carrier vesicles, is inhibited during mitosis in animal cells. At the same time, the Golgi complex and the nuclear envelope, as well as the endoplasmic reticulum in some cell types, become fragmented. Fragmentation of the Golgi is believed to facilitate its equal partitioning between daughter cells. In fact, it has been postulated that both the inhibition of membrane traffic and Golgi fragmentation during mitosis are due to an inhibition of vesicle fusion, while vesicle budding continues. Although less is known about the endocytic pathway, internalization and receptor recycling are also arrested during mitosis. We have now used a cell-free assay to show that the fusion of endocytic vesicles from baby hamster kidney cells is reduced in Xenopus mitotic cytosol when compared with interphase cytosol. We reconstituted this inhibition in interphase cytosol by adding a preparation enriched in the starfish homologue of the cdc2 protein kinase. Inhibition was greater than or equal to 90% when the added cdc2 activity was in the range estimated for that in mitotic Xenopus eggs, which indicates that during mitosis the cdc2 kinase mediates an inhibition of endocytic vesicle fusion, and possibly other fusion events in membrane traffic.     

Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry

Oscillations in cyclin-dependent kinase (CDK) activity drive the somatic cell cycle. After entry into mitosis, CDKs activate the anaphase-promoting complex (APC), which then promotes cyclin degradation and mitotic exit. The re-accumulation of cyclin A causes the inactivation of APC and entry into S phase, but how cyclin A can accumulate in the presence of active APC has remained unclear. Here we show that, during G1, APC autonomously switches to a state permissive for cyclin A accumulation. Crucial to this transition is the APC(Cdh1)-dependent autoubiquitination and proteasomal degradation of the ubiquitin-conjugating enzyme (E2) UbcH10. Because APC substrates inhibit the autoubiquitination of UbcH10, but not its E2 function, APC activity is maintained as long as G1 substrates are present. Thus, through UbcH10 degradation and cyclin A stabilization, APC autonomously downregulates its activity. This indicates that the core of the metazoan cell cycle could be described as a self-perpetuating but highly regulated oscillator composed of alternating CDK and APC activities.     

Cell transformation and activation of pp60c-src by overexpression of a protein tyrosine phosphatase.

The kinase activity of pp60c-src is specifically and transiently increased during mitosis and repressed during interphase. Loss of cell-cycle control of pp60c-src occurs on mutation of Tyr527 to Phe or when pp60c-src is associated with polyoma middle-T-antigen, and these conditions result in cell transformation or tumorigenesis. In both cases, pp60c-src has elevated kinase activity which is maintained throughout the cell cycle and accompanied by dephosphorylation of the carboxy-terminal negative regulatory Tyr527 site, or mimicry of Tyr527 dephosphorylation in the case of the mutant. Here we report that overexpression of the receptor-like protein tyrosine phosphatase PTP alpha results in persistent activation of pp60c-src kinase, with concomitant cell transformation and tumorigenesis. In PTP alpha-overexpressing cells, the pp60c-src kinase activation is accompanied by dephosphorylation at Tyr527, and direct dephosphorylation of this site by purified PTP alpha occurs in vitro. Our results suggest that PTP alpha is involved in the regulation of cell proliferation, exerting at least some of its effects through pp60c-src kinase, and has oncogenic capability when overexpressed.