Mitotic control.

1990 has been a year of continued exciting developments in cell cycle control. Progress has occurred in delineating the mechanism of activation of maturation-promoting factor during entry into mitosis and the mechanism of cyclin degradation responsible for exit from mitosis. Notable advances have also occurred in our understanding of the dependence of mitotic entry on completion of DNA synthesis. Both genetic and biochemical data link this crucial checkpoint to the function of the cdc25 gene product and the extent of phosphorylation of Tyr15 in cdc2 kinase.

Identification of an activator required for elevation of maturation-promoting factor (MPF) activity by gamma-S-ATP.

Maturation-promoting factor (MPF) is a cell cycle control element able to cause cells to enter M-phase upon microinjection and will induce metaphase in nuclei incubated in cell extracts. Previous work has shown that MPF is composed of a complex between p34cdc 2 protein kinase and a B-type cyclin. In the present work gamma-S-ATP was found to cause activation of MPF activity in partially purified preparations, but this activation was lost upon chromatography on Matrex Green gel A. Readdition of other Matrex Green fractions to purified MPF restored the ability of gamma-S-ATP to activate MPF for nuclear breakdown as well as phosphorylation of histone H1. Use of the system described here will facilitate study of p34cdc 2 kinase activation and identification of elements involved in MPF regulation.

Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts.

Incubation of demembranated sperm chromatin in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in nuclear envelope assembly, chromosome decondensation, and sperm pronuclear formation. In contrast, egg extracts made with EGTA-containing buffers induced the sperm chromatin to form chromosomes or irregularly shaped clumps of chromatin that were incorporated into bipolar or multipolar spindles. The 150,000 g supernatants of the EGTA extracts could not alone support these changes in incubated nuclei. However, these supernatants induced not only chromosome condensation and spindle formation, but also nuclear envelope breakdown when added to sperm pronuclei or isolated Xenopus liver or brain nuclei that were incubated in extracts made without EGTA. Similar changes were induced by partially purified preparations of maturation-promoting factor. The addition of calcium chloride to extracts containing condensed chromosomes and spindles caused dissolution of the spindles, decondensation of the chromosomes, and re-formation of interphase nuclei. These results indicate that nuclear envelope breakdown, chromosome condensation, and spindle assembly, as well as the regulation of these processes by Ca2+-sensitive cytoplasmic components, can be studied in vitro using extracts of amphibian eggs.

Activation of p34cdc2 kinase by cyclin A.

Functional clam cyclin A and B proteins have been produced using a baculovirus expression system. Both cyclin A and B can induce meiosis I and meiosis II in Xenopus in the absence of protein synthesis. Half-maximal induction occurs at 50 nM for cyclin A and 250 nM for cyclin B. Addition of 25 nM cyclin A to activated Xenopus egg extracts arrested in the cell cycle by treatment with RNase or emetine activates cdc2 kinase to the normal metaphase level and stimulates one oscillatory cell cycle. High levels of cyclin A cause marked hyperactivation of cdc2 kinase and a stable arrest at the metaphase point in the cell cycle. Kinetic studies demonstrate the concentration of cyclin A added does not affect the 10 min lag period required for kinase activation or the timing of maximal activity, but does control the rate of deactivation of cdc2 kinase during exit from mitosis. In addition, exogenous clam cyclin A inhibits the degradation of both A- and B-type endogenous Xenopus cyclins. These results define a system for investigating the biochemistry and regulation of cdc2 kinase activation by cyclin A.

Purification and cloning of a protein kinase that phosphorylates and activates the polo-like kinase Plx1.

The Xenopus polo-like kinase 1 (Plx1) is essential during mitosis for the activation of Cdc25C, for spindle assembly, and for cyclin B degradation. Polo-like kinases from various organisms are activated by phosphorylation by an unidentified protein kinase. A protein kinase, polo-like kinase kinase 1 or xPlkk1, that phosphorylates and activates Plx1 in vitro was purified to near homogeneity and cloned. Phosphopeptide mapping of Plx1 phosphorylated in vitro by recombinant xPlkk1 or in progesterone-treated oocytes indicates that xPlkk1 may activate Plx1 in vivo. The xPlkk1 protein itself was also activated by phosphorylation on serine and threonine residues, and the kinetics of activation of xPlkk1 in vivo closely paralleled the activation of Plx1. Moreover, microinjection of xPlkk1 into Xenopus oocytes accelerated the timing of activation of Plx1 and the transition from G2 to M phase of the cell cycle. These results define a protein kinase cascade that regulates several events of mitosis.

