Cyclin E
In human and murine cells, there are three known Cdc25 proteins: Cdc25A, Cdc25B, and Cdc25C. The three phosphatases share approximately 40 to 50% homology. Cdc25C and Cdc25A function at G2-M and G1-S transitions during the human cell cycle, respectively. Cdc25A associates with,
dephosphorylates, and activates the cell cycle kinase cyclin E-cdk2. p21CIP1 and p27
are cyclin-dependent kinase (cdk) inhibitors induced by growth-suppressive signals
(such as p53) and transforming growth factor beta (TGF-beta). A
cyclin binding motif has been identified near the N terminus of Cdc25A that is similar to the cyclin binding
Cy (or RR LFG) motif of the p21CIP1 family of cdk inhibitors and separate from the
catalytic domain. Mutations in this motif disrupt the association of Cdc25A with cyclin
E- or cyclin A-cdk2 in vitro and in vivo and selectively interfere with the
dephosphorylation of cyclin E-cdk2. A peptide based on the Cy motif of p21
competitively disrupts the association of Cdc25A with cyclin-cdks and inhibits the
dephosphorylation of the kinase. p21 inhibits Cdc25A-cyclin-cdk2 association and the
dephosphorylation of cdk2. Conversely, Cdc25A, which is itself an oncogene
up-regulated by the Myc oncogene, associates with cyclin-cdk and protects it from
inhibition by p21. Cdc25A also protects DNA replication in Xenopus egg extracts
from inhibition by p21. These results describe a mechanism by which the Myc- or
Cdc25A-induced oncogenic and p53- or TGF-beta-induced growth-suppressive
pathways counterbalance each other by competing for cyclin-cdks (Saha, 1997).
Cyclin E-Cdk2 interaction with p300 The p300 and CREB binding protein (CBP) transcriptional coactivators interact with a variety of transcription factors and regulate their
activity. Among the interactions that have been described, the COOH-terminal region of p300 binds to cyclin E-cyclin-dependent
kinase 2 (cyclin E-Cdk2) and TFIIB, as well as to the E1A gene products of adenovirus. Inhibition of Cdk activity by Cdk inhibitors,
such as p21 or p27, potentiates NF-kappaB activity and provides a mechanism to coordinate cell cycle progression with the
transcription of genes expressed during growth arrest. In this report, the specific domains of p300 required for the binding of p300 to cyclin E-Cdk2,
TFIIB, and E1A have been examined, as well as the ability of these proteins to interact with p300, either alone or in combination. 12S E1A, an inhibitor of p300-dependent transcription, reduces the
binding of TFIIB to p300, but not that of cyclin E-Cdk2, to p300. In contrast, 13S E1A, a pleiotropic transcriptional activator, does not inhibit TFIIB binding to p300,
although it enhances the interaction of cyclin E-Cdk2 with p300. Modification of cyclin E-Cdk2 is most likely required for association with p300 since the interaction
is observed only with cyclin E-Cdk2 purified from mammalian cells. Domain swap studies show that the cyclin homology domain of TFIIB is involved in interactions
with p300, although the homologous region from cyclin E does not mediate this interaction. These findings suggest that p300 or CBP function is regulated by
interactions of various proteins with a common coactivator domain (Felzien, 1999).
The observations of cooperative and competitive interactions of cyclin
E-Cdk2 and TFIIB with p300 provide mechanistic explanations for several
previously described functional activities of these proteins. For
instance, expression of the p21
cyclin-dependent kinase inhibitor activates human immunodeficiency virus transcription through NFkappa and p300. This
finding suggested that inhibition of cyclin E-Cdk2 complexes activates NF-kappaB through p300 and that active cyclin E-Cdk2 antagonizes this activation. It has also been shown that p21 specifically inhibits cyclin
E-Cdk2 complexes associated with p300-Rel A and CBP-Rel A complexes,
confirming that one mechanism for p21 activation of NF-kappaB through
p300-CBP is by means of its inhibition of associated cyclin E-Cdk2 complexes.
While active cyclin E-Cdk2 complexes seem to inhibit p300-CBP function,
TFIIB contributes to the activation of transcription by p300-CBP, as
demonstrated by its involvement in the recruitment of a CBP-containing
RNA polymerase II holoenzyme to the beta interferon enhancer. Thus, the observation reported in this paper that TFIIB and
cyclin E-Cdk2 complexes compete for binding to a common region of p300
provides an explanation for the opposing effects of TFIIB and cyclin
E-Cdk2 complexes on p300 activity. This example of competitive
interactions at the COOH terminus of p300 could be a mechanism that
occurs with additional regulatory proteins to control a variety of
promoters dependent on p300 (Felzien, 1999 and references).
Although the binding of TFIIB and cyclin E-Cdk2 to p300 is competitive,
cyclin E-Cdk2 and TFIIB differ in the biochemical basis for their
interaction with p300. The binding of TFIIB involves, in part, its
cyclin homology domain, but the corresponding region of cyclin E alone
cannot facilitate p300 binding. This result is consistent with the
findings that sequences within both the amino-
and carboxy-terminal regions of TFIIB are necessary for its
interaction with CBP. Intact cyclin E-Cdk2
complexes from nuclear extracts are required for interactions with
p300, suggesting that a specific conformation or
posttranslational modification of cyclin E-Cdk2 or additional polypeptides are needed to mediate interactions between cyclin E-Cdk2
and p300. Cdk2 function is modulated at specific cell cycle phases by
phosphorylation and dephosphorylation at certain threonine and tyrosine
residues. The requirement of the assembled cyclin
E-Cdk2 complex and an additional role of phosphorylation of critical
residues may ensure the formation of cyclin E-Cdk2-p300 complexes at
distinct times for proper regulation of certain genes (Felzien, 1999 and references).
Cyclin E nuclear import 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).
Mutation of Cyclin E Cyclins are regulatory subunits of cyclin-dependent kinases. Cyclin A, the first cyclin ever cloned, is thought to be an essential component of the cell-cycle engine. Mammalian cells encode two A-type cyclins, testis-specific cyclin A1 and ubiquitously expressed cyclin A2. This study tested the requirement for cyclin A function using conditional knockout mice lacking both A-type cyclins. Acute ablation of cyclin A in fibroblasts did not affect cell proliferation, but led to prolonged expression of another cyclin, cyclin E, across the cell cycle. However, combined ablation of all A- and E-type cyclins extinguished cell division. In contrast, cyclin A function was essential for cell-cycle progression of hematopoietic and embryonic stem cells. Expression of cyclin A is particularly high in these compartments, which might render stem cells dependent on cyclin A, whereas in fibroblasts cyclins A and E play redundant roles in cell proliferation (Kalaszczynska, 2009).
Transcriptional regulation of Cyclin E In the human, accumulation of G1 cyclins is regulated by E2F1. E2F binding sites are found in both the cyclin E and cyclin D1 promoters; both promoters are activated by E2F gene products, and at least for cyclin E, the E2F sites contribute to cell cycle control. The endogenous cyclin E gene is activated following expression of the E2F1 product encoded by a recombinant adenovirus vector. These results suggest the involvement of E2F1 and cyclin E in an autoregulatory loop that governs the accumulation of critical activities affecting the progression of cells through G1 (Ohtani, 1995).
In addition to the E2F-dependent activation of a number of genes encoding DNA replication activities such as DNA Pol alpha, the majority of genes encoding initiation proteins, including Cdc6 and the Mcm proteins, are activated following the stimulation of cell growth and are regulated by E2F. The transcription of a subset of these genes, which includes Cdc6, cyclin E, and cdk2, is also regulated during the cell cycle. Whereas overall E2F DNA-binding activity accumulates during the initial G1 following a growth stimulus, only E2F3-binding activity reaccumulates at subsequent G1/S transitions, coincident with the expression of the cell-cycle-regulated subset of E2F-target genes. Immunodepletion of E2F3 activity inhibits the induction of S phase in proliferating cells. It is proposed that E2F3 activity plays an important role during the cell cycle of proliferating cells, controlling the expression of genes whose products are rate limiting for initiation of DNA replication, thereby imparting a more dramatic control of entry into S phase than would otherwise be achieved by post-transcriptional control alone (Leone, 1998).
