Cyclin E
Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell
cycle progression in metazoans and is frequently overexpressed in cancer cells.
It is essential for entry to the cell cycle from G0 quiescent phase, for the
assembly of prereplication complexes and for endoreduplication in megakaryotes
and giant trophoblast cells. The crystal structure of pCDK2 in complex
with a truncated cyclin E1 (residues 81-363) is reported at 2.25 Å resolution. The
N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and
promotes identical changes in pCDK2 that lead to kinase activation. The
C-terminal cyclin box fold shows significant differences from cyclin A. It makes
additional interactions with pCDK2, especially in the region of the activation
segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic
analysis with model peptide substrates show a 1.6-fold increase in kcat for
pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and
pCDK2/cyclin A. The structural and kinetic results indicate no inherent
substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model
substrates (Honda, 2005 ).
Two classes of cyclins are expressed in mammalian cells during the G1 phase of the cell cycle: D-type cyclins (cyclins D1, D2,
and D3) and cyclin E. Although these proteins are collectively referred to as G1 cyclins, there are profound differences between the two G1
cyclin classes. While the three D-type cyclins share very similar amino acid sequences (over 70% identity), they display only weak sequence relatedness with cyclin E
(38% within the most conserved 'cyclin box' domain). In fact, cyclin E shares more similarity with the S
phase- and M phase-specific cyclins A, B1, and B2 than with the D-type cyclins. The expression of these two classes of G1 cyclins occurs at different points of cell cycle progression and is governed by distinct mechanisms. Thus, the expression of
D-type cyclins is controlled largely and perhaps entirely by extracellular signals. Cyclin D1 in particular is rapidly induced following mitogen challenge; its levels
rapidly decline when mitogens are withdrawn or when antimitogens are added. For this reason, D-cyclins are regarded as functional links between the extracellular
environment and the cell cycle machinery. In contrast to the regulation of the D-type cyclins, the expression of cyclin E is controlled by an autonomous mechanism and peaks suddenly at the G1/S boundary.
Once induced, D-cyclins bind and activate CDK4 and CDK6, while cyclin E associates with an entirely different catalytic partner, CDK2. Moreover, the cyclin D-
and cyclin E-associated CDK complexes are activated at different times during cell cycle progression, cyclin D-CDK4/6 complexes being active during the mid-G1
phase, whereas active cyclin E-CDK2 complexes are found at the G1/S boundary. The activity of cyclin D-CDK4/6 complexes is negatively
controlled by association with inhibitory molecules belonging to the INK4 family of CDK inhibitors. In contrast, cyclin E-CDK2 complexes appear to be immune to
inhibition by members of this family of proteins. A mouse strain has been generated in which the coding sequences of the
cyclin D1 gene (Ccnd1) have been deleted and replaced by those of human cyclin E (CCNE). In the tissues and cells of these mice, the expression pattern of human
cyclin E faithfully reproduces that normally associated with mouse cyclin D1. The replacement of cyclin D1 with cyclin E rescues all phenotypic manifestations of
cyclin D1 deficiency and restores normal development in cyclin D1-dependent tissues. Thus, cyclin E can functionally replace cyclin D1. These analyses suggest that
cyclin E is the major downstream target of cyclin D1 (Geng, 1999).
What then is the molecular basis of the observed rescue of cyclin D1 functions by cyclin E? Two possibilities are considered. (1) Knockin cyclin E-CDK2
complexes replace the functions normally executed by cyclin D1-CDK4/6 complexes. (2) The cyclin ED1 allele results in ectopic expression of the major
downstream target of cyclin D1, thereby obviating the need for cyclin D1 in cell cycle progression, revealing that cyclin E is the major rate-limiting target for cyclin D1. The evidence presented in this paper strongly favors the latter scenario. Thus, cyclin D1 is believed to play at least two functions in cell cycle progression: (1) it
drives the phosphorylation of the pRB; (indeed, cells that have lost pRB no longer require cyclin D1 for their growth, indicating that pRB is the major downstream
target of cyclin D1) and (2) cyclin D1 induction causes the redistribution of a cellular kinase inhibitor, p27Kip1, from the cyclin
E-CDK2 pool to the cyclin D-CDK4/6 pool, thereby liberating cyclin E-CDK2 complexes from inhibition and resulting in their activation. Both functions of cyclin D1 were examined in the tissues of cyclin ED1 mice. It was found that neither of these cyclin D1-dependent functions is replaced
by the ectopically expressed human cyclin E. Consequently, it is concluded that the ectopic cyclin ED1 allele rescues the proliferation of cyclin
D1-dependent tissues by bypassing the functions of cyclin D1 rather than by replacing them. This indicates, in turn, that cyclin E activity is the major downstream
rate-limiting target for cyclin D1 and that ectopic cyclin E expression, as achieved through the cyclin ED1 allele, bypasses the need for cyclin D1 in cell
cycle progression (Geng, 1999 and references).
