Cyclin B


Activation of cyclin-dependent kinases

Activation of the cyclin-dependent kinase to promote cell cycle progression requires their association with cyclins as well as phosphorylation of a threonine residue. This phosphorylation is carried out by the Cdk-activating kinase (CAK) (see Drosophila Cyclin-dependent kinase 7). Purification of CAK from mammals, starfish, and Xenopus has identified it as a heterotrimeric complex composed of a catalytic subunit, p40MO15/cdk7, a regulatory subunit, cyclin H, and an assembly factor, MAT1. CAK phosphorylates not only c34cdc2 (see Drosophila cdc2 but also other Cdks, including p33cdk2 and cdk4, which function earlier in the cell cycle. Additionally, the CAK subunits are components of TFIIH, a basal transcription factor involved in the initiation of transcription, phosphorylation of the C-terminal domain of the large subunit of RNA polymerase II and DNA repair. The cloning of the CAK from S. cerevisiae raises the possibility that the the predominant CAK in vertebrate cell extracts, may not function as a physiological CAK. S. cerevisiae CAK is active as a monomer and is not a component of the basal transcription factor (Kaldis, 1996 and references).

Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. These results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis (Long, 1998).

In starfish, fertilization occurs naturally at late meiosis I. In the absence of fertilization, however, oocytes complete meiosis I and II, resulting in mature eggs, which are still fertilizable, arrested at the pronucleus stage. In this study, cDNAs of starfish cyclin A and Cdc2 were isolated and the cell cycle dynamics of cyclin A and cyclin B levels and their associated Cdc2 kinase activity were monitored. Tyr phosphorylation of Cdc2, and Cdc25 phosphorylation states were examined throughout meiotic and early embryonic cleavage cycles in vivo. In meiosis I, cyclin A is undetectable and cyclin B/Cdc2 alone exhibits histone H1 kinase activity; thereafter, both cyclin A/Cdc2 and cyclin B/Cdc2 kinase activity oscillates along with the cell cycle. Cyclin B-associated Cdc2 (but not cyclin A-associated Cdc2) is subjected to regulation via Tyr phosphorylation. With some exceptions, phosphorylation states of Cdc25 correlate with cyclin B/Cdc2 kinase activity. Between meiosis I and II and at the pronucleus stage, cyclin A and B levels remain low, Cdc2 Tyr phosphorylation is undetectable, and Cdc25 remains phosphorylated depending on MAP kinase activity, showing a good correlation between these two stages. Upon fertilization of mature eggs, Cdc2 Tyr phosphorylation reappears and Cdc25 is dephosphorylated. In the first cleavage cycle, under conditions which prevent Cdc25 activity, cyclin A/Cdc2 is activated with a normal time course and then cyclin B/Cdc2 is activated with a significant delay, resulting in the delayed completion of M-phase. Thus, in contrast to meiosis I, both cyclin A and cyclin B appear to be involved in the embryonic cleavage cycles. It is proposed that regulation of cyclin A/Cdc2 and cyclin B/Cdc2 is characteristic of meiotic and early cleavage cycles (Okano-Uchida, 1998).

When treated with 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), a natural maturation-inducing hormone in fishes, fully grown zebrafish oocytes are induced to mature via the activation of the maturation-promoting factor (MPF), which consists of cdc2 (a catalytic subunit) and cyclin B (a regulatory subunit). In contrast, 17alpha,20beta-DP is unable to induce growing (previtellogenic and vitellogenic) oocytes to mature. To know the reason growing oocytes fail to mature upon 17alpha,20beta-DP treatment, changes in the components of machinery responsible for MPF activation were investigated during zebrafish oogenesis. Immunoblotting experiments using monoclonal antibodies against cdc2, cyclin B, and cdk7 (an activator of cdc2) have revealed that the concentrations of cdc2 and cdk7 are almost constant during oogenesis. Cyclin B is present in mature oocytes but absent in growing and fully grown immature oocytes. These results, which are identical to those in goldfish, strongly suggest that cyclin B is synthesized from stored (masked) mRNA after 17alpha,20beta-DP stimulation; cyclin B's binding to the preexisting cdc2 allows cdk7 to activate MPF. Microinjection of cyclin B protein induces MPF activation and germinal vesicle breakdown in growing oocytes, as well as in fully grown oocytes, indicating that cdk7 present in growing oocytes is already active. Northern blot analysis revealed the presence of cyclin B mRNA in both previtellogenic and fully grown oocytes. These results indicate that, as in fully grown oocytes, growing oocytes are already equipped with the catalytic subunit of MPF (cdc2) and its activator (cdk7) and that the appearance of the regulatory subunit of MPF (cyclin B) is sufficient for initiating maturation. Therefore, the unresponsiveness of growing oocytes to 17alpha,20beta-DP is attributable to a deficiency in the processes leading to cyclin B synthesis, which include 17alpha,20beta-DP reception on the oocyte surface, subsequent signal transduction pathways, and unmasking the stored cyclin B mRNA (Kondo, 1997).

