COMMENTARY
1811
Cyclin specificity: how many wheels do you need on a
unicycle?
Mary E. Miller and Frederick R. Cross*
The Rockefeller University, 1230 York Ave., New York, NY 10021, USA
*Author for correspondence (e-mail: fcross@rockvax.rockefeller.edu)
Journal of Cell Science 114, 1811-1820 © The Company of Biologists Ltd
Summary
Cyclin-dependent kinase (CDK) activity is essential for
eukaryotic cell cycle events. Multiple cyclins activate CDKs
in all eukaryotes, but it is unclear whether multiple cyclins
are really required for cell cycle progression. It has been
argued that cyclins may predominantly act as simple
enzymatic activators of CDKs; in opposition to this idea, it
has been argued that cyclins might target the activated
CDK to particular substrates or inhibitors. Such targeting
might occur through a combination of factors, including
temporal expression, protein associations, and subcellular
localization.
Introduction
The major events of the eukaryotic cell cycle depend on the
sequential function of cyclin-dependent kinases (CDKs;
Levine and Cross, 1995). CDK catalytic activity is dependent
upon physical association with cyclin regulatory subunits (De
Bondt et al., 1993; Jeffrey et al., 1995). In animals, multiple
CDKs exist and are activated by multiple cyclins. The many
different complexes make analysis in animal cells difficult. In
budding yeast, one CDK, Cdc28p, is present and activated by
different cyclins at different cell cycle positions (Fig. 1; Cross,
1995; Nasmyth, 1996). A single CDK also functions in cell
cycle control in fission yeast (Stern and Nurse, 1996).
Although the general principles of cell cycle control in yeast
and higher eukaryotes are similar, the single CDK makes
analysis simpler in yeast, and for this reason we focus primarily
on the yeast systems. We refer to results from animal systems
when clear comparisons can be made.
Different cyclin-CDK complexes are required for distinct
cell cycle events. In budding yeast, at cell cycle initiation, the
CDK Cdc28p is activated by the Cln cyclins; at initiation of
DNA replication Cdc28p is activated by the B-type Clb5p and
Clb6p cyclins, and at mitosis Cdc28p is activated by the B-type
Clb1p, Clb2p, Clb3p and Clb4p cyclins. Note that these
statements on the biological roles of different cyclins are based
mainly on knockout phenotypes. Since the different cyclins are
under distinct transcriptional and proteolytic controls, the
knockout phenotype does not distinguish between the absence
of any (generic) cyclin at a given time and the absence of a
specific cyclin. The question of whether the different cyclin
proteins are intrinsically specialized (apart from distinct
regulation of protein abundance) is one of the main subjects of
this Commentary.
regulated cyclin expression. The mitotic B-type cyclins are
degraded during or at the end of mitosis by the proteosome,
after ubiquitination by the highly conserved anaphasepromoting complex (APC; Irniger et al., 1995; King et al.,
1996; Zachariae et al., 1996). In budding yeast this control is
somewhat redundant with accumulation of the Sic1p inhibitor
of Clb-Cdc28p kinase activity (Schwab et al., 1997; Schwob
et al., 1994). It has been proposed that the resulting oscillation
of Clb-Cdc28p kinase activity is required for alternation of
DNA replication and mitosis (see below).
The budding yeast G1 cyclins (Cln proteins) are highly
unstable owing to the action of non-APC ubiquitinating
Skp1p–Cdc53p–F-box protein complex (SCF; Bai et al., 1996;
Willems et al., 1996). G1 cyclin instability is not thought to be
cell cycle controlled. Instability nevertheless imparts biological
significance on transcriptional control of G1 cyclins, since the
abundance of highly unstable proteins closely reflects recent
transcriptional activity.