Transient induction of the cyclin E gene in late G1 gates progression into S. This event is controlled via a cyclin E repressor module
(CERM), a novel bipartite repressor element located near the cyclin E transcription start site. CERM consists of a variant E2F-binding site and a
contiguous upstream AT-rich sequence that cooperate during G0/G1 to delay cyclin E expression until late G1. CERM binds the protein complex
CERC, which disappears upon progression through G0-G1 and reappears upon entry into the following G1. CERC disappearance correlates
kinetically with the liberation of the CERM module in vivo and cyclin E transcriptional induction. CERC contains E2F4/DP1 and a pocket protein,
and sediments faster than classical E2F complexes in a glycerol gradient, suggesting the presence of additional components in a novel high
molecular weight complex. Affinity purified CERC binds to CERM but not to canonical E2F sites, thus displaying behavior different from known
E2F complexes. In cells nullizygous for members of the Rb family, CERC is still detectable and CERM-dependent repression is functional. Thus
p130, p107 and pRb all function interchangeably in CERC. Notably, the CERC-CERM complex dissociates prematurely in pRb-/- cells in
correspondence with the premature expression of cyclin E. Thus, a new regulatory module has been idenfied that controls repression of G1-specific genes
in G0/G1 (Le Cam, 1999).
Rb forms a repressor complex containing histone deacetylase (HDAC) and the hSWI/SNF nucleosome remodeling complex, which inhibits
transcription of genes for cyclins E and A and arrests cells in the G1 phase of the cell cycle. Phosphorylation of Rb by cyclin D/cdk4 disrupts association with
HDAC, relieving repression of the cyclin E gene and G1 arrest. However, the Rb-hSWI/SNF complex persists and is sufficient to maintain repression of the cyclin A
and cdc2 genes, inhibiting exit from S phase. HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF then appear to maintain the order of cyclin E and A expression during the
cell cycle, which in turn regulates exit from G1 and from S phase, respectively (Zhang, 2000).
Association between Rb and PcG proteins forms a repressor complex that blocks entry of cells into mitosis. Also, evidence is provided that Rb colocalizes with nuclear PcG complexes and is important for association of PcG complexes with nuclear targets. The Rb-PcG complex may provide a means to link cell cycle arrest to differentiation events leading to embryonic pattern formation (Dahiya, 2001).
Rb can interact with HDAC-1-3, and histone deacetylase has been demonstrated to be required genetically for Rb function in C. elegans. HDAC activity is reportedly required for Rb family repression of the cyclin E gene. Also, it was proposed that this repression of cyclin E expression is relieved when growth factors signal through the Ras/MAP kinase pathway to induce expression of D cyclins, which in turn combine with cdk4/6 to form active kinases that phosphorylate Rb. This phosphorylation induces a conformational change in Rb that displaces HDAC, thereby relieving repression of cyclin E expression. Cyclin E then combines with cdk2 to form a kinase that is essential for progression of cells into S phase, at least in part because it is required for assembly of origins of DNA replication (Dahiya, 2001).
Rb repressor activity and growth suppression also require interaction with BRG1 or Brm, which are central components of SWI/SNF chromatin-remodeling complexes. Mice heterozygous for BRG1 or the SNF5 component of the SWI/SNF complex are prone to tumor formation, and BRG1 has been found to be mutated in multiple human tumor cell lines. Furthermore, genetic screens in Drosophila have identified Brm and two other SWI/SNF components as enhancers of E2F activity. These results are consistent with a role for the SWI/SNF complex as a tumor suppressor critical for the Rb family pathway. Accordingly, BRG1 has been found to be required for Rb to repress expression of cyclins E and A and cdc2. However, while this repression of cyclin E expression requires HDAC, repression of cyclin A and cdc2 expression does not. Cyclin A/cdk2 phosphorylation of the cdh1 subunit of the anaphase-promoting complex reportedly blocks its ubiquitin ligase-mediated turnover of cyclin B. Repression of cyclin A and cdc2 expression by the Rb family then prevents assembly of cyclin B/cdc2, which is required for entry into mitosis. Therefore, in addition to blocking the G1/S transition, this inhibition of cyclin B/cdc2 activity allows Rb to also block entry of cells into mitosis. The finding that repression of cyclin A and cdc2 expression is HDAC independent suggests that distinct Rb repressor complexes may block the cell cycle at G1/S (e.g., repression of cyclin E expression) and G2/M (e.g., repression of cyclin A and cdc2 expression) (Dahiya, 2001).
Cyclin E, a partner of the cyclin-dependent kinase Cdk2, has been implicated in positive control of the G1/S phase transition. Whereas degradation of cyclin E has been shown to be exquisitely regulated by ubiquitination and proteasomal action, little is known about posttranscriptional aspects of its biogenesis. In a yeast-based screen designed to identify human proteins that interact with human cyclin E, components of the eukaryotic cytosolic chaperonin CCT have been identified. The endogenous CCT complex in yeast is essential for the maturation of cyclin E in vivo. Under conditions of impaired CCT function, cyclin E fails to accumulate. Furthermore, newly translated cyclin E, both in vitro in reticulocyte lysate and in vivo in human cells in culture, is efficiently bound and processed by the CCT. In vitro, in the presence of ATP, the bound protein is folded and released in order to become associated with Cdk2. Thus, both the acquisition of the native state and turnover of cyclin E involve ATP-dependent processes mediated by large oligomeric assemblies (Won, 1998).
Cyclin E1 is essential for the G1-S transition during development in Xenopus embryos. Cyclin E1 is found to be abundant in eggs, and after fertilization, until the midblastula transition when levels of cyclin E1 protein and associated kinase activity are found to decline precipitously. The stability of the cyclin E1 protein appears to play a major role in reduction of cyclin E1 levels at this time. Since activation of turnover occurs independently of cell cycle progression, does not require ongoing protein synthesis, and is not triggered as a result of the ratio of nuclei to cytoplasm in embryonic cells, it is proposed that a developmental timing mechanism measures the 5 hour time period from the time of fertilization, and then allows activation of a protein degradative pathway that regulates cyclin E1. Characterization of the timer suggests that it might be held inactive in eggs by a mitogen-activated protein kinase signal transduction pathway (Howe, 1996).
During Xenopus development, the early cell cycles consist of rapid oscillations between DNA synthesis and mitosis until completion of the 12th mitotic division. Then the cycle lengthens and becomes asynchronous: zygotic transcription begins, and G phases are established, a period known as the midblastula transition (MBT). Some aspects of the MBT, such as zygotic transcription, depend on acquisition of a threshold nuclear to cytoplasmic (N/C) ratio, whereas others, such as maternal cyclin E degradation, are independent of nuclear events and appear to be controlled by an autonomous maternal timer. To investigate the function of cyclin E during the early cycles, cyclin E/Cdk2 kinase activity was specifically inhibited in fertilized eggs by injection of a truncated form of the Xenopus Cdk inhibitor, Xic1 (Delta34Xic1). Delta34Xic1 causes lengthening of the embryonic cell cycles; this correlates with increased levels of mitotic cyclins. However, DNA synthesis is not inhibited. Several hallmarks of the MBT are delayed for several hours in Delta34Xic1-injected embryos, including the disappearance of cyclins E and A, the initiation of zygotic transcription, and the reappearance of phosphotyrosine on Cdc2. In both control and Delta34Xic1-injected embryos, cyclin E is degraded after the 12th mitotic division, the point at which zygotic transcription begins, but experiments with alpha-amanitin show that cyclin E degradation is not dependent on zygotic transcription. Thus, the length of the early cycles and the timing of maternal cyclin degradation depend upon cyclin E/Cdk2 activity. Neither oscillations in cyclin E/Cdk2 activity during the early cycles nor the disappearance of cyclin E at the MBT are dependent on protein synthesis. These data suggest that cyclin E/Cdk2 is directly linked to an autonomous maternal timer that drives the early embryonic cell cycles until the MBT. No components of the cytoplasmic oscillator or of the developmental timer that controls its expression have been identified. However, it is possible that the centrosome duplication cycle can control the length of the early embryonic cell cycle (Hartley, 1997).