When full-grown oocytes of the newt Cynops pyrrhogaster are treated with progesterone in O-R2 solution containing antibiotics, approximately 85% of the oocytes complete meiosis synchronously. Maturation-promoting factor (MPF) activity appears just before germinal vesicle breakdown (GVBD) and the oocytes maintain high MPF activity throughout metaphase I and metaphase II of meiosis. A slight decrease of MPF activity is observed at the first polar body emission. The distribution of cyclin B1 was investigated with anti-cyclin B1 antibody. No cyclin B1 is found in the oocytes before progesterone treatment. Cyclin B1 appears in the cortex of animal hemispheres, especially around and inside the germinal vesicle just before GVBD. A large amount of cyclin B1 accumulates at metaphase I; approximately half disappears at the first polar body emission, and then cyclin B1 accumulates again at metaphase II. An inactive form of cdc2 kinase is observed in both the germinal vesicles and the oocyte cytoplasm, while an active form appears at the M phase. No MPF is observed in the oocytes from which the germinal vesicle has been removed. A cdk7-like molecule is localized in the germinal vesicle, but not in oocyte cytoplasm, indicating that inactive cdc2 kinase associated with cyclin B1 derived from cytoplasm is activated by phosphorylation in the germinal vesicle. The changes in the amount of cyclin B1 are synchronous with the first cell cycle after fertilization. Cyclin B1 is primarily localized in the cortex of the animal hemisphere. A shift in band mobility upon electrophoresis of cyclin B1 is observed from samples taken during the cell cycle; this shift is probably due to the protein's phosphorylation state (Sakamoto, 1998).

To study the mechanisms involved in the progression of meiotic maturation in the mouse, oocytes from two strains of mice, CBA/Kw and KE, were used which differ greatly in the rate at which they undergo meiotic maturation. CBA/Kw oocytes extrude the first polar body about 7 hours after breakdown of the germinal vesicle (GVBD), whilst the oocytes from KE mice take approximately 3-4 hours longer. In both strains, the kinetics of spindle formation are comparable. While the kinetics of MAP kinase activity are very similar in both strains (although slightly faster in CBA/Kw), the rise of cdc2 kinase activity is very rapid in CBA/Kw oocytes and slow and diphasic in KE oocytes. When protein synthesis is inhibited, the activity of the cdc2 kinase starts to rise but arrests shortly after GVBD with a slightly higher level in CBA/Kw oocytes, which may correspond to the presence of a larger pool of cyclin B1 in prophase CBA/Kw oocytes. After GVBD, the rate of cyclin B1 synthesis is higher in CBA/Kw than in KE oocytes, whilst the overall level of protein synthesis and the amount of messenger RNA coding for cyclin B1 are identical in oocytes from both strains. The injection of cyclin B1 messenger RNA in KE oocytes increases the H1 kinase activity and speeds up first polar body extrusion. Analysis of the rate of maturation in hybrids obtained after fusion of nuclear and cytoplasmic fragments of oocytes from both strains suggests that both the germinal vesicle and the cytoplasm contain factor(s) influencing the length of the first meiotic M phase. These results demonstrate that the rate of cyclin B1 synthesis controls the length of the first meiotic M phase and that a nuclear factor able to speed up cyclin B synthesis is present in CBA/Kw oocytes. It is possible that the CBA/Kw factor(s) may be involved in polyadenylation of cyclin B mRNA and that the rate of polyadenlyation may differ between the two strains. Alternatively, the CBA/Kw factor(s) may be regulating cyclin B1 translation at a different level, like at the level of the translation machinery itself (Polanski, 1998).