Yeast cyclin genes are transcriptionally controlled. The G1
cyclin genes CLN1 and CLN2 and the S-phase-acting CLB5
and CLB6 are activated by Cln3p-Cdc28p activity at cell cycle
Start (see below). CLB3 and CLB4 come on later for unknown
reasons. CLB1 and CLB2 (Clb2p is the major mitotic B-type
cyclin) are activated in a positive feedback loop involving
Mcm1p, Ndd1p, Fkh1p and Fkh2p, as well as Clb2p (Althoefer
et al., 1995; Amon et al., 1993; Koranda et al., 2000; Kumar
et al., 2000; Pic et al., 2000; Zhu et al., 2000). CLN3 expression
is moderately periodic owing to transcriptional activation late
in the cell cycle (McInerny et al., 1997).
Regulation of cyclin abundance
Control of cyclin gene transcription and cyclin proteolysis
governs cell cycle progression in many contexts in all
eukaryotes and gives rise to successive waves of cell-cycle-
Key words: Cyclin, CDK, Cell cycle, Targeting, Phosphorylation
Two central questions about cell cycle control by
CDKs: why does it matter that CDK activity
oscillates and does it matter which cyclin activates a
CDK?
Coupling between CDK activity cycles and periodic DNA
replication
A central aspect of cell cycle control is the alternation of DNA
1812
JOURNAL OF CELL SCIENCE 114 (10)
Cell growth
Mating-factor
pathway
Cln3p
Clb3p, Clb4p
Cln1p, Cln2p
Clb5p, Clb6p Clb1p, Clb2p
Fig. 1. Cyclins and control of the budding yeast cell cycle. All
cyclins shown interact with the Cdc28p CDK. The Cln G1 cyclins
are required for cell cycle Start, which entails bud emergence,
inhibition of the mating factor pathway and activation of Clb-Cdc28p
complexes. Cln3p is primarily a transcriptional activator of CLN1,
CLN2, CLB5 and CLB6 and other genes (Breeden, 1996; Dirick et
al., 1995; Levine et al., 1996; Stuart and Wittenberg, 1995; Tyers et
al., 1993). The CLN1 CLN2 gene pair may act directly to drive bud
emergence and morphogenesis, as well as Clb-Cdc28p activation
(reviewed in Cross, 1995). Cell cycle Start is modulated by cell
growth and by the mating-factor pathway, probably through direct
effects on Cln-Cdc28p function (Cross, 1995; Jeoung et al., 1998;
Nasmyth, 1993; Nasmyth, 1996). Clb-Cdc28p complexes are directly
responsible for activation of DNA replication and mitotic initiation
(Nasmyth, 1996). Shaded circles represent nuclei undergoing DNA
replication.
replication and mitosis (Rao and Johnson, 1970; Stillman,
1996). A model was proposed to account for this alternation
(Nasmyth, 1996). High Clb-Cdc28p kinase activity was
proposed to inhibit loading of proteins required for origin
formation. Once replicated through direct use or passive
replication, an origin was proposed to ‘unload’, preventing
another initiation from this origin until reloading. Thus, cells
locked in a low Clb-Cdc28p activity state will not replicate,
because Clb-Cdc28p activity is required for replication
(Schwob et al., 1994), whereas cells locked in a high ClbCdc28p activity state will not re-replicate, because they cannot
reload replication origins. The model thus proposes an
obligatory oscillation of Clb-Cdc28p kinase activity for
consecutive and alternating rounds of mitosis and replication.
Independently of the requirement for low Clb-Cdc28p
activity for reloading origins, a drop in Clb-Cdc28p activity is
required for completion of mitosis. Cells in which Clb2pCdc28p activity is maintained at a high level by overexpression
of undegradable Clb2p block in anaphase; separated
chromosomes remain on a long anaphase spindle, and cells fail
to complete cytokinesis or nuclear division (Surana et al.,
1993).
As described below, a similar model was proposed based on
work in fission yeast (Stern and Nurse, 1996). In this model, a
moderate level of the B-type cyclin-CDK complex activity is
required to begin S phase, and an increased level is required to
trigger mitosis, thus ensuring that DNA replication occurs
before mitosis. Again, high activity is proposed to inhibit the
loading of proteins on origins, so that, once replicated, they
will not replicate again and B-type cyclin-CDK activity must
drop for cells to exit mitosis (Stern and Nurse, 1996).
