cdc2
Cdc2 and the G2-->M transition MPF (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 has five sites of 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 is still 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 to abolish phosphorylation, the mutant cyclin B1Ala is inactivated; activity can be
enhanced by mutation of these residues to Glu, which mimics 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).
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).
MPF amplification in Xenopus oocyte extracts depends on a two-step activation of Cdc25 phosphatase. The activation of Cdc2 kinase induces the entry into M-phase of all eukaryotic cells. A cell-free system prepared from
prophase-arrested Xenopus oocytes has been developed to analyze the mechanism initiating the all-or-none activation of Cdc2 kinase. Inhibition of phosphatase 2A, the
major okadaic acid-sensitive Ser/Thr phosphatase in these extracts, provokes Cdc2 kinase amplification and concomitant hyperphosphorylation of
Cdc25 phosphatase, with a lag of about 1 h. Polo-like kinase (Plx1 kinase) is activated slightly after Cdc2. All these events are totally inhibited by the
cdk inhibitor p21(Cip1), demonstrating that Plx1 kinase activation depends on Cdc2 kinase activity. Addition of a threshold level of recombinant Cdc25
induces a linear activation of Cdc2 and Plx1 kinases and a partial phosphorylation of Cdc25. It is proposed that the Cdc2 positive feedback loop involves
two successive phosphorylation steps of Cdc25 phosphatase: the first one is catalyzed by Cdc2 kinase and/or Plx1 kinase but it does not modify Cdc25
enzymatic activity; the second one requires a new kinase counteracted by phosphatase 2A. Under the conditions of this assay, Cdc2
amplification and MAP kinase activation are two independent events (Kara, 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).
The mammalian MCM protein family, presently with six members, exists in the nuclei in two forms: chromatin-bound
and unbound. The former dissociates from chromatin with progression through the S phase. A procedure has been established to isolate chromatin-bound and unbound complexes containing all six human MCM (hMCM)
proteins by immunoprecipitation. This procedure has been applied to HeLa cells synchronized in each of
the G1, S, and G2/M phases; hMCM heterohexameric complexes could be detected in all three phases. In addition, depending
on the cell cycle and the state of chromatin association, hMCM2 and 4 in the complexes were found to variously
change their phosphorylation states. Concentrating attention on G2/M phase hyperphosphorylation, hMCM2
and 4 in the complexes have been found to be good substrates for cdc2/cyclin B in vitro. Furthermore, when cdc2 kinase is inactivated
in temperature-sensitive mutant murine FT210 cells, the G2/M hyperphosphorylation of the murine MCM2 and MCM4
and the release of the MCMs from chromatin in the G2 phase are severely impaired. Taken together, the data suggest
that the six mammalian MCM proteins function and undergo cell cycle-dependent regulation as heterohexameric
complexes and that phosphorylation of the complexes by cdc2 kinase may be one of mechanisms negatively regulating
the MCM complex-chromatin association (Fujita, 1999).
Dissociation of CDC2 and Cyclin B at the end of M phase Inactivation of cyclin B-Cdc2 kinase at the exit from M phase depends on the specific proteolysis of the cyclin B subunit, whereas
the Cdc2 subunit remains present at nearly constant levels throughout the cell cycle. It is unknown how Cdc2 escapes degradation
when cyclin B is destroyed. In Xenopus egg extracts that reproduce the exit from M phase in vitro, dissociation
of the cyclin B-Cdc2 complex occurs under conditions where cyclin B is tethered to the 26S proteasome but not yet degraded.
The dephosphorylation of Thr 161 on Cdc2 is unlikely to be necessary for the dissociation of the two subunits. However, the
dissociation is dependent on the presence of a functional destruction box in cyclin B. Cyclin B ubiquitination is also, by itself, not sufficient for separation of
Cdc2 and cyclin B. The 26S proteasome, but not the 20S proteasome, is capable of dissociating the two subunits. These results indicate that the cyclin B and
Cdc2 subunits are separated by the proteasome through a mechanism that precedes proteolysis of cyclin B and is independent of proteolysis. As a result, cyclin
B levels decrease on exit from M phase but Cdc2 levels remain constant (Nishiyama, 2000).