Cyclin E is a mammalian G1 cyclin that is both required and rate limiting for entry into S phase. The expression of cyclin E is periodic, peaking at the G1-S transition and then decaying as S phase progresses. To understand the mechanisms underlying cyclin E periodicity, the regulation of cyclin E degradation was investigated. Cyclin E is degraded by the ubiquitin-proteasome system, and this degradation is regulated by both cdk2 binding and cdk2 catalytic activity. Free cyclin E is readily ubiquitinated and degraded by the proteasome. Binding to cdk2 protects cyclin E from ubiquitination: this protection is reversed by cdk2 activity in a process that involves phosphorylation of cyclin E itself. The data are most consistent with a model in which cdk2 activity initiates cyclin E degradation by promoting the disassembly of cyclin E-cdk2 complexes, followed by the ubiquitination and degradation of free cyclin E (Clurman, 1996).
Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome-dependent pathway. Cyclin
E ubiquitination is stimulated by two different pathways in vivo: one functions when it is free of its CDK partner, and the other when it is phosphorylated on threonine 380. The first
of these pathways has been pursued by using a two-hybrid screen to identify proteins that bind only to free cyclin E. This resulted in the
isolation of human Cul-3, a member of the cullin family of E3 ubiquitin-protein ligases. Cul-3 is bound to cyclin
E but not to cyclin E-Cdk2 complexes in mammalian cells; overexpression of Cul-3 increases ubiquitination of cyclin E
but not other cyclins. Conversely, deletion of the Cul-3 gene in mice causes increased accumulation of cyclin E protein, and has cell-type-specific effects on
S-phase regulation. In the extraembryonic ectoderm, in which cells undergo a standard mitotic cycle, there is a greatly increased number of cells in S phase.
In the trophectoderm, in which cells go through endocycles, there is a block to entry into S phase. The SCF pathway, which targets cyclins for
ubiquitination on the basis of their phosphorylation state, and the Cul-3 pathway, which selects cyclin E for ubiquitination on the basis of its assembly into
CDK complexes, may be complementary ways to control cyclin abundance (Singer, 1999).
Thus ubiquitination of cyclin E depends on two parameters: its binding to a CDK, and its state of phosphorylation on threonine 380. These two pathways may be governed by distinct ubiquitinating enzymes, one of which recognizes a feature unique to unbound
cyclin E, and the other that directly recognizes phosphorylated T380. Phosphorylation of T380 is required for ubiquitination
of cyclin E bound to Cdk2, but not for ubiquitination of unbound cyclin E. Ubiquitination of a budding yeast G1 cyclin, Cln2, occurs by means of a
phosphorylation-triggered pathway in which an E3 protein-ubiquitin ligase, the SCF complex, binds directly to the phosphorylated cyclin. The fact that phosphorylation of T380 in cyclin E promotes the
ubiquitination of cyclin E is consistent with the SCF paradigm, but direct evidence for the involvement of this pathway in the turnover of cyclin E is, thus far,
lacking (Singer, 1999).
A second pathway, one which involves ubiquitination and degradation of proteins that are separated from their normal binding partners, may also be critically
important. This pathway was initially recognized as being crucial for the rapid turnover of proteins within the endoplasmic reticulum that are either misfolded or
incorrectly assembled into multiprotein complexes. One example is the rapid
destruction of T-cell receptor alpha chains that fail to assemble into complexes with other receptor subunits. This general idea has been extended to include nuclear proteins including cyclin E, which is protected from ubiquitination when assembled with Cdk2. Another example is that of E2F-1, which is protected from ubiquitination when bound to Rb. In the case of cyclin E, Cul-3 recognizes and stimulates the ubiquitination of unbound cyclin E, but not cyclin E within cyclin
E-Cdk2 complexes. Cyclin E might contain an instability determinant that is masked in the cyclin E-Cdk2 complex. Alternatively, it is
possible that other features of the cyclin E-Cdk2 complex, such as its kinase activity, might downregulate the interaction between cyclin E and Cul-3. It is
important to emphasize that this pathway for cyclin E ubiquitination is unlikely to be limited to the destruction of unfolded or otherwise nonfunctional protein.
Cells lacking Cul-3 appear to accumulate excess, biologically active cyclin E as evidenced by the misregulation of S phase in Cul-3/
embryos. It therefore seems that the unbound cyclin E is at least potentially active, and it is crucial for the cell to limit the size of this pool (Singer, 1999 and references therein).
The ubiquitin-proteasome pathway plays an important role in control of the abundance of cell cycle regulators. Mice lacking Skp2, an
F-box protein and substrate recognition component of an Skp1-Cullin-F-box protein (SCF) ubiquitin ligase, were generated. Although
Skp2-/- animals are viable, cells in the mutant mice contain markedly enlarged nuclei with polyploidy and multiple centrosomes, and show
a reduced growth rate and increased apoptosis. Skp2-/- cells also exhibit increased accumulation of both cyclin E and p27Kip1. The
elimination of cyclin E during S and G2 phases is impaired in Skp2-/- cells, resulting in loss of cyclin E periodicity. Biochemical studies
have shown that Skp2 interacts specifically with cyclin E and thereby promotes its ubiquitylation and degradation both in vivo and in vitro.
These results suggest that specific degradation of cyclin E and p27Kip1 is mediated by the SCFSkp2 ubiquitin ligase complex, and that Skp2 may control chromosome
replication and centrosome duplication by determining the abundance of cell cycle regulators. Although the precise mechanisms responsible for monitoring centrosome number and function are unknown, recent studies with cell-free assays have suggested that
cyclin E-dependent kinase activity is required for centrosome duplication. Permanent centrosome duplication is observed during early
embryogenesis, in which, as in Skp2-/- cells, cyclin E is not degraded periodically. Thus, the overduplication of
centrosomes apparent in Skp2-/- cells is likely to be attributable to the overexpression and retention of cyclin E (Nakayama, 2000).
Cyclin E, one of the activators of the cyclin-dependent kinase Cdk2, is expressed near the G1-S phase transition and is thought to be critical for the initiation of DNA replication and other S-phase functions. Accumulation of cyclin E at the G1-S boundary is achieved by periodic transcription coupled with regulated proteolysis linked to autophosphorylation of cyclin E. The proper timing and amplitude of cyclin E expression seem to be important, because elevated levels of cyclin E have been associated with a variety of malignancies and constitutive expression of cyclin E leads to genomic instability. Turnover of phosphorylated cyclin E depends on an SCF-type protein-ubiquitin ligase that contains the human homolog of yeast Cdc4, which is an F-box protein containing repeated sequences of WD40 (a unit containing about 40 residues with tryptophan (W) and aspartic acid (D) at defined positions). The gene encoding hCdc4 (Drosophila homolog: archipelago) was found to be mutated in a cell line derived from breast cancer that expressed extremely high levels of cyclin E (Strohmaier, 2001).
Mutations in parkin (see Drosophila parkin), which encodes a RING domain protein associated with ubiquitin ligase activity, lead to autosomal recessive Parkinson's disease characterized by midbrain dopamine neuron loss. Parkin functions in a multiprotein ubiquitin ligase complex that includes the F-box/WD repeat protein hSel-10 and Cullin-1. HSel-10 serves to target the parkin ubiquitin ligase activity to cyclin E, an hSel-10-interacting protein previously implicated in the regulation of neuronal apoptosis. Consistent with the notion that cyclin E is a substrate of the parkin ubiquitin ligase complex, parkin deficiency potentiates the accumulation of cyclin E in cultured postmitotic neurons exposed to the glutamatergic excitotoxin kainate and promotes their apoptosis. Furthermore, parkin overexpression attenuates the accumulation of cyclin E in toxin-treated primary neurons, including midbrain dopamine neurons, and protects them from apoptosis (Staropoli, 2003).
Autophosphorylation-triggered ubiquitination has been proposed to be the major pathway regulating cyclin E protein abundance: phosphorylation of cyclin E on T380 by its associated CDK allows binding to the receptor subunit, Fbw7, of the SCFFbw7 ubiquitin ligase. This model has been tested in vivo; it has been found to be an inadequate representation of the pathways that regulate cyclin E degradation. Assembly of cyclin E into cyclin E-Cdk2 complexes is shown (1) to be required in vivo for turnover by the Fbw7 pathway, (2) Cdk2 activity is required for cyclin E turnover in vivo because it phosphorylates S384, (3) phosphorylation of T380 in vivo does not require Cdk2 and is mediated primarily by GSK3, and (4) two additional phosphorylation sites, T62 and S372, are also required for turnover. Thus, cyclin E turnover is controlled by multiple biological inputs and cannot be understood in terms of autophosphorylation alone (Welcker, 2003).