A investigation was carried out to see whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppresses its activation (not its activity) at G2/M, it has been shown that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts (Abrieu, 1998).

In the Xenopus oocyte system mitogen treatment triggers the G2/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, a cell-free oocyte extract was used that carries out the signaling events of oocyte maturation after addition of PKI, the heat-stable inhibitor of PKA. PKI stimulates the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos does not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delays, but does not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide has a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibits activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21Cip1 has no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity. It is evident that Plx1 is an essential trigger kinase for Cdc25C activation at the G2/M transition. No other kinase appears to be able to substitute for this function of Plx1 in G2, although, once activated, cyclin B-Cdc2 is capable of activating Cdc25C in a positive feedback loop (Qian, 2001).

Cyclin B1-Cdk1 is the key initiator of mitosis, but when and where activation occurs has not been precisely determined in mammalian cells. Activation may occur in the nucleus or cytoplasm; just before nuclear envelope breakdown Polo-like kinase1 (Plk1) is proposed to phosphorylate cyclin B1 in its nuclear export sequence (NES), to trigger rapid nuclear import. Phospho-specific antibodies were raised against cyclin B1 that primarily recognise the active form of the complex. Cyclin B1 was shown to be initially phosphorylated on centrosomes in prophase; Plk1 phosphorylates cyclin B1, but not in the NES. Furthermore, phosphorylation by Plk1 does not cause cyclin B1 to move into the nucleus. It is concluded that cyclin B1-Cdk1 is first activated in the cytoplasm and that centrosomes may function as sites of integration for the proteins that trigger mitosis (Jackman, 2003).

Interaction with cAMP-PKA pathway

Cell cycle progression in cycling Xenopus egg extracts is accompanied by fluctuations in the concentration of adenosine 3',5'-monophosphate (cAMP) and in the activity of the cAMP-dependent protein kinase (PKA). The concentration of cAMP and the activity of PKA decrease at the onset of mitosis and increase at the transition between mitosis and interphase. Blocking the activation of PKA at metaphase prevents the transition into interphase; the activity of M phase-promoting factor (MPF, the cyclin B-p34cdc2 complex) remains high, and mitotic cyclins are not degraded. The arrest in mitosis is reversed by the reactivation of PKA. The inhibition of protein synthesis prevents the accumulation of cyclin and the oscillations of MPF, PKA, and cAMP. Addition of recombinant nondegradable cyclin B activates p34cdc2 and PKA and induces the degradation of full-length cyclin B. Addition of cyclin A activates p34cdc2 but not PKA, nor does it induce the degradation of full-length cyclin B. These findings suggest that cyclindegradation and exit from mitosis require MPF-dependent activation of the cAMP-PKA pathway (Grieco, 1996).

Nuclear import of Cyclin B

M-phase promoting factor or maturation promoting factor, a key regulator of the G2-->M transition of the cell cycle, is a complex of cdc2 and a B-type cyclin. Xenopus cyclin B1 contains five sites for Ser phosphorylation, four of which map to a recently identified cytoplasmic retention signal (CRS). The CRS appears to be responsible for the cytoplasmic localization of B-type cyclins, although the underlying mechanism remains unclear. Phosphorylation of cyclin B1 is not required for cdc2 binding or cdc2 kinase activity. However, when all of the Ser phosphorylation sites in the CRS are mutated to Ala, thereby abolishing phosphorylation, the mutant cyclin B1Ala is inactivated; activity can be enhanced by mutation of these residues to Glu in order to mimic phosphoserine, suggesting that phosphorylation of cyclin B1 is required for its biological activity. Biological activity can be restored to cyclin B1Ala by appending either a nuclear localization signal (NLS), or a second CRS domain with the Ser phosphorylation sites mutated to Glu, while fusion of a second CRS domain with the Ser phosphorylation sites mutated to Ala inactivates wild-type cyclin B1. Nuclear histone H1 kinase activity is detected in association with cyclin B1Ala targeted to the nucleus by a wild-type NLS, but not by a mutant NLS. These results demonstrate that nuclear translocation mediates the biological activity of cyclin B1 and suggest that phosphorylation within the CRS domain of cyclin B1 plays a regulatory role in this process. Given the similar in vitro substrate specificity of cyclin-dependent kinases, this investigation provides direct evidence for the hypothesis that the control of subcellular localization of cyclins plays a key role in regulating the biological activity of cyclin-dependent kinase-cyclin complexes (Li, 1997).