Because previous studies have demonstrated that the phosphorylation of Thr 161 on Cdc2 increases the stability of the cyclin B-Cdc2 complex, one might have anticipated that the dephosphorylation of Thr 161 might contribute to its dissociation. However, contrary to this premise, these results suggest a possibility that dephosphorylation on Thr 161 is not necessary for dissociation of the cyclin B-Cdc2 complex at exit from M phase. This possibility is consistent with the previous report that okadaic acid prevents both the dephosphorylation of Cdc2 and the drop in H1 kinase
activity, but not cyclin B degradation. The fact that simply reversing the step that stabilizes the cyclin B-Cdc2 complex does not cause dissociation of the complex once it has formed, provides support for the notion that a positive-acting mechanism releases the two subunits at the end of M phase (Nishiyama, 2000).
The present results indicate that a functional destruction box is required for dissociation of cyclin B-Cdc2. Considering that the destruction box in cyclin
B is known to be involved in polyubiquitination of cyclin B, one could postulate that polyubiquitination of cyclin B itself might cause
the release of Cdc2 from cyclin B. In accordance with this notion, it has been suggested that the polyubiquitin chain helps to unfold the target proteins. However, in the present study polyubiquitination is not by itself sufficient to dissociate Cdc2 from cyclin B. It is not known to what
extent cyclin B is unfolded as a result of ubiquitination. What then is the role of the destruction box in the dissociation of cyclin B-Cdc2? Although the possibility that the destruction box contributes to a function other than polyubiquitination of cyclin B, it is reasonable that polyubiquitination of cyclin
B is a way for targeting cyclin B-Cdc2 to the dissociation because the proteasome is required for this dissociation in the present study and because a
multi-ubiquitin chain is a well-known targeting signal to the proteasome. Thus, polyubiquitination of cyclin B is likely to target the
complex to the proteasome where Cdc2 is released from cyclin B. Whether or not ubiquitination also plays a role in facilitating the disassembly of the complex
by the proteasome remains to be resolved (Nishiyama, 2000).
How does the proteasome dissociate Cdc2 from cyclin B? The 19S
regulatory particle is composed of two subcomplexes, the 'base' and
the 'lid'. The base is located proximal
and the lid distal to the 20S core particle. The lid is essential
for the recognition, and possibly binding, of polyubiquitinated substrate proteins, whereas the base is likely to promote substrate unfolding through its six distinct AAA (ATPases
associated with a variety of cellular
activities)-type ATPase components in a chaperone-like
manner that is independent of polyubiquitin chains.
In addition, the 20S proteolytic core is composed of two alpha- and two
beta-rings. The alpha-rings discriminate
between unfolded and folded proteins, and the beta-rings, which
constitute the central catalytic core, can cleave substrates
proteolytically. On the basis of the above and the results presented in
this paper, the following model for degradation of cyclin B: Initially, polyubiquitin chains
of cyclin B that have been added by the APC/C tether the cyclin B-Cdc2
complex to the lid of the 19S particle. Then, because of a
chaperone-like function of the base of the 19S particle with the aid of
the alpha-rings in the 20S component, the tethered cyclin B-Cdc2 complex
is unfolded. Consequently, Cdc2 dissociates from cyclin B, while cyclin
B is translocated into the catalytic core of the 20S component. Lastly,
cyclin B is degraded within the lumen of the 20S component by the
proteolytic activity of the beta-rings. At present, however, it cannot be discriminated whether Cdc2 is passively separated from cyclin B as a
consequence of cyclin B unfolding or whether Cdc2 is actively
dissociated from the cyclin B-Cdc2 complex by a chaperone-like
activity of the proteasome components (Nishiyama, 2000).
A model is presented for release of Cdc2 from cyclin B by the 26S
proteasome. (1) Initially, cyclin B complexed with Cdc2 is polyubiquitinated by
an APC/C-dependent pathway at exit from M phase. (2) Next, the lid of the
19S regulatory particle recognizes and tethers the polyubiquitinated cyclin
B that remains associated with Cdc2. (3) The cyclin B-Cdc2 complex
is unfolded and dissociated by the chaperone-like activity of the base
of the 19S regulatory particle with the aid of the alpha ring of the 20S
proteasome. The translocation of unfolded cyclin B to the 20S
proteasome may be required for further unfolding of cyclin B, and both
processes may be coupled to one another. (4) Finally, the unfolded cyclin B
is translocated and degraded in the hollow center (beta rings of the
20S proteasome) in which all catalytic sites are located (Nishiyama, 2000).