Requirement for Cdk2 in cytostatic factor-mediated metaphase II arrest.

The unfertilized eggs of vertebrates are arrested in metaphase of meiosis II because of the activity of cytostatic factor (CSF). Xenopus CSF is thought to contain the product of the Mos proto-oncogene, but other proteins synthesized during meiosis II are also required for arrest induced by CSF. In Xenopus oocytes, ablation of synthesis of cyclin-dependent kinase 2 (Cdk2) during meiosis resulted in absence of the metaphase II block, even though the Mosxe protein kinase was fully active at metaphase. Introduction of purified Cdk2 restored metaphase II arrest, and increasing the amount of Cdk2 during meiosis I (when Mosxe is present) led to metaphase arrest at meiosis I. These data indicate that metaphase arrest is a result of cooperation between a proto-oncogene kinase and a cyclin-dependent kinase and illustrate the interaction of a cell growth regulator with a cell cycle control element.

Requirement of Cdk2-cyclin E activity for repeated centrosome reproduction in Xenopus egg extracts.

The abnormally high number of centrosomes found in many human tumor cells can lead directly to aneuploidy and genomic instability through the formation of multipolar mitotic spindles. To facilitate investigation of the mechanisms that control centrosome reproduction, a frog egg extract arrested in S phase of the cell cycle that supported repeated assembly of daughter centrosomes was developed. Multiple rounds of centrosome reproduction were blocked by selective inactivation of cyclin-dependent kinase 2-cyclin E (Cdk2-E) and were restored by addition of purified Cdk2-E. Confocal immunomicroscopy revealed that cyclin E was localized at the centrosome. These results demonstrate that Cdk2-E activity is required for centrosome duplication during S phase and suggest a mechanism that could coordinate centrosome reproduction with cycles of DNA synthesis and mitosis.

Induction of metaphase arrest in cleaving Xenopus embryos by the protein kinase p90Rsk.

Before fertilization, vertebrate eggs are arrested in metaphase of meiosis II by cytostatic factor (CSF), an activity that requires activation of the mitogen-activated protein kinase (MAPK) pathway. To investigate whether CSF arrest is mediated by the protein kinase p90Rsk, which is phosphorylated and activated by MAPK, a constitutively activated (CA) form of Rsk was expressed in Xenopus embryos. Expression of CA Rsk resulted in cleavage arrest, and cytological analysis showed that arrested blastomeres were in M phase with prominent spindles characteristic of meiotic metaphase. Thus, Rsk appears to be the mediator of MAPK-dependent CSF arrest in vertebrate unfertilized eggs.

Induction of metaphase arrest in cleaving Xenopus embryos by MAP kinase.

The natural arrest of vertebrate unfertilized eggs in second meiotic metaphase results from the activity of cytostatic factor (CSF). The product of the c-mos(xe) proto-oncogene is thought to be a component of CSF and can induce metaphase arrest when injected into blastomeres of two-cell embryos. The c-Mos(xe) protein can directly activate the mitogen-activated protein kinase kinase (MAP kinase kinase) in vitro, leading to activation of MAP kinase. MAP kinase and c-Mos(xe) are active in unfertilized eggs and are rapidly inactivated after fertilization. Microinjection of thiophosphorylated MAP kinase into one blastomere of a two-cell embryo induced metaphase arrest similar to that induced by c-Mos(xe). However, only arrest with c-Mos(xe) was associated with activation of endogenous MAP kinase. These results indicate that active MAP kinase is a component of CSF in Xenopus and suggest that the CSF activity of c-Mos(xe) is mediated by MAP kinase.