Phosphorylations within N- and C-terminal degrons independently control the binding of cyclin E to the SCFFbw7 and thus its ubiquitination and proteasomal degradation. This study determined the physiologic significance of cyclin E degradation by this pathway. A knockin mouse was constructed in which both degrons were mutated by threonine to alanine substitutions (cyclin ET74A T393A); ablation of both degrons abolished regulation of cyclin E by Fbw7. The cyclin ET74A T393A mutation disrupted cyclin E periodicity and causes cyclin E to continuously accumulate as cells reentered the cell cycle from quiescence. In vivo, the cyclin ET74A T393A mutation greatly increased cyclin E activity and caused proliferative anomalies. Cyclin ET74A T393A mice exhibited abnormal erythropoiesis characterized by a large expansion of abnormally proliferating progenitors, impaired differentiation, dysplasia, and anemia. This syndrome recapitulates many features of early stage human refractory anemia/myelodysplastic syndrome, including ineffective erythropoiesis. Epithelial cells also proliferated abnormally in cyclin E knockin mice, and the cyclin ET74A T393A mutation delayed mammary gland involution, implicating cyclin E degradation in this anti-mitogenic response. Hyperproliferative mammary epithelia contained increased apoptotic cells, suggesting that apoptosis contributes to tissue homeostasis in the setting of cyclin E deregulation. Overall these data show the critical role of both degrons in regulating cyclin E activity and reveal that complete loss of Fbw7-mediated cyclin E degradation causes spontaneous and cell type-specific proliferative anomalies (Minella, 2008).
Cyclin E
regulates the cell cycle transition from G1 to S phase and is degraded before entry into G2 phase. This study shows that in mice RhoBTB3, a Golgi-associated, Rho-related ATPase, regulates the S/G2 transition of the cell cycle by targeting Cyclin E for ubiquitylation. Depletion of RhoBTB3 arrested cells in S phase, triggered Golgi fragmentation, and elevated Cyclin E levels. On the Golgi, RhoBTB3 bound Cyclin E as part of a Cullin3 (CUL3)-dependent RING-E3 ubiquitin ligase complex comprised of RhoBTB3, CUL3 (see Drosophila Cullin-3), and RBX1. Golgi association of this complex is required for its ability to catalyze Cyclin E ubiquitylation and allow normal cell cycle progression. These experiments reveal a novel role for a Ras superfamily member in catalyzing Cyclin E turnover during S phase, as well as an unexpected, essential role for the Golgi as a ubiquitylation platform for cell cycle control (Lu, 2013).
Germ cells, the cells that give rise to sperm and egg, maintain the potential to recreate all cell types in a new individual. This wide developmental potential, or totipotency, is manifested in unusual tumors called teratomas, in which germ cells undergo somatic differentiation. Although recent studies have implicated RNA regulation, the mechanism that normally prevents the loss of germ cell identity remains unexplained. In C. elegans, a teratoma is induced in the absence of the conserved RNA-binding protein GLD-1. Here, this study demonstrates that GLD-1 represses translation of CYE-1/cyclin E during meiotic prophase, which prevents germ cells from re-entering mitosis and inducing embryonic-like transcription. A mechanism is described that prevents precocious mitosis in germ cells undergoing meiosis, it is proposed that this mechanism maintains germ cell identity by delaying the onset of embryonic gene activation until after fertilization, and a paradigm is provided for the possible origin of human teratomas (Biedermann, 2009).
The C. elegans germline provides an excellent model for analyzing the regulation of stem cell activity and the decision to differentiate and undergo meiotic development. The distal end of the adult hermaphrodite germline contains the proliferative zone, which includes a population of mitotically cycling cells and cells in meiotic S phase, followed by entry into meiotic prophase. The proliferative fate is specified by somatic distal tip cell (DTC) niche-germline GLP-1 Notch signaling through repression of the redundant GLD-1 and GLD-2 pathways that promote entry into meiosis. This study describes characteristics of the proliferative zone, including cell cycle kinetics and population dynamics, as well as the role of specific cell cycle factors in both cell cycle progression and the decision between the proliferative and meiotic cell fate. Mitotic cell cycle progression occurs rapidly, continuously, with little or no time spent in G1, and with cyclin E (CYE-1) levels and activity high throughout the cell cycle. In addition to driving mitotic cell cycle progression, CYE-1 and CDK-2 also play an important role in proliferative fate specification. Genetic analysis indicates that CYE-1/CDK-2 promotes the proliferative fate downstream or in parallel to the GLD-1 and GLD-2 pathways, and is important under conditions of reduced GLP-1 signaling, possibly corresponding to mitotically cycling proliferative zone cells that are displaced from the DTC niche. Furthermore, GLP-1 signaling was found to regulate a third pathway, in addition to the GLD-1 and GLD-2 pathways and also independent of CYE-1/CDK-2, to promote the proliferative fate/inhibit meiotic entry (Fox, 2011).
Considerable evidence points to a role for G1 cyclin-dependent kinase (CDK) in allowing the accumulation of E2F transcription factor activity and induction of the S phase of the cell cycle. Numerous experiments have also demonstrated a critical role for both Myc and Ras (See Drosophila Ras1) activities in allowing cell-cycle progression. Inhibition of Ras activity blocks the normal growth-dependent activation of G1 CDK, prevents activation of the target genes of E2F, and results in cell-cycle arrest in G1. Ras is essential for entry into the S phase in Rb+/+ fibroblasts but not in Rb-/- fibroblasts, establishing a link between Ras and the G1 CDK/Rb/E2F pathway. However, although expression of Ras alone will not induce G1 CDK activity or S phase, coexpression of Ras with Myc allows the generation of cyclin E-dependent kinase activity and the induction of S phase, coincident with the loss of the p27 cyclin-dependent kinase inhibitor (CKI). These results suggest that Ras, along with the activation of additional pathways, is required for the generation of G1 CDK activity, and that activation of cyclin E-dependent kinase in particular depends on the cooperative action of Ras and Myc (Leone, 1997).
Estrogen-induced progression through G1 phase of the cell cycle is preceded by increased expression of the G1-phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, clonal MCF-7 breast cancer cell lines were derived in which c-Myc or cyclin D1 is expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 is sufficient for S-phase entry in cells previously arrested in G1 phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression is not accompanied by increased cyclin D1 expression or Cdk4 activation, nor is cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 is sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21 following estrogen treatment. Interestingly, this is accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130 (a pRB-related pocket protein), identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis, which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21 (Prall, 1998).
Specific cyclin-dependent kinases are found to regulate transcriptional activation by NF-kappaB (Drosophila homolog Dorsal) transcription factor through interactions with the transcriptional coactivator p300. The transcriptional activation domain of RelA(p65), a subunit of NF-kappaB, interacts with an amino-terminal region of p300 that is distinct from a carboxyl-terminal region of p300 required for binding to the cyclin E-Cdk2 complex. The CDK inhibitor p21 (Drosophila homolog Dacapo) or a dominant negative Cdk2, which inhibits p300-associated cyclin E-Cdk2 activity, stimulates NF-kappaB-dependent gene expression, which is also enhanced by expression of p300 in the presence of p21. The interaction of NF-kappaB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression (Perkins, 1997).