MAP kinase and CDC2 at the G2-M transition The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2
complexes (prematuration-promoting factor [MPF]) be kept in an inactive form, due largely to the
inhibitory phosphorylation of this pre-MPF. The role of mitogen-activated protein
(MAP) kinase (see Drosophila Rolled) has been investigated in the activation of this pre-MPF. Mitogen-activated protein kinase activation down-regulates a
mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested
oocytes. The cytoplasm of both frog and starfish oocytes
contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of either G2-arrested
starfish or Xenopus oocytes is prematurely activated by microinjection of c-mos or Ste-11 delta N
fusion proteins, the rate and extent of MPF inactivation is much reduced. Both effects are
suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that
MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes.
In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown,
much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a
sensitive response to hormonal stimulation that does not require MAP kinase activation (Abrieu, 1997a).
Down-regulation of MAP kinase (MAPK) is a universal consequence of fertilization in the animal
kingdom, although its role is not known. MAPK inactivation is essential for
embryos, both vertebrate and invertebrate, to enter first mitosis. Suppressing down-regulation of
MAPK at fertilization, for example by constitutively activating the upstream MAPK cascade,
specifically suppresses cyclin B-cdc2 kinase activation and its consequence, entry into first mitosis. It
thus appears that MAPK functions in meiotic maturation by preventing unfertilized eggs from
proceeding into parthenogenetic development. The most general effect of artificially maintaining
MAPK activity after fertilization is prevention of the G2 to M-phase transition in the first mitotic cell
cycle, even though inappropriate reactivation of MAPK after fertilization may lead to metaphase arrest
in vertebrates. Advancing the time of MAPK inactivation in fertilized eggs does not, however, speed
up their entry into first mitosis. Thus, sustained activity of MAPK during part of the first mitotic cell
cycle is not responsible for late entry of fertilized eggs into first mitosis (Abrieu, 1997).
Constitutively active MAP kinase/ERK kinase (MEK), an activator of the
mitogen-activated protein kinase (MAPK) signaling pathway, was added to cycling Xenopus egg extracts at
various times during the cell cycle. p42MAPK activation during entry into M-phase arrests the cell
cycle in metaphase. p42MAPK activation during
interphase inhibits entry into M-phase. In these interphase-arrested extracts, H1 kinase activity
remains low; Cdc2 is tyrosine phosphorylated, and nuclei continued to enlarge. The interphase
arrest is overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or
by Mos inhibits Cdc2 activation by cyclin B. A specific inhibitor of MEK blocks the
effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK does not inhibit DNA replication.
These results indicate that in addition to the established role of p42MAPK activation in M-phase
arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into
M-phase (Bitangcol, 1998).
The lethal toxin (LT) from Clostridium sordellii is a glucosyltransferase that modifies and inhibits small
G proteins of the Ras family, Ras and Rap, as well as Rac proteins. LT induces cdc2 kinase activation
and germinal vesicle breakdown (GVBD) when microinjected into full-grown Xenopus oocytes. Toxin
B from Clostridium difficile, that glucosylates and inactivates Rac proteins, does not induce cdc2
activation, indicating that proteins of the Ras family, Ras and/or Rap, negatively regulate cdc2 kinase
activation in Xenopus oocyte. In oocyte extracts, LT catalyzes the incorporation of [14C]glucose into a
group of proteins of 23 kDa and into one protein of 27 kDa. The 23-kDa proteins are recognized by
anti-Rap1 and anti-Rap2 antibodies, whereas the 27-kDa protein is recognized by several anti-Ras
antibodies and probably corresponds to K-Ras. Microinjection of LT into oocytes together with
UDP-[14C]glucose results in a glucosylation pattern similar to that found in in vitro glucosylation, indicating that
the 23- and 27-kDa proteins are in vivo substrates of LT. In vivo time-course analysis reveals that the
27-kDa protein glucosylation is completed within 2 h, well before cdc2 kinase activation, whereas the
23-kDa proteins are partially glucosylated at GVBD. This observation suggests that the 27-kDa Ras
protein could be the in vivo target of LT, allowing cdc2 kinase activation. Interestingly, inactivation of
Ras proteins does not prevent the phosphorylation of c-Raf1 and the activation of MAP kinase that
occurs normally around GVBD (Rime, 1998).
