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).
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).
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).
LATS/Warts, a negative regulator of cdc2 The cyclin dependent kinase CDC2 has been shown to interact with LATS1, a human homolog of Drosophila Warts. In immunoprecipitation experiments, the amount of co-precipitated CDC2 varies with the cell cycle. Co-precipitated CDC2 is most abundant at early mitosis, after which the amount of co-precipitated CDC2 progressively decreases as the cell cycle progresses. No co-precipitated CDC2 is detected in quiescent cells. Using the yeast two-hybrid assay, full-length LATS1 and the N-terminal region of LATS interacts with CDC2. The C-terminal kinase domain of LATS1 does not interact with CDC2. Consistent with the notion that the CDC2-associated N-terminal domain is essential for LATS function, a transgene lacking this domain does not rescue warts mutant flies. Neither full-length LATS1 nor the N-terminal region of LATS1 shows any interaction with the G1 cell-cycle kinases CDK2 and CDK4, indicating that the association between LATS1 and CDC2 is specific. The association of LATS1 and CDC2 suggests that LATS1 could act as a tumor suppressor by negatively regulating CDC2 kinase activity, although such a negative effect could not be detected in vitro (Tao, 1999).
Yeast two-hybrid experiments show that the N-terminal region of human LATS1 interacts with CDC2 more strongly than does full-length LATS1. Furthermore, the C-terminal LATS1 kinase domain does bind to the N-terminal region of LATS1 in the two-hybrid assay. These observations raise the possibility that the LATS1 kinase domain may function as a negative regulatory domain that interferes with the CDC2/LATS1 association via intramolecular binding to its N-terminal region. The association of LATS1 with CDC2 is correlated with LATS1's state of phosphorylation. This correlation could be coincidental, however, as phosphorylation is a common mechanism regulating protein activities during the cell cycle. Phosphorylation of LATS1 may be a prerequisite for its binding to DCD2 and may change its conformation, disrupting the intramolecular association between the N and C termini of LATS1 and freeing the N-terminal domain of LATS1 for CDC2 binding (Tao, 1999).
LATS1 is phosphoryated, and the phosphorylation state oscillates with the cell cycle. All LATS1 protein is phosphorylated at late prophase and remains phosphorylated through metaphase. Dephosphorylated LATS can be detected when cells begin to enter anaphase, and by the start of telophase, most LATS1 is dephosphorylated. These observations suggest that LATS1 undergoes two major phosphorylation changes during the cell cycle: LATS1 is phosphorylated at the G2/M boundary or in early prophase, and becomes dephosphorylated at the metaphase/anaphase boundary (Tao, 1999).
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