M-phase-promoting factor (MPF), a complex of cdc2 and a B-type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. Cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)-dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES-dependent transport, results in nuclear accumulation of cyclin B1 in G2 phase. Disruption of a newly identified conserved cyclin B1 NES abolishes the nuclear export of this protein, and consequently the NES-disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Expression of the NES-disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage-induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage-induced G2 checkpoint (Toyoshima, 1998).

Activation of the Cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Cyclin B1 localization changes dramatically during the cell cycle, precipitously transiting from the cytoplasm to the nucleus at the beginning of mitosis. Presumably, this relocalization promotes the phosphorylation of nuclear targets critical for chromatin condensation and nuclear envelope breakdown. The previously characterized cytoplasmic retention sequence of Cyclin B1, responsible for its interphase cytoplasmic localization, is actually an autonomous nuclear export sequence, capable of directing nuclear export of a heterologous protein, and able to bind specifically to the recently identified export mediator, CRM1. It is proposed that the observed cytoplasmic localization of Cyclin B1 during interphase reflects the equilibrium between ongoing nuclear import and rapid CRM1-mediated export. In support of this hypothesis, it was found that treatment of cells with leptomycin B, which disrupts Cyclin B1-CRM1 interactions, leads to a marked nuclear accumulation of Cyclin B1. In mitosis, Cyclin B1 undergoes phosphorylation at several sites, a subset of which have been proposed to play a role in Cyclin B1 accumulation in the nucleus. Both CRM1 binding and the ability to direct nuclear export are affected by mutation of these phosphorylation sites; thus, it is proposed that phosphorylation of Cyclin B1 at the G2/M transition prevents its interaction with CRM1, thereby reducing nuclear export and facilitating nuclear accumulation (Yang, 1998).

Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes was examined in digitonin-permeabilized mammalian cells. Also examined were the potential physical interactions between these Cdks, cyclins, and soluble import factors. The nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from the site used to bind importin-alpha (Moore, 1999).

The key regulator of G2-M transition of the cell cycle is M-phase promoting factor (MPF), a complex composed of cdc2 and a B-type cyclin. Cyclin B1 nuclear localization involves phosphorylation within a region called the cytoplasmic retention signal, which also contains a nuclear export signal. The mechanism of MPF nuclear localization remains unclear since it contains no functional nuclear localization signal (NLS). The yeast two-hybrid screen was used to find protein(s) potentially mediating localization of cyclin B1 and a novel interaction between cyclin B1 and cyclin F was identified. Cyclin F is the largest known protein of the cyclin family with a predicted mol. wt of 87 kDa, although it migrates as a 100-110 kDa protein. Examination of the amino acid sequence of cyclin F indicates the presence of a cyclin box, F-box, PEST sequences and two putative NLS regions. Cyclin F protein accumulates in interphase, reaches maximal levels at G2-M phase, and decreases at mitosis just prior to the destruction of cyclin B1. Cdc2, cyclin B1 and cyclin F form a complex that exhibits histone H1 kinase activity. Cyclin B1 and cyclin F also colocalize through immunofluorescence studies. Additionally, deletion analysis reveals that each putative NLS of cyclin F is functional. Taken together, the data suggest that the NLS regions of cyclin F regulate cyclin B1 localization to the nucleus. The interaction between cyclin B1 and cyclin F represents the first example of direct cyclin-cyclin binding, and elucidates a novel mechanism that regulates MPF localization and function (Kong, 2000).

Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes 1 and 2. RNA interference (RNAi) was used to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. It was found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear (Gong, 2007).

Although the functional analysis of cyclins in animal cells stretches back nearly two decades, as yet no simple consensus has emerged on which cyclins are important for mitosis. In Xenopus egg extracts, cyclins B1 and B2 appear to redundantly drive NEB; it is less clear whether an A-type cyclin is required. In Drosophila embryos, cyclins B and B3 appear to play redundant roles in NEB. Cyclin A is required as well, although recent work suggests that this is because cyclin A is required for the inactivation of Cdh1 and the accumulation of cyclin B, cyclin B3, and Cdc25, rather than because of a direct role in mitosis. The situation may be different in HeLa cells, since cyclin B1 and B2 levels are not low in cyclin A2 knockdown cells, as would be expected if cyclin A2 were required to suppress Cdh1 activation in G2 phase or prophase. Nevertheless, these studies underscore the importance of cyclin A2 in NEB in HeLa cells, in part through regulating the localization of cyclin B1 and in part through cyclin B1/B2-independent effects (Gong, 2007 and references therein).