New insight into metaphase arrest by cytostatic factor: from establishment to release.

Since the discovery of cytostatic factor (CSF) 35 years ago, significant progress has been made in identifying molecular components of CSF activity and the mechanism of CSF-induced metaphase II arrest (CSF arrest). This short review focuses on recent discoveries in the field and discusses the implication of these results for a general picture of CSF establishment and release. One recent focus is on the cyclin E/Cdk2 pathway. The discovery of a downstream target for cyclin E/Cdk2, the spindle checkpoint protein Mps1, provides insight into how cyclin E/Cdk2 contributes to CSF arrest. The anaphase promoting complex/cyclosome (APC/C) inhibitor Emi2 is another recent focus of work in the field. It is now clear that not only is degradation of Emi2 critical for CSF release, but its abrupt accumulation during meiosis II (M II) is also required for the establishment of CSF arrest. Thus, by discrete pathways of APC/C inhibition operative during CSF arrest, the stability of cell cycle arrest in the egg appears to be reinforced by multiple mechanisms.

Developmental regulation of MCM replication factors in Xenopus laevis.

At the midblastula transition (MBT) during Xenopus laevis development, zygotic transcription begins [1], and the rapid, early cleavage cycles are replaced by cell-division cycles that lengthen and acquire G (gap) phases [2] and checkpoints [3-5]. This cell-cycle remodeling may result from either a loss of maternal products, the transcription of zygotic genes, or the replacement of maternal proteins by zygotic gene products. We have identified an example of the third possibility: distinct maternal and zygotic genes encoding a member of the minichromosome maintenance (MCM) protein family. The mcm genes were identified in yeast by mutations that blocked replication of artificial chromosomes or perturbed the G1/S transition in the cell cycle [6,7]. In Xenopus eggs, the MCM2-MCM7 proteins assemble as multimeric complexes at chromosomal origins of replication [8-14]. The sequential, cell-cycle-dependent assembly of the origin replication complex (ORC), CDC6 protein and the MCM complex at origins of replication ensures that DNA replicates only once per cell cycle [15,16]. The periodic association of the MCM complex with chromatin may be regulated via phosphorylation by cyclin-dependent kinases (Cdks) [11]. We have cloned the first example of a developmentally regulated mcm gene, zygotic mcm6 (zmcm6), expressed only after gastrulation when the cell cycle is remodeled. The zMCM6 protein assembles into MCM complexes and differs from maternal MCM6 (mMCM6) in having a carboxy-terminal extension and a consensus cyclin-Cdk phosphorylation site. There may also be maternal-zygotic pairs of other MCMs. These data suggest that MCMs are critical for cell-cycle remodeling during early Xenopus development.

Activation of the anaphase-promoting complex and degradation of cyclin B is not required for progression from Meiosis I to II in Xenopus oocytes.

Sister chromatid separation and cyclin degradation in mitosis depend on the association of the anaphase-promoting complex (APC) with the Fizzy protein (Cdc20), leading to the metaphase/anaphase transition and exit from mitosis [1--3]. In Xenopus, after metaphase of the first meiotic division, only partial cyclin degradation occurs, and chromosome segregation during anaphase I proceeds without sister chromatid separation [4--7]. We investigated the role of xFizzy during meiosis using an antisense depletion approach. xFizzy accumulates to high levels in Meiosis I, and injection of antisense oligonucleotides to xFizzy blocks nearly all APC-mediated cyclin B degradation and Cdc2/cyclin B (MPF) inactivation between Meiosis I and II. However, even without APC activation, xFizzy-ablated oocytes progress to Meiosis II as shown by cyclin E synthesis, further accumulation of cyclin B, and evolution of the metaphase I spindle to a metaphase II spindle via a disc-shaped aggregate of microtubules known to follow anaphase I [8]. Inhibition of the MAPK pathway by U0126 in antisense-injected oocytes prevents cyclin B accumulation beyond the level that is present at metaphase I. Full synthesis and accumulation can be restored in the presence of U0126 by the expression of a constitutively active form of the MAPK target, p90(Rsk). Thus, p90(Rsk) is sufficient not only to partially inhibit APC activity [7], but also to stimulate cyclin B synthesis in Meiosis II.