CDC2, meiosis, egg maturation and egg activation 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).
MAP kinase activation occurs during meiotic maturation
of oocytes from all animals, but the requirement for MAP
kinase activation in reinitiation of meiosis appears to vary
between different organisms. In particular, it has become
accepted that MAP kinase activation is necessary for
progesterone-stimulated meiotic maturation of Xenopus
oocytes, while this is clearly not the case in other systems.
While MAP
kinase increases the efficiency of Cdc2 activation, it is the
intrinsic sensitivity of oocytes to levels of Cdc2 kinase that
determines whether or not oocytes undergo initiation of meiotic
maturation in the absence of MAP kinase activity.
MAP kinase activation
in Xenopus oocytes is an early response to progesterone and
can be temporally dissociated from MPF activation. MAP kinase activation can be suppressed by
treatment with geldanamycin or by overexpression of the
MAP kinase phosphatase Pyst1. A transient and low-level
early activation of MAP kinase increases the efficiency of
cell cycle activation later on, when MAP kinase activity is
no longer essential. Many oocytes can still undergo
reinitiation of meiosis in the absence of active MAP kinase.
Suppression of MAP kinase activation does not affect the
formation or activation of Cdc2-cyclin B complexes, but
reduces the level of active Cdc2 kinase.
Even in species that do not require cyclin B synthesis for
germinal vesicle breakdown (GVBD), MAP kinase regulates cyclin B synthesis after GVBD. For example, oocytes from mos -/- mice can undergo initiation
of meiotic maturation but do not activate MAP kinase,
indicating that other activators of the MAP kinase cascade,
such as Raf-1, cannot replace Mos function. The rate of formation of Cdc2 kinase activity after
GVBD in mouse oocytes is controlled by cyclin B1 synthesis, while cyclin B does not accumulate in
mouse oocytes injected with Mos antisense oligonucleotides. The results in Xenopus oocytes (this
paper) show striking parallels to these results and lead to a
suggestion that oocyte meiotic initiation might indeed be
controlled in a universally similar way. Quantitative differences
may well exist between different oocyte systems, such as the
sensitivity of GVBD to Cdc2 kinase levels, which means that
meiosis is organized along the same principles but
quantitatively different ways (Fisher, 1999).
Regulatory mechanisms assure that during meiosis, cells undergo a cell cycle transition from M phase to M phase without entering S. In order to study the regulation of this transition, a Xenopus oocyte
extract that performs the M-M transition has been developed. Using the meiotic extract, it was found that a low level of Cdc2 activity
remains at the exit of meiosis I (MI), due to incomplete degradation of cyclin B. The inactivation of the residual Cdc2 activity induces
both entry into S phase and tyrosine phosphorylation on Cdc2 after MI. Quantitative analysis has demonstrated that a considerable amount of
Wee1 is present at the MI exit and Cdc2 inhibitory phosphorylation during this period is suppressed by the dominance of Cdc2 over
Wee1. Consistently, the addition of more than a critical amount of Wee1 to the extract induces Cdc2 inhibitory phosphorylation, changing
the M-M transition into an M-S-M transition. Thus, the Cdc2 activity remaining at MI exit is required for suppressing entry into S phase during the meiotic M-M
transition period (Iwabuchi, 2000).