Link between CyclinB/cdk2 kinase and cell cycle progression

The generation of calcium oscillations at fertilization and during mitosis appears to be controlled by the cell cycle machinery. For example, the calcium oscillations in oocytes and embryos occur during metaphase and terminate upon entry into interphase. Reported here is the manipulation of sperm-triggered calcium oscillations by cyclin-dependent kinase (CDK) activity, the major component of maturation/M phase promoting factor (MPF). To control the CDK activity mRNAs encoding full-length GFP-tagged cyclin B1 or a truncated and therefore stabilized form of cyclin B1 (D90) were microinjected into unfertilized oocytes. In the presence of full-length cyclin B1, the calcium oscillations terminate when cyclin B1 levels fall along with the concomitant fall, in the associated CDK activity. In addition, when the CDK activity is elevated indefinitely with D90 cyclin B1, the calcium oscillations also continue indefinitely. Finally, in oocytes that contain low mitogen-activated protein (MAP) kinase activity and elevated CDK activity, the sperm-triggered calcium oscillations are again prolonged. It is concluded that the CDK activity of the ascidian oocyte can be regarded as a positive regulator of sperm-triggered calcium oscillations, a finding that may apply to other oocytes that display sperm-triggered calcium oscillations at fertilization. Furthermore, these findings may have a bearing upon the mitotic calcium signals of early embryos (Levasseur, 2000).

These results does not differentiate between the two hypotheses of egg activation: the receptor hypothesis and the sperm factor hypothesis. The discovery that the calcium-releasing second messenger InsP3 increases at fertilization is a central starting point of fertilization. The receptor hypothesis takes as the starting point that sperm trigger the calcium signal in the egg through interaction between factors on the sperm and egg surfaces. This interaction is thought to lead to the activation of an egg phospholipase C (PLC) and the generation of InsP3 in the egg. However, the molecular identity of the interacting ligands remains unknown. The sperm factor hypothesis takes as the starting point sperm-egg fusion, and the subsequent delivery of a sperm factor into the egg cytosol. Sperm cytosolic extracts faithfully mimic the pattern of calcium oscillations triggered by sperm at fertilization in mammals, nemerteans and ascidians. Such a factor has been proposed to be a high molecular mass protein. All attempts to determine the identity of such a molecule have so far proved unsuccessful, although PLC and a truncated form of the c-kit tyrosine kinase present in spermatozoa remain ongoing candidates (Levasseur, 2000 and references therein).

In higher eukaryotes, cdk2 kinase plays an essential role in regulating the G1-S transition. Cycling Xenopus egg extracts have been used to examine the requirement of cdk2 kinase on progression into mitosis. When cdk2 kinase activity is inhibited by the cdk-specific inhibitor (p21Cip1), a block to mitosis occurs, and inactive cdc2-cyclin B accumulates. This block occurs in the absence of nuclei and is not due to direct inhibition of cdc2 by Cip. This block to mitosis is reversible by restoring cdk2-cyclin E kinase activity to a Cip-treated cycling extract. Immunodepletion of cdk2 from interphase extracts prevents activation of cdc2 upon the addition of exogenous cyclin B. Guadagno's study shows that cdk2 kinase is a positive regulator of cdc2-Cyclin B complexes and establishes a link between cdk2 kinase and cell cycle progression into mitosis (Guadagno, 1996).