The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk).

BACKGROUND: During oocyte maturation in Xenopus, progesterone induces entry into meiosis I, and the M phases of meiosis I and II occur consecutively without an intervening S phase. The mitogen-activated protein (MAP) kinase is activated during meiotic entry, and it has been suggested that the linkage of M phases reflects activation of the MAP kinase pathway and the failure to fully degrade cyclin B during anaphase I. To analyze the function of the MAP kinase pathway in oocyte maturation, we used U0126, a potent inhibitor of MAP kinase kinase, and a constitutively active mutant of the protein kinase p90(Rsk), a MAP kinase target. RESULTS: Even with complete inhibition of the MAP kinase pathway by U0126, up to 90% of oocytes were able to enter meiosis I after progesterone treatment, most likely through activation of the phosphatase Cdc25C by the polo-like kinase Plx1. Subsequently, however, U0126-treated oocytes failed to form metaphase I spindles, failed to reaccumulate cyclin B to a high level and failed to hyperphosphorylate Cdc27, a component of the anaphase-promoting complex (APC) that controls cyclin B degradation. Such oocytes entered S phase rather than meiosis II. U0126-treated oocytes expressing a constitutively active form of p90(Rsk) were able to reaccumulate cyclin B, hyperphosphorylate Cdc27 and form metaphase spindles in the absence of detectable MAP kinase activity. CONCLUSIONS: The MAP kinase pathway is not essential for entry into meiosis I in Xenopus but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. Moreover, one substrate of MAP kinase, p90(Rsk), is sufficient to mediate these effects during oocyte maturation.

Cip1 blocks the initiation of DNA replication in Xenopus extracts by inhibition of cyclin-dependent kinases.

BACKGROUND: Cip1 is a 21 kD protein that interacts with and inhibits cyclin-dependent kinases (cdks). Expression of Cip1 is induced by the tumour suppressor p53, and tumour cells have greatly reduced levels of Cip1. As cdks are required for normal progression through the cell cycle, their inhibition by Cip1 may mediate the ability of p53 to block cell proliferation. Cip1 has also been shown to inhibit the DNA polymerase delta auxiliary factor PCNA (proliferating cell nuclear antigen), which is required for replication-fork elongation, and this could be an alternative mechanism by which p53-induced Cip1 blocks cell proliferation. RESULTS: We have investigated the effect of Cip1 protein on chromosomal DNA replication, using cell-free extracts of Xenopus eggs that initiate and complete chromosome replication under normal cell-cycle control. Cip1 protein strongly inhibited an early stage of DNA replication in this system, and this inhibition was not complemented by extracts that had been affinity-depleted of cdks. In contrast, Cip1 did not inhibit the elongation of replication forks that had accumulated in the presence of aphidicolin. Cip1 inhibition of DNA replication was fully rescued by addition of cyclins A or E, but not cyclin B, cdk2 or PCNA. CONCLUSIONS: Our results suggest that Cip1 specifically blocks the initiation of DNA replication by inhibition of a cyclin-dependent kinase (cdk2), but has no major effect on the elongation of preassembled replication forks. The ability of cyclin A or cyclin E to rescue the Cip1 inhibition suggests that these cyclins may play a direct role in the initiation of replication in the Xenopus system.

Bub1 is activated by the protein kinase p90(Rsk) during Xenopus oocyte maturation.

BACKGROUND: The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle. In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC. However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk) involves components of the spindle assembly checkpoint. RESULTS: xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bub1 in the presence of U0126. Moreover, purified p90(Rsk) phosphorylates Bub1 in vitro and increases its protein kinase activity. CONCLUSIONS: Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90(Rsk), is sufficient to activate Bub1 in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90(Rsk) and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk).

Phosphorylation and protein synthetic events in Xenopus laevis oocytes microinjected with pp60v-src.