The fact that the M-M transition could be inhibited by increasing the amount of Wee1 in MI implies that the amount of Wee1 must be strictly controlled during
MI-MII transition. The amount of Wee1 is unlikely to be regulated by degradation, because His tagged Wee1 injected into oocytes and added to oocyte extracts is
stable throughout the oocyte maturation period. Like the mRNAs of c-Mos, cyclins A1 and B1 and
Cdk2, Wee1 mRNA contains cytoplasmic polyadenylation elements in the 3' untranslated region, suggesting that Wee1 mRNA is
translationally activated during oocyte maturation, depending on the hormonal stimulation. In addition, rapid
accumulation of Wee1 during the post-germinal vesicle breakdown period depends on the germinal-vesicle contents, suggesting the synthesis of Wee1 is
regulated differently from that of c-Mos and cyclin B1, whose synthesis starts before GVBD, independent of the germinal-vesicle contents. The mechanisms regulating Wee1 expression during oocyte maturation remain to be elucidated. In conclusion, in Xenopus oocytes, the residual Cdc2 activity remaining at MI exit is required to suppress Wee1 during the MI-MII
transition period, thereby ensuring the meiotic transition from M phase to M phase (Iwabuchi, 2000).
Xenopus oocytes arrested in prophase I resume meiotic division in response to progesterone and arrest at metaphase II. The meiotic maturation
period can be divided into three phases: (1) a pre-GVBD period, starting after hormonal addition and lasting between 3 and 5 h until GVBD;
(2) metaphase I/metaphase II transition, or interkinesis, wherein
nuclear envelopes do not reform, chromosomes remain
condensed, and DNA replication does not occur; (3)
metaphase II arrest. Entry into meiosis I depends on the activation of Cdc2 kinase [M-phase promoting factor (MPF)]. To better understand the
role of Cdc2, MPF activity was specifically inhibited by injection of the CDK inhibitor, Cip1. When Cip1 is injected at
germinal vesicle breakdown (GVBD) time, Cdc25 and Plx1 are both dephosphorylated and Cdc2 is rephosphorylated on tyrosine. The autoamplification loop characterizing MPF is therefore not only required for MPF generation before GVBD,
but also for its stability during the GVBD period. The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C),
responsible for cyclin degradation, is also under the control of Cdc2; therefore, Cdc2 activity itself induces its own
inactivation through cyclin degradation, allowing the exit from the first meiotic division (Frank-Vaillant, 2001).
In contrast, cyclin accumulation, responsible for subsequent Cdc2 activity increase allowing entry into metaphase II, is independent of Cdc2. The c-Mos/mitogen-activated protein kinase (MAPK) pathway remains active when Cdc2 activity is inhibited at GVBD time. This pathway could be responsible for the sustained cyclin neosynthesis. In contrast, during the metaphase II block, the c-Mos/MAPK pathway depends on Cdc2. Therefore, the metaphase II block depends on a dynamic interplay between MPF and CSF (cytostatic function of the c-Mos/MAPK pathway during the metaphase II arrest), the c-Mos/MAPK pathway stabilizing cyclin B, whereas in turn, MPF prevents c-Mos degradation (Frank-Vaillant, 2001).
Mammalian eggs are arrested in metaphase II of meiosis until fertilization. Arrest is maintained by cytostatic factor (CSF)
activity, which is dependent on the MOS-MEK-MAPK pathway. Inhibition of MEK1/2 with a specific inhibitor, U0126, parthenogenetically activates mouse eggs, producing phenotypes similar to Mos-/- parthenogenotes (premature, unequal cleavages and large polar bodies). U0126 inactivates MAPK in eggs within 1 h, in contrast to the 5 h required after fertilization, while the time course of MPF inactivation is similar in U0126-activated and fertilized eggs. Inactivation of MPF by the cdc2 kinase inhibitor roscovitine induces parthenogenetic activation. Inactivation of MPF
by roscovitine results in the subsequent inactivation of MAPK with a time course similar to that following fertilization. Notably, roscovitine also produces some Mos-/--like phenotypes, indistinguishable from U0126 parthenogenotes. Simultaneous inhibition of both MPF and MAPK in eggs treated with roscovitine and U0126 produces a very high proportion of
eggs with the more severe phenotype. These findings confirm that MEK is a required component of CSF in mammalian eggs
and imply that the sequential inactivation of MPF followed by MAPK inactivation is required for normal spindle function and polar body emission (Philips, 2002).