poly(A) polymerase (PAP) adds a poly(A) tail to nearly every newly transcribed pre-mRNA, and is a part of a multisubunit complex that tightly couples precise pre-mRNA cleavage to subsequent poly(A) addition. p34cdc2/cyclin B (mitosis/meisois promoting factor, or MPF) hyperphosphorylates poly(A) polymerase during M-phase of the cell cycle, causing repression of its enzymatic activity. Mutation of three cyclin-dependent kinase (cdk) consensus sites in the PAP C-terminal regulatory domain prevent complete phosphorylation and MPF-mediated repression. PAP also contains four nearby non-consensus cdk sites that are phosphorylated by MPF. Remarkably, full phosphorylation of all these cdk sites is required for repression of PAP activity, and partial phosphorylation has no detectable effect. The consensus sites are phosphorylated in vitro at a 10-fold lower concentration of MPF than the non-consensus sites. Consistent with this, during meiotic maturation of Xenopus oocytes, consensus sites are phosphorylated prior to the non-consensus sites at metaphase of meiosis I, and remain so throughout maturation, while the non-consensus sites do not become fully phosphorylated until after 12 h of metaphase II arrest. It is proposed that PAP's multiple cdk sites, and their differential sensitivity to MPF, provide a mechanism to link repression specifically to late M-phase. This reflects a general means to control the timing of cdk-dependent regulatory events during the cell cycle. Progressive phosphorylation of PAP, first on consensus cdk sites and later on non-consensus sites, provides a mechanism for linking repression of PAP activity exclusively to late M-phase (Colgan, 1998)

The initiation of mitosis requires the activation of M-phase promoting factor (MPF). MPF activation and its subcellular localization are dependent on the phosphorylation state of its components, cdc2 and cyclin B1. In a two-hybrid screen using a bait protein to mimic phosphorylated cyclin B1, a novel interaction was detected between cyclin B1 and patched1 (ptc1), a tumor suppressor associated with basal cell carcinoma (BCC). Ptc1 interacts specifically with constitutively phosphorylated cyclin B1 derivatives and is able to alter their normal subcellular localization. Furthermore, addition of the ptc1 ligand, sonic hedgehog (shh), disrupts this interaction and allows cyclin B1 to localize to the nucleus. Expression of ptc1 in 293T cells is inhibitory to cell proliferation; this inhibition could be relieved by coexpression of a cyclin B1 derivative that constitutively localizes to the nucleus and that could not interact with ptc1 due to phosphorylation-site mutations to Ala. In addition, endogenous ptc1 and endogenous cyclin B1 interact in vivo. The findings reported here demonstrate that ptc1 participates in determining the subcellular localization of cyclin B1 and suggest a link between the tumor suppressor activity of ptc1 and the regulation of cell division. Thus, it is proposed that ptc1 participates in a G2/M checkpoint by regulating the localization of MPF (Barnes, 2001).

The cyclosome/anaphase-promoting complex is a multisubunit ubiquitin ligase that targets for degradation mitotic cyclins and some other cell cycle regulators in exit from mitosis. It becomes enzymatically active at the end of mitosis. The activation of the cyclosome is initiated by its phosphorylation, a process necessary for its conversion to an active form by the ancillary protein Cdc20/Fizzy. Previous reports have implicated either cyclin-dependent kinase 1-cyclin B or polo-like kinase as the major protein kinase that directly phosphorylates and activates the cyclosome. These conflicting results could be due to the use of partially purified cyclosome preparations or of immunoprecipitated cyclosome, whose interactions with protein kinases or ancillary factors may be hampered by binding to immobilized antibody. To examine this problem, cyclosome has been purified from HeLa cells by a combination of affinity chromatography and ion exchange procedures. With the use of purified preparations, it was found that both cyclin-dependent kinase 1-cyclin B and polo-like kinase directly phosphorylate the cyclosome, but the pattern of the phosphorylation of the different cyclosome subunits by the two protein kinases is not similar. Plk1 and Cdk1/cyclin-B have additive effects in phosphorylating and activating the APC/C; the former preferentially phosphorylates Cdc16 and Cdc23, and the latter preferentially phosphorylates Cdc27. Each protein kinase can restore only partially the cyclin-ubiquitin ligase activity of dephosphorylated cyclosome. However, following phosphorylation by both protein kinases, an additive and nearly complete restoration of cyclin-ubiquitin ligase activity is observed. It is suggested that this joint activation may be due to the complementary phosphorylation of different cyclosome subunits by the two protein kinases (Golan, 2002).

Cyclin B-Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase-independent mechanism>

Entry into M phase is governed by cyclin B-Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B-Cdk1-dependent inhibition of the protein phosphatase 2A (PP2A)-B55, which antagonizes cyclin B-Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B-Cdk1 to phosphorylate and activate alpha-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B-Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. This study shows that cyclin B-Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B-Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles (Okumura, 2014).