Transforming growth factor beta (TGF-beta)-mediated G1 arrest specifically targets inactivation of cyclin D:cyclin-dependent kinase (Cdk) 4/6 complexes. TGF-beta-treated human HepG2 hepatocellular carcinoma cells arrest in G1, but retain continued cyclin D:Cdk4/6 activity and active, hypophosphorylated retinoblastoma tumor suppressor protein. Consistent with this observation, TGF-beta-treated cells fail to induce p15(INK4b), down-regulate CDC25A, or increase levels of p21(CIP1), p27(KIP1), and p57(KIP2). However, TGF-beta treatment results in the specific inactivation of cyclin E:Cdk2 complexes caused by absence of the activating Thr(160) phosphorylation on Cdk2. Whole-cell lysates from TGF-beta-treated cells show inhibition of Cdk2 Thr(160) Cdk activating kinase (CAK) activity; however, cyclin H:Cdk7 activity, a previously assumed mammalian CAK, is not altered. Saccharomyces cerevisiae contains a genetically and biochemically proven CAK gene, CAK1, that encodes a monomeric 44-kDa Cak1p protein unrelated to Cdk7. Anti-Cak1p antibodies cross-react with a 45-kDa human protein with CAK activity that is specifically down-regulated in response to TGF-beta treatment. Taken together, these observations demonstrate that TGF-beta signaling mediates a G(1) arrest in HepG2 cells by targeting Cdk2 CAK and suggests the presence of at least two mammalian CAKs: one specific for Cdk2 and one for Cdk4/6 (Nagahara, 1999).
Mitosis requires activity of the cyclin B cyclin-dependent kinase 1 (cdc2) heterodimer. Exit from mitosis depends on the inactivation of the complex by the degradation of cyclin B. Cdk2 is also active during mitosis. In Xenopus egg extracts, cdk2 is primarily in complex with cyclin E, which is stable. At the end of mitosis, downregulation of cdk2-cyclin E activity is accompanied by inhibitory phosphorylation of cdk2. Cdk2-cyclin E activity maintains cdk1-cyclin B during mitosis. At mitosis exit, cdk2 is inactivated prior to cdk1. The loss of cdk2 activity follows and depends upon an increase in protein kinase A (PKA) activity. Prematurely inactivating cdk2 advances the time of cyclin B degradation and cdk1 inactivation. Blocking PKA, instead, stabilizes cdk2 activity and inhibits cyclin B degradation and cdk1 inactivation. The stabilization of cdk1-cyclin B is also induced by a mutant cdk2-cyclin E complex that is resistant to inhibitory phosphorylation. P21-Cip1, which inhibits both wild-type and mutant cdk2-cyclin E, reverses mitotic arrest under either condition. These findings indicate that the proteolysis-independent downregulation of cdk2 activity at the end of mitosis depends on PKA and is required to activate the proteolysis cascade that leads to mitosis exit (D'Angiolella, 2001).
SWI-SNF complexes have been implicated in transcriptional regulation by chromatin remodeling. An interaction has been identified between two components of the mammalian SWI-SNF complex and cyclin E, an essential cell cycle regulatory protein required for G1/S transition. BRG1 and BAF155, mammalian homologs of yeast SWI2 (Drosophila homolog: Brahma) and SWI3 (Drosophila homolog: Moira), respectively, are found in cyclin E complexes and are phosphorylated by cyclin E-associated kinase activity. Overexpression of BRG1 causes growth arrest and induction of senescence-associated beta-galactosidase activity, which can be overcome by cyclin E. These results suggest that cyclin E may modulate the activity of the SWI-SNF apparatus to maintain the chromatin in a transcriptionally permissive state (Shanahan, 1999).
The helix-loop-helix (HLH) protein Id2 is thought to affect the balance between cell growth and differentiation by negatively regulating the function of basic helix-loop-helix (bHLH) transcription factors. Id2 acts by forming heterodimers that are unable to bind to specific (E-box) DNA sequences. This activity can be overcome by phosphorylation of a serine residue within a consensus target site for cyclin-dependent kinases (Cdks). In vitro, Id2 can be phosphorylated by either cyclin E-Cdk2 or cyclin A-Cdk2 but not by cyclin D-dependent kinases. Analogous phosphorylation occurs in serum-stimulated human diploid fibroblasts at a time in late G1 consistent with the appearance of active cyclin E-Cdk2. These data provide a link between cyclin-dependent kinases and bHLH transcription factors that may be critical for the regulation of cell proliferation and differentiation (Hara, 1997).
The functions of basic helix-loop-helix (bHLH) transcription factors in activating differentiation-linked gene expression and in inducing G1 cell cycle arrest are negatively regulated by members of the Id family of HLH proteins. These bHLH antagonists are induced during a mitogenic signaling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences. Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser5) in Id2 has been shown to occur during late G1-to-S phase transition of the cell cycle; this neutralizes the function of Id2 in abrogating E-box-dependent bHLH homo- or heterodimer complex formation in vitro. An analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH homo- or hetero-dimer complex formation in vitro and E-box-dependent reporter gene function in vivo. Whereas unphosphorylated Id3 abrogates an E12 homodimer complex but not the E12-MyoD heterodimer, phosphorylation at serine 5 results in a switch to abrogation of the E12-MyoD heterodimer complex. Compared with wild-type Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to promote cell cycle S phase entry in transfected fibroblasts, whereas an Id3 Ala5 mutant (ablating phosphorylation) displays an activity significantly greater than that of wild-type Id3 protein. The Asp5 Id3 mutant is completely devoid of any activity in promoting S phase, implying that Cdk-dependent phosphorylation inactivates the G1-to-S cell cycle regulatory function of this Id protein. Therefore, Cdk2-dependent phosphorylation provides a switch during late G1-to-S phase that both nullifies an early G1 cell cycle regulatory function of Id3 and modulates its target bHLH specificity. These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not simply a function of its ability to modulate bHLH heterodimer-dependent gene expression: these data establish a biologically important mechanism through which Cdk2 and Id-bHLH functions are integrated in the coordination of cell proliferation and differentiation (Deed, 1997).
To understand the mechanisms by which CDKs regulate cell cycle progression, it is necessary to identify and characterize the physiological substrates of these kinases. A screening method was developed to identify novel CDK substrates. One of the cDNAs identified in the screen is identical to the recently isolated NPAT gene. NPAT associates with cyclin E-CDK2 in vivo and can be phosphorylated by this CDK. The protein level of NPAT peaks at the G1/S boundary. Overexpression of NPAT accelerates S-phase entry, and this effect is enhanced by coexpression of cyclin E-CDK2. These results suggest that NPAT is a substrate of cyclin E-CDK2 and plays a role in S-phase entry (Zhao, 1998).
In eukaryotic cells, histone gene expression is one of the major events that marks entry into S phase. While this process is tightly linked to cell cycle position, how it is regulated by the cell cycle machinery is not known. NPAT, a novel substrate of the cyclin E-Cdk2 complex, is associated with human replication-dependent histone gene clusters on both chromosomes 1 and 6 in S phase. NPAT activates histone gene transcription and this activation is dependent on the promoter elements (SSCSs) previously proposed to mediate cell cycle-dependent transcription. Cyclin E is also associated with the histone gene loci, and cyclin E-Cdk2 stimulates the NPAT-mediated activation of histone gene transcription. Thus, NPAT is involved in a key S phase event and provides a link between the cell cycle machinery and activation of histone gene transcription (Zhao, 2000).
Cyclin E/Cdk2 acts at the G1/S-phase transition to promote the E2F transcriptional program and the initiation of DNA synthesis. How cyclin E/Cdk2 controls S-phase events was examined, as well as the subcellular localization of the cyclin E/Cdk2 interacting protein p220NPAT and its regulation by phosphorylation. p220 is localized to discrete nuclear foci. Diploid fibroblasts in Go and G1 contain two p220 foci, whereas S- and G2-phase cells contain primarily four p220 foci. Cells in metaphase and telophase have no detectable focus. p220 foci contain cyclin E and are coincident with Cajal bodies (CBs), subnuclear organelles that associate with histone gene clusters on chromosomes 1 and 6. Interestingly, p220 foci associate with chromosome 6 throughout the cell cycle and with chromosome 1 during S phase. Five cyclin E/Cdk2 phosphorylation sites in p220 have been identified. Phospho-specific antibodies against two of these sites react with p220 within CBs in a cell cycle-specific manner. The timing of p220 phosphorylation correlates with the appearance of cyclin E in CBs at the G1/S boundary, and this phosphorylation is maintained until prophase. Expression of p220 activates transcription of the histone H2B promoter. Importantly, mutation of Cdk2 phosphorylation sites to alanine abrogates the ability of p220 to activate the histone H2B promoter. Collectively, these results strongly suggest that p220NPAT links cyclical cyclin E/Cdk2 kinase activity to replication-dependent histone gene transcription (Ma, 2000).