During oogenesis, the Xenopus oocyte is blocked in prophase of meiosis I. It becomes competent to resume meiosis in response to progesterone at the end of its growing period (stage VI of oogenesis). Stage IV oocytes contain a store of inactive pre-MPF (Tyr15-phosphorylated Cdc2 bound to cyclin B2); the Cdc25 phosphatase that catalyzes Tyr15 dephosphorylation of Cdc2 is also present. However, the positive feedback loop that allows MPF autoamplification is not functional at this stage of oocyte growth. When cyclin B is overexpressed in stage IV oocytes, MPF autoamplification does not occur and the newly formed cyclin B-Cdc2 complexes are inactivated by Tyr15 phosphorylation, indicating that Myt1 kinase remains active and that Cdc25 is prevented from being activated. Plx1 kinase (or polo-like kinase), which is required for Cdc25 activation and MPF autoamplification in full grown oocytes is not expressed at the protein level in small stage IV oocytes. In order to determine if Plx1 could be the missing regulator that prevents MPF autoamplification, polo kinase was overexpressed in stage IV oocytes. Under these conditions, the MPF-positive feedback loop was restored. Moreover, acquisition of autoamplification competence does not require the Mos/MAPK pathway (Karaiskou, 2004).
Thus, Plx1 protein, crucial for the function of the auto-amplification feedback loop in full-grown oocytes is not expressed in small oocytes. Both Cdc25 and Myt1 are direct substrates of Plk1 during M phase. The results indicate that overexpression of Plk1 in stage IV oocytes authorizes cyclin B1 to form active complexes with Cdc2. This observation shows that in oocytes, Plk1 participates in the formation of an active MPF trigger by downregulating Myt1. Moreover, it indicates that progesterone unresponsiveness of small oocytes depends on a stable activity of Myt1 kinase, because of Plx1 absence. Although Plk1 expression prevents Tyr15 phosphorylation of Cdc2 after cyclin B overexpression, Cdc25 is not fully activated. This shows that full activation of Cdc25 requires a further regulatory mechanism. Indeed, Xenopus Cdc25 can be negatively regulated through Ser287 phosphorylation by several kinases, including Chk1 and PKA. Cdc25C, which is phosphorylated on Ser287 in Xenopus prophase oocytes, is dephosphorylated by type 1 phosphatase (PP1) at GVBD. Since the PP1 inhibitor I prevents meiotic maturation, PP1 could participate in the regulation of the MPF autoamplification loop by catalyzing the removal of the inhibitory Ser287 phosphate, and could therefore be involved in the regulation of Cdc25 during oogenesis (Karaiskou, 2004).
In competent oocytes, Plx1 action on Cdc25 is antagonized by an okadaic acid-sensitive phosphatase, involving PP2A activity. This explains why the auto-amplification mechanism can be artificially activated by okadaic acid. However, okadaic acid is unable to promote Cdc2 activation in small incompetent oocytes, showing that the loop implying Cdc2, Cdc25, Plx1 and PP2A is not functional in growing oocytes. The most probable explanation for this defect is the absence of Plx1 in stage IV oocytes. Indeed, it has been shown, both in vivo and in vitro, that expression of Plk1 is sufficient to restore the activation of MPF in response to okadaic acid in incompetent oocytes. Plx1 is therefore the missing factor explaining why the auto-amplification of MPF is defective in small oocytes (Karaiskou, 2004).
Altogether, these experiments show that the incompetence of small oocytes to resume meiosis is ensured by the absence of Plx1 resulting in a double negative control on MPF activation. (1) The formation of active complexes between Cdc2 and newly synthesized cyclins is prevented by a sustained activity of Myt1 that escapes downregulation by Plx1. (2) Cdc25 activation that is normally achieved through a feedback loop involving Plx1 is also prevented. Further investigation will be necessary to discover (1) how Plx1 expression is controlled by cell size at the end of oogenesis; (2) how PP2A controls Cdc25 activity in small oocytes, and (3) how the initial steps of the progesterone transduction pathway connect to MPF regulators, allowing the female germ cell to resume meiosis when oocyte growth is completed (Karaiskou, 2004).