Currently available information on the mammalian cell cycle enables one to postulate how this functional replacement might occur. pRB is the major target for cyclin
D-CDK4/6 complexes. When hypophosphorylated, pRB binds and sequesters several proteins, most notably transcription
factors of the E2F family. The phosphorylation of pRB leads to the release of these captive proteins, which then proceed to transactivate target genes. Alternatively,
the dissociation of pRB from E2Fs removes a transcription-repressing factor from E2Fs bound to control elements of certain promoters.
One of the genes regulated by the E2Fs is cyclin E, leading to the hypothesis that
cyclin D1 serves to control the activity of cyclin E via pRB and E2Fs. An elaboration of this model predicts that cyclin D1-CDK4/6 complexes bring about the initial,
preparatory pRB phosphorylation, leading to the induction of small amounts of cyclin E. The end consequence of these
steps is the activation of cyclin E, whose ectopic expression, as described here, appears to obviate the function of cyclin D1 (Geng, 1999 and references).
Apart from its role as a partner of CDK4 and CDK6 kinases, cyclin D1 has been reported to play a unique role as an Estrogen receptor (ER) coactivator. This might suggest that the observed defect in the mammary development of cyclin D1-/- mice derives from inadequate activation of the
ER in this tissue. The evidence presented in this paper argues against this explanation: (1) this paper shows that cyclin E, which was reported not to bind the ER and
not to serve as ER coactivator, can replace cyclin D1 in driving normal mammary epithelial development, and (2) this paper shows that
the induction of a major mammary epithelial ER-responsive gene, the PR, proceeds normally in cyclin D1-deficient mice. These
findings have led to the conclusion that the crucial role for cyclin D1 in mammary development reflects the activity of cyclin D1 toward other targets, such as pRB (Geng, 1999 and references).
The studies described in this paper strongly suggest that cyclin E is the major rate-limiting target for cyclin D1 in mammalian cells. This, together with the
well-established fact that the D-type cyclins are controlled by extracellular signals, while E cyclins are controlled internally, might suggest that the cyclin D-pRB
pathway has evolved in order to enable metazoan cells to connect extracellular signals with cyclin E activation, thereby controlling the entrance into S phase. Since
this cyclin D-pRB pathway is subverted in essentially all human cancers, the elucidation of its role in driving normal cell proliferation is essential to understanding the
molecular basis of malignant cell growth (Geng, 1999).
Overexpression of human cyclin E shortens G1, causing a premature entry into S. A target for the cyclin E-cdk2 complex is the Retinoblastoma protein (See Drosophila Retinoblastoma-family protein). Coexpression of Rb with cyclin E induces Rb hyperphosphorylation and overrides the ability of Rb to suppress G1 exit. Hypophosphorylated Rb can interact with the transcription factor E2F during G1 and this complex can bind to DNA and repress transcription of E2F target genes. Conversely, hyperphosphorylation of Rb prevents its interaction with E2F, releasing it from an inhibitory constraint and enabling it to promote gene expression (Sherr, 1993 and references).
The retinoblastoma protein (pRb) acts to constrain the G1-S transition in mammalian cells.
Phosphorylation of pRb in G1 inactivates its growth-inhibitory function, allowing for cell cycle
progression. Phosphorylation of S780 results in a lose of Rb's ability to bind to E2F. Phosphorylation of S807 and/or S811 is required to abolish Rb binding to c-Abl, while modification of threonine 821 and or T826 is required to abolish Rb binding to LXCXE-containing proteins such as simian virus 40 large T antigen. Although several cyclins and associated cyclin-dependent kinases (cdks) have been
implicated in pRb phosphorylation, the precise mechanism by which pRb is phosphorylated in vivo
remains unclear. By selectively inhibiting either cdk4/6 or cdk2, it has been shown that endogenous D-type cyclins, acting with cdk4/6, are able to phosphorylate pRb only partially, a process that is likely to be
completed by cyclin E-cdk2 complexes. Cyclin E-cdk2 is unable to phosphorylate pRb in
the absence of prior phosphorylation by cyclin D-cdk4/6 complexes. Complete phosphorylation of pRb,
inactivation of E2F binding, and activation of E2F transcription occur only after the sequential action of at
least two distinct G1 cyclin kinase complexes (Lundberg, 1998).
Cyclin E is necessary and rate limiting for the passage of mammalian cells through the G1 phase of the
cell cycle. Control of cell cycle progression by cyclin E involves cdk2 kinase, which requires cyclin E
for catalytic activity. Expression of cyclin E/cdk2 leads to an activation of cyclin A gene expression, as
monitored by reporter gene constructs derived from the human cyclin A promoter. Promoter activation
by cyclin E/cdk2 requires an E2F binding site in the cyclin A promoter. Cyclin
E/cdk2 kinase can directly bind to E2F/p107 complexes formed on the cyclin A promoter-derived E2F
binding site, and this association is controlled by p27KIP1, most likely through direct protein-protein
interaction. These observations suggest that cyclin E/cdk2 associates with E2F/p107 complexes in late
G1 phase, once p27KIP1 has decreased below a critical threshold level. Since a kinase-negative mutant
of cdk2 prevents promoter activation, it appears that transcriptional activation of the cyclin A gene
requires an active cdk2 kinase tethered to its promoter region (Zerfass-Thome, 1997).