Cyclin E-cdk2 is a critical regulator of cell cycle progression from G1 into S phase in mammalian cells. Despite this important function, little is known about the downstream targets of this cyclin-kinase complex. Components of the pre-mRNA processing machinery have been identified as potential targets of cyclin E-cdk2. Cyclin E-specific antibodies coprecipitate a number of cyclin E-associated proteins from cell lysates, among which are the spliceosome-associated proteins, SAP 114, SAP 145, and SAP 155, as well as the snRNP core proteins B' and B. The three SAPs are all subunits of the essential splicing factor SF3, a component of U2 snRNP. Cyclin E antibodies also specifically immunoprecipitated U2 snRNA and the spliceosome from splicing extracts. SAP 155 serves as a substrate for cyclin E-cdk2 in vitro and its phosphorylation in the cyclin E complex can be inhibited by the cdk-specific inhibitor p21. SAP 155 contains numerous cdk consensus phosphorylation sites in its N terminus and is phosphorylated prior to catalytic step II of the splicing pathway, suggesting a potential role for cdk regulation. These findings provide evidence that pre-mRNA splicing may be linked to the cell cycle machinery in mammalian cells (Seghezzi, 1998).
Transcription of rRNA genes by RNA polymerase I increases following serum stimulation of quiescent NIH 3T3 fibroblasts. To elucidate the mechanism underlying transcriptional activation during progression through the G1 phase of the cell cycle, the activity and phosphorylation pattern of the nucleolar transcription factor upstream binding factor (UBF) has been analyzed. Using a combination of tryptic phosphopeptide mapping and site-directed mutagenesis, Ser484 has been identified as a direct target for cyclin-dependent kinase 4 (cdk4)-cyclin D1- and cdk2-cyclin E-directed phosphorylation. Mutation of Ser484 impairs rDNA transcription in vivo and in vitro. The data demonstrate that UBF is regulated in a cell cycle-dependent manner and suggest a link between G1 cdks-cyclins, UBF phosphorylation and rDNA transcription activation (Voit, 1999).
Androgens play an important role in the growth of prostate cancer, but the molecular mechanism that underlies development of resistance to antiandrogen therapy remains unknown. Cyclin E has now been shown to increase the transactivation activity of the human androgen receptor (AR) in the presence of its ligand dihydrotestosterone. The AR exhibits two transactivation functions that are mediated by ligand-independent (AF-1) and ligand-dependent (AF-2) activation domains located in the NH2-terminal AB region and in the COOH-terminal ligand-binding EF region, respectively. The enhancement of AR activity by cyclin E is resistant to inhibition by the antiandrogen 5-hydroxyflutamide. Cyclin E has been shown to bind directly to the COOH terminus portion of the AB domain of the AR, and to enhance its AF-1 transactivation function. These results suggest that cyclin E functions as a coactivator of the AR, and that aberrant expression of cyclin E in tumors may contribute to persistent activation of AR function, even during androgen ablation therapy (Yamamoto, 2000).
Centrosomes nucleate microtubules and duplicate once per cell cycle. This duplication and subsequent segregation in mitosis results in maintenance of the one centrosome/cell ratio. Centrosome duplication occurs during the G1/S transition in somatic cells and must be coupled to the events of the nuclear cell cycle; failure to coordinate duplication and mitosis results in abnormal numbers of centrosomes and aberrant mitoses. Using both in vivo and in vitro assays, it has been shown that centrosome duplication in Xenopus laevis embryos requires cyclin/cdk2 kinase activity. Injection of the cdk (cyclin-dependent kinase) inhibitor p21 into one blastomere of a dividing embryo blocks centrosome duplication in that blastomere; the related cdk inhibitor p27 has a similar effect. An in vitro system using Xenopus extracts carries out separation of the paired centrioles within the centrosome. This centriole separation activity is dependent on cyclin/cdk2 activity; depletion of either cdk2 or of the two activating cyclins, cyclin A and cyclin E, eliminates centriole separation activity. In addition, centriole separation is inhibited by the mitotic state, suggesting a mechanism of linking the cell cycle to periodic duplication of the centrosome (Lacey, 1999).
Centrosome duplication is indispensable for the formation of the bipolar mitotic spindle. Surprisingly, even if DNA replication or mitosis is inhibited, centrosome duplication can still occur. Thus, it remains unknown how centrosome duplication is coordinated with the cell cycle. Centrosome duplication is shown to require cyclin-dependent kinase 2 (Cdk2) in mammalian cells. In Chinese hamster ovary (CHO) cells, whereas centrosome duplication is not inhibited by hydroxyurea (HU) treatment, which arrests the cells in S phase, it is inhibited by mimosine treatment, which arrests the cells in late G1 phase. Cdk2 activity is higher in HU-treated cells than in mimosine-treated cells. Remarkably, inhibition of the Cdk2 activity in HU-treated cells with butyrolactone I or roscovitine, or by expression of the Cdk inhibitor p21(Waf1/Cip1), blocks the continued centrosome duplication. Moreover, overexpression of Cdk2 reversed the inhibition of centrosome duplication by mimosine treatment. These results indicate a requirement of Cdk2 activity for centrosome duplication and therefore suggest an underlying mechanism for the coordination of centrosome duplication with the cell cycle. Because Cdk2 is activated as a Cdk2-cyclin E complex at the G1-S boundary and as a Cdk2-cyclin A complex during S phase progression, it is unclear from this work whether the cyclin E and/or the cyclin A partner of Cdk2 is involved in regulating centrosome duplication (Matsumoto, 1999).
Centrosome hyperamplification and the consequential mitotic defects contribute to chromosome instability in cancers. Loss or mutational inactivation of p53 has been shown to induce chromosome instability through centrosome hyperamplification. It has recently been found that Cdk2-cyclin E is involved in the initiation of centrosome duplication, and that constitutive activation of Cdk2-cyclin E results in the uncoupling of the centrosome duplication cycle and the DNA replication cycle. Cyclin E overexpression and p53 mutations occur frequently in tumors. Cyclin E overexpression and loss of p53 synergistically increase the frequency of centrosome hyperamplification in cultured cells as well as in tumors developed in p53-null, heterozygous, and wildtype mice. Through examination of cells derived from Waf1-null mice, it has been further found that Waf1, a potent inhibitor of Cdk2-cyclin E and a major target of p53's transactivation function, is involved in coordinating the initiation of centrosome duplication and DNA replication, suggesting that Waf1 may act as a molecular link between p53 and Cdk2-cyclin E in the control of the centrosome duplication cycle (Mussman, 2000).
Excess cyclin E-Cdk2 accelerates entry into S phase of the cell cycle and promotes polyploidy, which may contribute to genomic instability in cancer cells. 20 amino acids in cyclin E have been identified as a centrosomal localization signal (CLS) essential for both centrosomal targeting and promoting DNA synthesis. Expressed wild-type, but not mutant, CLS peptides localizes on the centrosome, preventes endogenous cyclin E and cyclin A from localizing to the centrosome, and inhibits DNA synthesis. Ectopic cyclin E localizes to the centrosome and accelerates S phase entry even with mutations that abolish Cdk2 binding, but not with a mutation in the CLS. These results suggest that cyclin E has a modular centrosomal-targeting domain essential for promoting S phase entry in a Cdk2-independent manner (Matsumoto, 2004).
Strong loss-of-function mutations have been identified in the C. elegans cyclin E gene, cye-1. Mutations in cye-1 lead to the underproliferation of many postembryonic blast lineages as well as defects in fertility and gut-cell endoreduplication. In addition, cye-1 is required maternally, but not zygotically for embryonic development. An analysis of vulval development in cye-1 mutants suggests that a timing mechanism may control the onset of vulval cell terminal differentiation: once induced, these cells appear to differentiate after a set amount of time, rather than a specific number of division cycles. cye-1 mutants also show an increase in the percentage of vulval precursor cells (VPCs) that adopt vulval cell fates, indicating that cell-cycle length can play a role in the proper patterning of vulval cells. By analyzing cul-1 mutants, coding for a protein degradation pathway component known to generate an opposite, hyperproliferation phenotype, it is further demonstrated that vulval cell terminal differentiation can be uncoupled from associated changes in vulval cell division planes (Fay, 2000).