In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin-dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes. Activation of PKB and CDK1 was monitored during maturation of mouse oocytes. PKB phosphorylation and activation preceded GVBD (germinal vesicle breakdown) in oocytes maturing either in vitro or in vivo. Activation was transient and PKB activity was markedly reduced when virtually all of the oocytes had undergone GVBD. PKB activation was independent of CDK1 activity, because although butyrolactone I prevented CDK1 activation and GVBD, PKB was nevertheless transiently phosphorylated and activated. LY-294002, an inhibitor of phosphoinositide 3-kinase-PKB signalling, suppressed activation of PKB and CDK1 as well as resumption of meiosis. OA (okadaic acid)-sensitive phosphatases are involved in PKB-activity regulation, because OA induced PKB hyperphosphorylation. During resumption of meiosis, PKB phosphorylated on Ser(473) is associated with nuclear membrane and centrosome, whereas PKB phosphorylated on Thr(308) is localized on centrosome only. The results of the present paper indicate that PKB is involved in CDK1 activation and resumption of meiosis in mouse oocytes. The presence of phosphorylated PKB on centrosome at the time of GVBD suggests its important role for an initial CDK1 activation (Kalous, 2005).
Female meiosis is driven by the activities of two major kinases, cyclin-dependent kinase 1 (Cdk1) and mitogen-activated protein kinase (MAPK). To date, the role of MAPK in control of meiosis is thought to be restricted to maintaining metaphase II arrest through stabilizing Cdk1 activity. This study finds that MAPK and Cdk1 play compensatory roles to suppress the anaphase-promoting complex/cyclosome (APC/C) activity early in prometaphase, thereby allowing accumulation of APC/C substrates essential for meiosis I. Furthermore, inhibition of MAPK around the onset of APC/C activity at the transition from meiosis I to meiosis II led to accelerated completion of meiosis I and an increase in aneuploidy at metaphase II. These effects appear to be mediated via a Cdk1/MAPK-dependent stabilization of the spindle assembly checkpoint, which when inhibited leads to increased APC/C activity. These findings demonstrate new roles for MAPK in the regulation of meiosis in mammalian oocytes (Nabti, 2014).
The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Data is presented indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. These data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, it was found that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, it is reported that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy (Chu, 2007).
p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 310-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases (Grimmler, 2007).
p27Kip1 is an intrinsically unstructured protein (IUP) that controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Usually, binding of p27 inactivates the kinase; however, p27 was surprisingly also found to be associated with active cyclin D holoenzymes. p27 level is frequently controlled by regulated translation and proteolysis. The protein is abundant in quiescent (G0) cells and is relatively stable in G0 and early G1 phase. p27 becomes unstable as cells progress toward S phase. p27 degradation is initiated by different ubiquitin ligases. Among these, the KPC1 complex ubiquitinates free unphosphorylated p27, whereas Skp2-dependent E3 ligase complexes target p27 only after phosphorylation on threonine 187 (T187). Active cyclin E/Cdk2 can phosphorylate T187 of cyclin/Cdk-bound p27. While free and active cyclin/Cdk2 efficiently phosphorylates Cdk-bound p27, p27-bound Cdk2 is catalytically inactive due to p27-mediated remodeling of the catalytic cleft and displacement of ATP. This has suggested that degradation by the SCF-Skp2 pathway may require p27-free cyclin E/Cdk2 and has led to the puzzle of how p27 degradation can be initiated in G1 (Grimmler, 2007).
This study reports that p27 can be phosphorylated on a tyrosine residue at position 88 (Y88) within its Cdk-binding domain. This phosphorylation caused the inhibitory 310-helix of p27 to be ejected from the ATP-binding pocket of Cdk2. Y88-phosphorylated p27 still binds to cyclin/Cdk complexes, but the associated kinase retains significant catalytic activity. Furthermore, Y88-phosphorylated p27 becomes an efficient substrate for phosphorylation on T187 by Cdk2 within the trimeric complex. Thus, Y88 phosphorylation may trigger p27 ubiquitination in the absence of free cyclin/Cdk2 and may initiate SCF-Skp2-dependent p27 degradation at the G1/S transition (Grimmler, 2007).
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