Microinjection of purified pp60v-src, the transforming protein of Rous sarcoma virus, into Xenopus laevis oocytes accelerated the rate of progesterone- or insulin-induced meiotic maturation. This acceleration was abolished by incubating the oocytes with cycloheximide or puromycin during a 2-h interval between pp60v-src microinjection and progesterone addition. In contrast, exposure to actinomycin D did not alter the acceleration of maturation by microinjected pp60v-src. Associated with progesterone treatment and pp60v-src microinjection were a number of qualitative changes in phosphoproteins; a few of these changes are common to both stimuli. These results indicate that the action of pp60v-src in oocytes involves both phosphorylation and protein synthetic events that affect oocyte maturation.

Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions.

The cdc2 kinase and B-type cyclins are known to be components of maturation- or M-phase-promoting factor (MPF). Phosphorylation of cyclin B has been reported previously and may regulate entry into and exit from mitosis and meiosis. To investigate the role of cyclin B phosphorylation, we replaced putative cdc2 kinase phosphorylation sites in Xenopus cyclins B1 and B2 by using oligonucleotide site-directed mutagenesis. We found that Ser-90 of cyclin B2 and Ser-94 or Ser-96 of cyclin B1 are the main phosphorylation sites both in functional Xenopus egg extracts and after phosphorylation with purified MPF in vitro. Microtubule-associated protein (MAP) kinase from Xenopus eggs phosphorylated cyclin B1 significantly at Ser-94 or Ser-96, whereas it was largely inactive against cyclin B2. The substitutions that ablated phosphorylation at these sites, however, resulted in no functional differences between mutant and wild-type cyclin, as judged by the kinetics of M-phase degradation, induction of mitosis in egg extracts, or induction of oocyte maturation. These results indicate that the phosphorylation of Xenopus B-type cyclins by cdc2 kinase or MAP kinase is not required for the hallmark functions of cyclin.

Transforming ras proteins accelerate hormone-induced maturation and stimulate cyclic AMP phosphodiesterase in Xenopus oocytes.

Transforming Harvey (Ha) ras oncogene products accelerated the time course of Xenopus oocyte maturation induced by insulin, insulinlike growth factor 1, or progesterone. The transforming constructs, [Val-12]Ha p21 and [Val-12, Thr-59]Ha p21, displayed equal potency and efficacy in their abilities to accelerate the growth peptide-induced response. Normal Ha p21 was only 60% as powerful and one-fifth as potent as the mutants containing valine in the 12 position. In contrast, two nontransforming constructs, [Val-12, Ala-35, Leu-36, Thr-59]Ha p21 and [Val-12, Thr-59]Ha(term-174) p21, had no effect on the time course of hormone-induced maturation. Effects of the transforming ras proteins on hormone-induced maturation correlated with their abilities to stimulate in vivo phosphodiesterase activity measured after microinjection of 200 microM cyclic [3H] AMP. When p21 injection followed 90 min of insulin treatment, there was no increase in phosphodiesterase activity over that measured after hormone treatment or p21 injection alone, but additive effects of p21 and insulin on enzyme activity were observed during the first 90 min of insulin treatment. Even though normal Ha p21 and transforming [Val-12, Thr-59]Ha p21 stimulated oocyte phosphodiesterase to equal levels when coinjected with substrate at the initiation of the in vivo assay, the transforming protein elicited a more sustained stimulation of enzyme activity. These results suggest that stimulation of a cyclic AMP phosphodiesterase activity associated with insulin-induced maturation is involved in the growth-promoting actions of ras oncogene products in Xenopus oocytes.

Metaphase protein phosphorylation in Xenopus laevis eggs.

Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior.

Microinjection of pp60v-src into Xenopus oocytes increases phosphorylation of ribosomal protein S6 and accelerates the rate of progesterone-induced meiotic maturation.

Microinjection of purified pp60v-src into Xenopus oocytes caused the phosphorylation of ribosomal protein S6 on serine residues and also increased total protein phosphorylation, with almost a two-fold increase in the percentage of phosphotyrosine present. In addition, pp60v-src accelerated the time course of progesterone-induced oocyte maturation, suggesting that the biochemical pathway influenced by pp60v-src is related to that induced by progesterone.