A proposal for the minimal cell cycle
It is likely that the cell cycle requires an alternation between a
low B-type cyclin-Cdk kinase activity state and a high kinase
activity state, and this must be coupled to an alternation in the
activity of the APC ubiquitinating machinery (Fig. 2A). Active
Cdk stimulates DNA replication and spindle assembly, and
may also promote activation of the form of the APC that is
active with Cdc20p. Cdc20p is a ‘specificity factor’ that
induces the APC to ubiquitinate Pds1p and some Clb proteins
in budding yeast (Rudner et al., 2000; Rudner and Murray,
2000). The low Cdk activity state is largely self-reinforcing in
budding yeast, because it is characterized by accumulation of
the Sic1p inhibitor of Clb kinase, by high activity of Hct1pdependent Clbp proteolysis and by low CLB transcription.
Hct1p is a Cdc20p relative that is predominantly involved in
Clb2p proteolysis in the low Clb activity state. All of these
conditions are reversed by the Cln kinases, since they are
immune to Sic1p and Hct1p, and their transcription is
independently controlled. Cln-dependent phosphorylation of
Sic1p leads to its degradation, and Cln-dependent
phosphorylation of Hct1p may lead to its inactivation (Amon,
1997; Visintin et al., 1998; Zachariae et al., 1998). The high
Clb activity state is also self-reinforcing, even without Cln
kinase activity, because Sic1p and Hct1p phosphorylation can
be carried out by Clb kinase activity, and Clb2p, at least,
stimulates its own transcription (see Fig. 2B; Amon, 1997;
Amon et al., 1993).
CDK-cyclin activity: quantitative vs qualitative
models for specific functions
There are two extreme viewpoints on the question of functional
specialization of cyclins: the particular cyclin might be
irrelevant to function except as a general activator of CDK
enzymatic activity (Stern and Nurse, 1996); alternatively,
cyclin identity might be essential for interaction of a CDK with
appropriate (cyclin-specific) targets. Intermediate scenarios are
possible, in which some cell cycle events require specific
cyclins for targeting but others simply require a given level of
activated CDK and are independent of the activating cyclin.
The case against the importance of cyclin specificity
On the basis of work in fission yeast, it has been proposed that
there is no requirement for cyclin-specific targeting of CDK
activity to appropriate substrates (Stern and Nurse, 1996).
Although there are multiple cyclins in this organism, deletion
of all but a single cyclin, Cdc13p, yields a viable strain with
coordinated DNA replication and mitosis (Fisher and Nurse,
1996). Although strains lacking other cyclins cig1, cig2 and
puc1 exhibit a significant delay in initiation of replication, this
delay can be eliminated by removal of the Rum1p inhibitor
(Martin-Castellanos et al., 2000).
On the basis of these observations, it was proposed that
different cell cycle events occur as a consequence of the
accumulation of different quantities of active cyclin-CDK
complex. DNA replication was proposed to have a lower
threshold for CDK activity than does mitosis; as with the model
discussed above (Nasmyth, 1996; Stern and Nurse, 1996), it
was proposed that high CDK activity also blocks reloading of
replication origins to block re-replication before mitosis.
According to this ‘quantitative’ model, a periodic rise and fall
Cyclin specificity: how many wheels do you need on a unicycle?