The Retinoblastoma-related protein p107, like the p21 family of cdk inhibitors, can inhibit the phosphorylation of target substrates by cyclin A/cdk2 and cyclin E/cdk2 complexes. The associations of p107 and p21 with cyclin/cdk2 rely on a structurally and functionally related interaction domain. Interactions between p107 and p21 are mutually exclusive: p21 causes a dissociation of p107/cyclin/cdk2 complexes to yield p21/cyclin/cdk2 complexes. The activation of the p107-bound cyclin/cdk kinases leads to dissociation of p107 from the transcription factor E2F. It has been suggested that p107 functions similarly to Rb, causing growth arrest of sensitive cells in the G1 phase of the cell cycle. The p107 molecule can be dissected into two domains, either of which is able to independently block cell cycle progression. One domain corresponds to the sequences needed for interaction with transcription E2F, and the other corresponds to the interaction domain for cyclin A or cyclin E complexes (Zhu, 1995 a and b).
The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Growth suppression by two of these proteins, p107 and p130, could result from the inhibition
of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism by which
inhibition occurs. In vivo and in vitro evidence suggests that p107 is a bona fide inhibitor of both cyclin
A-cdk2 and cyclin E-cdk2. p107 exhibits an inhibitory constant (Ki) comparable to that of the cdk inhibitor p21/WAF1. In
contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the
amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes,
although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the
previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either
kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and
growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to
cyclin-cdk2 complexes and kinase inhibition. The ability to distinguish between p107 and p130 as inhibitors rather than
simple substrates suggests that these proteins may represent true inhibitors of cdks (Castano, 1998).
Eukaryotic DNA replication requires the previous formation of a prereplication complex containing the ATPase Cdc6 and the minichromosome maintenance (Mcm) complex. Although considerable insight has been gained from in vitro studies and yeast genetics, the functional analysis of replication proteins in intact mammalian cells has been lacking. Adenoviral vectors have been used to express normal and mutant forms of Cdc6 in quiescent mammalian cells to assess function. Cdc6 expression alone is sufficient to induce a stable association of endogenous Mcm proteins with chromatin in serum-deprived cells where cyclin-dependent kinase (cdk) activity is low. Moreover, endogenous Cdc6 is sufficient to load Mcm proteins onto chromatin in the absence of cdk activity in p21-arrested cells. Cdc6 synergizes with physiological levels of cyclin E/Cdk2 to induce semiconservative DNA replication in quiescent cells whereas cyclin A/Cdk2 is unable to collaborate with Cdc6. Cdc6 that cannot be phosphorylated by cdks is fully capable of inducing Mcm chromatin association and replication. Mutation of the Cdc6 ATP-binding site severely impairs the ability of Cdc6 to induce Mcm chromatin loading and reduces its ability to induce replication. Nevertheless, the ATPase domain of Cdc6 in the absence of the noncatalytic amino terminus is not sufficient for either Mcm chromatin loading or DNA replication, indicating a requirement for this domain of Cdc6 (Cook, 2002).
The best-characterized substrates of cyclin E/Cdk2 are the
retinoblastoma family proteins, Rb, p130, and p107. Phosphorylation of
Rb by cyclin D/Cdk4 and cyclin E/Cdk2 dissociates Rb from E2F and
allows the induction of E2F target genes. The synergy between
low-level cyclin E/Cdk2 expression and Cdc6 is only
seen when cyclin E/Cdk2 activity is low enough to induce
endogenous cdc6 expression minimally. Thus one function of cyclin E/Cdk2 in the induction of S phase is its well-documented role in transcriptional
control of E2F target genes such as cdc6. The role of cyclin E/Cdk2 in Mcm chromatin loading is restricted to its function in E2F-dependent transcriptional control of the cdc6 gene because expression
of Cdc6 in the absence of cdk activity (either by ectopic expression or
by induction of the endogenous gene by E2F) bypasses the need for cdk activity in Mcm chromatin loading. Cyclin E/Cdk2 activity is not required for prereplication complex formation as long as Cdc6 is produced (Cook, 2002).
Clearly, cyclin E/Cdk2 plays additional roles in replication
initiation downstream of Mcm chromatin loading because Cdc6-mediated Mcm chromatin loading is not sufficient for replication without cyclin
E/Cdk2, and Cdk2 activity is still required for initiation in
X. laevis extracts in which transcriptional control is not important. At least one of those functions is likely to be the loading of the Cdc45 protein onto the newly formed prereplication complex, although the precise mechanism of this aspect of cyclin E/Cdk2 function remains to be elucidated (Cook, 2002).
Continued: Cyclin E: Evolutionary Homologs part 2/3
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