Mitotic CDKs control the metaphase-anaphase transition and trigger spindle elongation

Mitotic cyclin-dependent kinases (CDKs) control entry into mitosis, but their role during mitotic progression is less well understood. This study characterizes the functions of CDK activity associated with the mitotic cyclins Clb1, Clb2, and Clb3. Clb-CDKs are important for the activation of the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C)-Cdc20 that triggers the metaphase-anaphase transition. Furthermore, an essential role is defined for Clb-CDK activity in anaphase spindle elongation. Thus, mitotic CDKs serve not only to initiate M phase, but are also needed continuously throughout mitosis to trigger key mitotic events such as APC/C activation and anaphase spindle elongation (Rahal, 2008).

It has been suggested that increasing amounts of CDKs establish consecutive cell cycle events, with low CDK levels promoting S phase and high levels of CDKs triggering M phase. The data indicate that increasing amounts of Clb-CDKs are also responsible for triggering consecutive mitotic events. Inactivation of CLB1 and CLB2 causes no delay in cell cycle progression prior to metaphase, whereas inactivation of CLB2 and CLB3 causes a modest 30-min delay. In contrast, inactivation of CLB1, CLB2, CLB3, and CLB4 causes cell cycle arrest in G2, with replicated DNA and unseparated SPBs. Furthermore, Clb-CDK activity rises as cells progress through mitosis. Based on these observations, it is proposed that higher amounts of mitotic CDK activity are needed for entry into anaphase than for entry into mitosis. This steady rise in mitotic CDK activity helps establish the order of events during early mitosis, with a lower threshold of Clb-CDK activity triggering entry into mitosis and a second, higher one triggering entry into anaphase. Finally, once anaphase entry has been initiated, Clb proteolysis causes a decline in Clb-CDK activity, triggering exit from mitosis (during which thresholds may also play a role. Interestingly, increasing amounts of mitotic CDK activity may also govern progression through early stages of mitosis in mammalian cells. Mitotic CDK activity rises as cells progress from G2 into metaphase. Furthermore, complete inactivation of Cdk1 by RNAi-based methods prevents entry into mitosis, whereas partial Cdk1 inactivation delays entry into anaphase. Therefore, requiring a steady rise in mitotic CDK activity for mitotic progression may be a general mechanism by which all eukaryotic cells ensure that chromosome segregation occurs only after chromosomes have condensed and a mitotic spindle has formed (Rahal, 2008).

The END network couples spindle pole assembly to inhibition of the anaphase-promoting complex/cyclosome in early mitosis

Cyclin-dependent kinase 1 (Cdk1) initiates mitosis and later activates the anaphase-promoting complex/cyclosome (APC/C) to destroy cyclins. Kinetochore-derived checkpoint signaling delays APC/C-dependent cyclin B destruction, and checkpoint-independent mechanisms cooperate to limit APC/C activity when kinetochores lack checkpoint components in early mitosis. The APC/C and cyclin B localize to the spindle and poles, but the significance and regulation of these populations remain unclear. This study describes a critical spindle pole-associated mechanism, called the END (Emi1/NuMA/dynein-dynactin) network, that spatially restricts APC/C activity in early mitosis. The APC/C inhibitor Emi1 binds the spindle-organizing NuMA/dynein-dynactin complex to anchor and inhibit the APC/C at spindle poles, and thereby limits destruction of spindle-associated cyclin B. Cyclin B/Cdk1 activity recruits the END network and establishes a positive feedback loop to stabilize spindle-associated cyclin B critical for spindle assembly. The organization of the APC/C on the spindle also provides a framework for understanding microtubule-dependent organization of protein destruction (Ban, 2007).

Progression through mitosis depends on the periodic accumulation and destruction of cyclins. Cyclin B accumulates and activates the cyclin-dependent kinase 1 (Cdk1) in mitosis to form mitosis-promoting factor (MPF). MPF drives chromosome reorganization and formation of the mitotic spindle. Later in mitosis, MPF downregulates its own activity by initiating the ubiquitination and destruction of cyclins by the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase. The delay between activation of the APC/C by MPF at the beginning of mitosis and the destruction of cyclin B at mitotic exit is critical to allow sufficient time for the spindle to form and direct chromosome congression. Inhibition of the APC/C during this period is linked to kinetochore-dependent activation of spindle assembly checkpoint components, notably the APC/C inhibitors Mad2 and BubR1. However, the potential role for other APC/C inhibitors, including Emi1, in contributing to APC/C regulation in mitosis is poorly understood (Ban, 2007).