Wild-type vulvae undergo terminal differentiation during the third round of VPC divisions. This differentiation process can be described as having three sequential components: (1) the execution of characteristic cell division planes; (2) the cessation of further cell divisions, and (3) the acquisition of 'mature' vulval-cell traits including cell shape changes, cell movements and the expression of terminal differentiation markers. Interestingly, terminal differentiation of vulval cells in cye-1 mutants initiates during the second division cycle. While this event is premature with respect to the normal number of cell divisions, the slower cell-cycle rate observed for vulval cells in cye-1 mutants means that terminal differentiation is initiated with roughly the same timing as in wild type, approximately 4 hours after the induction of VPCs. Vulval lineage defects reminiscent of cye-1 mutants have been observed in animals treated with hydroxyurea (HU), indicating that disruption of the cell cycle by an alternate means can lead to a similar phenotype (Fay, 2000).
It is proposed that a timing mechanism, independent of the cell cycle, controls the onset of vulval cell terminal differentiation. Following the initial induction event, vulval cells are instructed to divide with characteristic terminal division planes after a specified amount of elapsed time. This differentiation 'stopwatch' could function intrinsically within vulval cells, possibly through the steady accumulation of a 'differentiation-promoting-factor'. Alternatively, differentiation could be triggered by the controlled degradation (or dilution) of a 'differentiation-inhibitor'. A variation on this model places the actual timing mechanism in surrounding tissues, which would then emit a 'differentiation signal'. In support of an intrinsic mechanism, cell ablation experiments indicate that following induction, vulval cell divisions are unaffected by the absence of the somatic gonad including the anchor cell (Fay, 2000).
Entry into an alternative larval stage known as the 'dauer' can under certain circumstances reset the identities of dividing vulval cells back to a pluripotent state. Using a specialized genetic background it has been found that dividing vulval cells can revert to a multipotential state ('reversal of fate') following a forced entry into the dauer stage, provided that vulval cells have not already undergone terminal divisions. These previously committed cells can then be re-induced to form vulval tissue at a later stage through the normal inductive pathway (Fay, 2000 and references therein).
This finding leads to an interesting implication for the timing model. Namely, the clock controlling vulval cell differentiation can apparently be set back to zero, provided it is still 'ticking'. However, once terminal differentiation has been initiated, there is no possibility of going back. Importantly, this result also implies that the proposed timer would necessarily be mechanistically linked somehow to factors controlling life cycle stages such as genes identified through screens for heterochronic mutants (Fay, 2000).
An analysis of vulval development in cul-1 mutants reveals that the execution of terminal division planes can be uncoupled from both cell-cycle withdrawal and the expression of terminal differentiation markers. It is therefore proposed that further divisions are normally prevented by the action of CUL-1 protein, presumably by degrading cyclins that promote transit through G1. A failure to withdraw from the cell cycle in turn prevents normal differentiation processes. The finding that cul-1 can variably suppress cye-1 vulval and (to a lesser extent) germline defects, can be explained in several ways. Suppression could result in part from an increase in the stability of cyclin E protein (transcribed from maternal cye-1 mRNA), or possibly other G1-phase cyclins, leading to faster cell-cycle transit times and an increased number of divisions. Consistent with this hypothesis, cullins are required for the degradation of G1 cyclin family members. It is also possible that cell-cycle rates are largely unaltered in the double mutants but, by preventing vulval cells from exiting the cell cycle, a greater number of divisions can occur. The poor health of the marked cye-1;cul-1 double mutant strain precludes direct measurement of cell-cycle times in these animals. However, given that cell-cycle division rates are roughly normal in cul-1 single mutants and that life-cycle stages are extended, it is likely that this second mechanism plays a significant role in the observed suppression. The finding that the cye-1 underproliferation defect is often epistatic to cul-1 indicates that, even in the absence of cul-1, cyclin E levels are generally limiting. Given that vulval cells in cul-1 mutants eventually stop dividing and appear to differentiate, it is likely that additional factors may control cell-cycle exit. Obvious candidates include members of the p21Cip/27Kip family of CDK inhibitory proteins (CKIs). Consistent with a role for CKI proteins in vulval differentiation, the C. elegans p27 homolog, cki-1, is expressed in vulval cells shortly after terminal divisions take place. However, no uncoupling of vulval cell terminal division events could be detected using cki-1(RNAi). This suggests that cki-1 may act redundantly with other cell-cycle inhibitors, or could simply reflect a limitation of the RNAi technique in reducing gene activity in the vulva (Fay, 2000 and references therein).
The expression and function of C. elegans cye-1 gene encoding the G1 cell cycle regulator cyclin E has been studied by using monoclonal antibodies directed against CYE-1 protein, cye-1::GFP reporter genes, and a cye-1 chromosomal deletion mutation. A ubiquitous embryonic pattern of expression becomes restricted and dynamic during postembryonic development. Promoter analysis reveals a relatively small region of cis-acting sequences that are necessary for the complex pattern of expression of this gene. Two other G1 cell cycle genes, encoding cyclin D and CDK4/6, have similarly compact promoter requirements. This suggests that a relatively simple mechanism of regulation may underlie the dynamic developmental patterns of expression exhibited by these three G1 cell cycle genes. Analysis of a new cye-1 deletion allele confirms and extends previous studies of two point mutations in the gene (Brodigan, 2003).
Analysis of cye-1 promoter elements necessary for expression has identified a conserved sequence element (element -2) containing two consensus E2F binding sites. E2F is a heterodimeric transcription factor (comprised of E2F and DP) that regulates the expression of cyclin E and many genes required for DNA synthesis. The disassociation of E2F from Rb is thought to be a major regulatory step in promoting the G1/S transition. There are two E2F-like genes (efl-1 and efl-2) and one DP-like gene (dpl-1) in C. elegans. Mutations in efl-1 and dpl-1 affect Ras signaling in embryogenesis and vulval development. Both efl-1 and dpl-1 have roles in regulating G1 cell cycle progression when assayed in a cyclin D mutant background. Given the conservation of factors regulating G1/S progression, it seems likely that E2F regulation of cyclin E gene expression will also be conserved in C. elegans. Further studies will be required to directly test this notion (Brodigan, 2003).
The GC-rich element -2 also contains a conserved, consensus Sp1 binding site and two StuA binding sites. Sp1 has been implicated in both the activation and repression of several cell cycle genes in tissue culture studies and is thought to have an important role in the G1 phase of the cell cycle. Sp1 is a good candidate for another factor involved in cye-1 regulation and warrants further study. StuA is a transcription factor identified in Aspergillus with multiple roles during development, including the regulation of G1/S target genes. StuA is related to SWI4 and MBP1 of Saccharomyces cerevisiae, components of a G1/S transcriptional regulator providing intriguing links to cell cycle control. A gene (C31H1.1) encoding a protein with limited similarity to MBP1 has been identified in C. elegans, although its function is unknown, and further study will be required to determine whether it has a role in G1/S cell cycle progression in the nematode (Brodigan, 2003).
In mammalian cells, cyclin E-CDK2 complexes are activated in the late G1 phase of the cell cycle and are believed to have an essential role in promoting S-phase entry. The murine genes CCNE1 and CCNE2, encoding cyclins E1 and E2, were subjected to targeted mutagenesis. Whereas single knockout mice are viable, double knockout embryos die around midgestation. Strikingly, however, these embryos show no overt defects in cell proliferation. Instead, developmental phenotypes are observed consistent with placental dysfunction. Mutant placentas had an overall normal structure, but the nuclei of trophoblast giant cells, which normally undergo endoreplication and reach elevated ploidies, showed a marked reduction in DNA content. Trophoblast stem cells were derived from double knockout E3.5 blastocysts. These cells retain the ability to differentiate into giant cells in vitro, but are unable to undergo multiple rounds of DNA synthesis, demonstrating that the lack of endoreplication is a cell-autonomous defect. Thus, during embryonic development, the needs for E-type cyclins can be overcome in mitotic cycles but not in endoreplicating cells (Parisi, 2003).