In S and G2 phases of vertebrate cells, Emi1 prevents the premature destruction of cyclins A and B, thereby allowing cells to progress into mitosis. As cells commit to mitosis at nuclear envelope breakdown (NEBD), the bulk of Emi1 is destroyed by the SCFβTRCP E3 ligase, thus permitting selective activation of the APC/C to degrade substrates in prometaphase, such as cyclin A. The APC/C is restrained from destroying cyclin B, in part by establishment of the spindle checkpoint. In early mitosis, however, checkpoint-independent mechanisms cooperate to restrain APC/C activity when kinetochore signaling is not yet fully established. Indeed, a population of Emi1 localizes to the spindle poles in early mitosis, suggesting that this inhibitor could function to regulate the APC/C during this critical period when spindles are forming (Ban, 2007).

MPF activation drives spindle assembly by initiating NEBD, which in turn permits Ran-GTP-dependent release of microtubule-organizing activities to nucleate microtubule asters. MPF activity also promotes microtubule organization by phosphorylating microtubule-associated proteins and motor proteins. Spindle microtubules are integrated and focused at spindle poles by several factors, including the minus end-directed dynein-dynactin motor complex and the spindle protein NuMA (nuclear mitotic apparatus), as a critical step in the formation of the bipolar spindle. Because MPF regulates both spindle assembly and APC/C activation, a mechanism coupling the processes would ensure proper timing of cyclin accumulation and destruction (Ban, 2007).

The mitotic spindle itself may organize and regulate APC/C and MPF activity, thus providing a link between the processes. Both cyclin B and the APC/C are localized to the spindle and poles in mitosis. Moreover, cyclin B appears to be destroyed at the poles and spindles at the metaphase-to-anaphase transition. These studies have suggested that the organization of MPF and the APC/C on the spindle potentially contribute to maintaining the spindle structure and facilitating cyclin destruction during mitotic exit. How these components are organized and regulated on the spindle is unclear (Ban, 2007).

This study, carried out in mammalian cells, describes an essential regulatory network that physically and functionally links the NuMA and dynein-dynactin spindle-organizing components to the APC/C and its associated inhibitor Emi1. A network of Emi1, NuMA, and dynein-dynactin (END) spatially regulates the APC/C on the mitotic spindle to prevent premature cyclin B destruction on the spindle. Stabilization of cyclin B/Cdk1 activity promotes NuMA-dependent assembly of microtubules at spindle poles and reinforces the recruitment of the END network to poles. It is proposed that the END network establishes a positive feedback loop in early mitosis that sustains localized cyclin B/Cdk1 activity on the spindle critical for maintaining spindle integrity (Ban, 2007).

BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/C(Cdc20) in interphase

BubR1 is an essential mitotic checkpoint protein with multiple functional domains. It has been implicated in mitotic checkpoint control, as an active kinase at unattached kinetochores, and as a cytosolic inhibitor of APC/C(Cdc20) activity, as well as in mitotic timing and stable chromosome-spindle attachment. Using BubR1-conditional knockout cells and BubR1 domain mutants, it was demonstrated that the N-terminal Cdc20 binding domain of BubR1 is essential for all of these functions, whereas its C-terminal Cdc20-binding domain, Bub3-binding domain, and kinase domain are not. The BubR1 N terminus binds to Cdc20 in a KEN box-dependent manner to inhibit APC/C activity in interphase, thereby allowing accumulation of cyclin B in G(2) phase prior to mitosis onset. Together, these results suggest that kinetochore-bound BubR1 is nonessential and that soluble BubR1 functions as a pseudosubstrate inhibitor of APC/C(Cdc20) during interphase to prevent unscheduled degradation of specific APC/C substrates (Malureanu, 2009).

A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest

Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. This study reports the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 binds to the core APC, and this binding is disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 is antagonized by PP2A, which can bind to Emi2 and promote Emi2-APC interactions. It is concluded that constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest (Wu, 2007)

Cyclin B/cdc2 link to DNA replication

Continued: CyclinB Evolutionary homologs  part 3/3 | back to  part 1/3

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

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