Stimulatory and inhibitory signals regulate cell proliferation through the activity of specific enzymes that operate in distinct phases of the cell cycle. Cell cycle progression, arrest, and withdrawal has been studied in the oligodendrocyte progenitor (OP) cell model system, focusing on the G1 phase and G1-S transition. Not only were proliferating OPs found to display higher protein levels of cyclin E and D and cyclin-dependent kinases (cdk) 2, 4, and 6 than cells that had permanently withdrawn from the cycle, but the kinase activities of both cyclin D-cdk4/6 and cyclin E-cdk2 are also higher in dividing OPs. This is associated with a decrease in the formation of the cyclin E-cdk2 and cyclin D-cdk4/cyclin D-cdk6 complexes in differentiated oligodendrocytes that have permanently withdrawn from the cell cycle. However, reversible cell cycle arrest in G1 induced by glutamatergic and beta-adrenergic receptor activation or cell depolarization does not modify cyclin E and cdk2 protein expression compared with proliferating OPs. Instead, these agents cause a selective decrease in cdk2 activity and an impairment of cyclin E-cdk2 complex formation. Although cyclin D protein levels are higher than in proliferating cells, cyclin D-associated kinase activity is not modified in G1-arrested OPs. Analysis in corpus callosum in vivo has shown that cyclin E-cdk2 activity increases between postnatal days 3 and 15 and decreases between postnatal days 15 and 30. These results indicate that the cyclin E-cdk2 complex is a major regulator of OP cell cycle progression and that the cdks involved in reversible cell cycle arrest are distinct from those implicated in permanent cell cycle withdrawal (Ghiani, 2001).
Cyclin E, in conjunction with its catalytic partner cdk2, is rate limiting for entry into the S phase of the cell cycle. Cancer cells frequently contain mutations within the cyclin D-Retinoblastoma protein pathway that lead to inappropriate cyclin E-cdk2 activation. Although deregulated cyclin E-cdk2 activity is believed to directly contribute to the neoplastic progression of these cancers, the mechanism of cyclin E-induced neoplasia is unknown. The consequences of deregulated cyclin E expression have been studied in primary cells; cyclin E was found to initiate a p53-dependent response that prevents excess cdk2 activity by inducing expression of the p21Cip1 cdk inhibitor. The increased p53 activity is not associated with increased expression of the p14ARF tumor suppressor. Instead, cyclin E leads to increased p53 serine15 phosphorylation that is sensitive to inhibitors of the ATM/ATR family. When either p53 or p21cip1 is rendered nonfunctional, then the excess cyclin E becomes catalytically active and causes defects in S phase progression, increased ploidy, and genetic instability. It is concluded that p53 and p21 form an inducible barrier that protects cells against the deleterious consequences of cyclin E-cdk2 deregulation. A response that restrains cyclin E deregulation is likely to be a general protective mechanism against neoplastic transformation. Loss of this response may thus be required before deregulated cyclin E can become fully oncogenic in cancer cells. Furthermore, the combination of excess cyclin E and p53 loss may be particularly genotoxic, because cells cannot appropriately respond to the cell cycle anomalies caused by excess cyclin E-cdk2 activity (Minella, 2002).
How might deregulated cyclin E cause S phase abnormalities that activate an S phase checkpoint? In yeast, S phase-promoting cyclins inhibit the transition of replication origins to the prereplicative state. Furthermore, when early-firing origins are inhibited by hydroxyurea, then the stalled early origins inhibit late origins through a checkpoint that requires the Mec1 protein (the budding yeast ATM/ATR homolog. Similarly, inhibition of ATR function in a human cell line by a kinase-inactive ATR mutant renders these cells hypersensitive to treatments that prolong DNA synthesis, and cyclin E overexpression is synthetically lethal with ATR inhibition. Thus, perhaps cyclin E deregulation leads to aberrant licensing of replication origins, and the resultant S phase progression defect may be sensed by a protein such as ATR, which then enforces an S phase checkpoint. Furthermore, the stalled replication origins associated with this prolonged S phase may be fragile and constitute the precursors to genetic instability. Another mechanism through which enforced cyclin E expression might impair normal cell cycle progression is by cyclin A-cdk2 inhibition, since cyclin A-cdk2 activity (and cyclin A expression) drops substantially in cells with ectopic cyclin E expression. However, cyclin E-induced cell cycle anomalies persist in E6-expressing cells with high levels of cyclin A-cdk2 kinase activity, so cyclin A-cdk2 activity cannot be the principle cause of the cyclin E-associated S phase phenotype (Minella, 2002).
Cellular origins and genetic factors governing the genesis and maintenance of glioblastomas (GBM) are not well understood. This study reports a pathogenetic role of the developmental regulator Id4 (inhibitor of differentiation 4) in GBM. In primary murine Ink4a/Arf(-/-) astrocytes, and human glioma cells, evidence is provided that enforced Id4 can drive malignant transformation by stimulating increased cyclin E to produce a hyperproliferative profile and by increased Jagged1 expression with Notch1 activation to drive astrocytes into a neural stem-like cell state. Thus, Id4 plays an integral role in the transformation of astrocytes via its combined actions on two-key cell cycle and differentiation regulatory molecules (Jeon, 2008).
The impact was examined of activated Notch signaling on the immature differentiation profiles and neurosphere-forming capacity of Id4-transduced Ink4a/Arf-/- astrocytes. Notch1, but not cyclin E, knockdown resulted in a marked decrease in the expression of NSC markers, Nestin, Cd133, and Hes1, and correspondingly, NIC overexpression in Ink4a/Arf-/- astrocytes induced expression of these immature markers. In the neurosphere assay, Notch1 knockdown resulted in a significant decrease in neurosphere number from the Id4-transduced Ink4a/Arf-/- astrocytes compared with a modest decrease in the cyclin E knockdown cultures. Furthermore, NIC overexpression, but not cyclin E, was comparable with Id4 in promoting neurosphere formation following their transduction into Ink4a/Arf-/- astrocytes. Pharmacological inhibition of Notch signaling (DAPT, a γ-secretase inhibitor) or Jagged1 knockdown in Id4-transduced Ink4a/Arf-/- astrocytes resulted in decreased neurosphere-forming capacity and NSC marker expression. Thus, Id4-induced activation of Jagged-Notch axis in Ink4a/Arf-/- astrocytes plays an essential role in promoting the neural stem cell-like phenotype (Jeon, 2008).
Next, attempts were made to corroborate the murine findings in human glioma cells. shRNA-mediated depletion of Id4 in human LN229 glioma cells (which express the highest levels of Id4) resulted in down-regulation of cyclin E, Jagged1, NIC, Notch-downstream target genes (Hes1, Hey1, and Hey2), and Notch transcriptional activity, as well as a marked decrease in cell proliferation. Conversely, Id4 overexpression in human A172 glioma cells (which express low endogenous levels of Id4) induced up-regulation of Jagged1, NIC, and cyclin E; Notch transcriptional activity; cell proliferation; and neurosphere formation. It was also found that expression levels of Id4, NIC, Jagged1, and cyclin E were markedly increased in the primary human glioma stem cell line, NCI0822, as compared with NHA and HB1.F3 cells (Jeon, 2008).
Furthermore, a Tet-On-inducible gene expression system was used to assess whether Id4 directly leads to induction of cyclin E and Notch signaling. Id4 was markedly increased in the Ink4a/Arf-/- astrocytes transduced with rtTA and Rev-TRE-Id4 grown in the presence of doxycycline (Dox) for 2 d compared with Dox-untreated counterpart cells. It was also found that Dox-treated cells showed marked elevations in the levels of cyclin E, Jagged1, and NIC. These results strengthen the link between Id4 and the control of cyclin E and Notch signaling in the astrocytes (Jeon, 2008).
In conclusion, Id4 can drive the malignant transformation of astrocytes via disregulation of cell cycle and differentiation control, achieved through the up-regulation of cyclin E and activation of Jagged-Notch1 signaling. These findings of Id4-induced developmental plasticity have implications for both the cellular origins of GBM as well as its prominent renewal potential in experimental and clinical trials setting. Thus, these observations may inform the rational development of anti-Id4 agents that may impede the insuperable nature of GBM recurrence (Jeon, 2008).
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