1813
of CDK activity can yield qualitatively different responses at
specificity could be due to extrinsic regulatory controls that
different levels. The observation that (as in budding yeast) large
differ for different cyclins. For example, the APC degrades
numbers of different cyclins have apparently distinct biological
mitotic (Clb) cyclins but not G1 (Cln) cyclins, and the Sic1p
roles could simply be due to the phased activation of different
inhibitor and the Swe1p inhibitory kinase inhibit Clb-Cdc28p
genes, which have been selected during evolution for their
but not Cln-Cdc28p complexes (Booher et al., 1993; Irniger et
ability to achieve the appropriate overall quantity of activated
al., 1995; King et al., 1996; Schwob et al., 1994). In contrast,
CDK.
the inhibitor Far1p, when activated by the mating-factor
In fission yeast, loss of Cdc13p-Cdc2p activity (either
pathway, inhibits the G1 Cln-Cdc28p but not Clb-Cdc28p
through deletion of cdc13 or overexpression of the Cdc13pcomplexes (Gartner et al., 1998; Jeoung et al., 1998; Peter and
Cdc2p inhibitor rum1+) induces multiple rounds of reHerskowitz, 1994). In fission yeast, the Rum1p inhibitor
replication of DNA (Hayles et al., 1994; Sanchez-Diaz et al.,
inhibits the kinase activity of Cdc13p-Cdc2p and Cig2p-Cdc2p
1998). This could fit with the idea of high cyclin-Cdk activity
complexes, whereas Puc1p-Cdc2p and Cig1p-Cdc2p
blocking reuse of origins of replication. The model still appears
complexes are insensitive to inhibition. Additionally, Rum1p
to require some mechanism to allow cyclical inactivation of the
itself is phosphorylated and targeted for degradation by Puc1premaining functional cyclins in these contexts (since otherwise,
Cdc2p and Cig1p-Cdc2p complexes (Benito et al., 1998).
replication origins should remain unloaded after one round of
Thus a biological requirement for a specific cyclin could be
replication). This has not been addressed experimentally to our
due not to differences in output of different cyclin-CDK
knowledge.
complexes but rather to differences in regulatory input to the
According to this view, the apparent functional specificity of
cyclin-CDK system. For example, the specific requirement for
different cyclins (based on deletion analysis)
could simply be due to differences in the
timing of cyclin expression. In this case, the
cyclin-CDK complex would be available only
Origin use
at certain times in the cell division cycle and Origin
Spindle
Anaphase
Telophase
(DNA
therefore have access to only a subset of loading
assembly
substrates. Certainly, a correlation between
replication)
expression patterns and the timing of
functions exists (as described above). The
question becomes, in the absence of this
temporal regulation, do cyclins continue to
Clb kinase
confer functional specificity on the CDK?
Overexpression of the B-type cyclin CLB5
Hct1p
Pds1p
can rescue deletion of the three G1-type
Cln kinase
Cdc20p
Sic1p
cyclins (Epstein and Cross, 1992).
Additionally, constitutive overexpression of
Hct1p,
the B-type cyclin CLB1 rescues a strain
Cdc20p
lacking all remaining B-type cyclins (Haase
(APC activators)
Cell
growth
Cdc14p
and Reed, 1999). In Drosophila, no single
deletion of cyclin A, cyclin B or cyclin B3
caused striking defects, but deletion of both
cyclin B and cyclin B3, or of cyclin A and
cyclin B3, gave mitotic defects (Jacobs et al.,
Cln
1998), suggesting substantial overlap among
Low Clb
High Clb
the function of these cyclins. Such results,
activity
Cdc14p, activity
coupled with the fact that, in fission yeast,
Cdc20p
cdc13 is sufficient as the sole B-type cyclin
(Fisher and Nurse, 1996), are not consistent
High [Sic1p]
Low [Sic1p]
with the idea that cyclin targeting is essential
Hct1 active
Hct1p inactive
for appropriate CDK activity.
A dramatic result suggesting that specific
Low CLB
High CLB
cyclins are not required for targeting Cdk
transcription
transcription
activity was the finding that appropriate
‘activating’ mutations in the budding yeast Fig. 2. The minimal cell cycle. (A) Regulatory machinery responsible for the minimal
Cdc28p CDK eliminate all detectable cyclin cell cycle. Note that inhibition (indicated by a flat line) occurs by multiple mechanisms:
requirements for cell cycle Start, which stoichiometric inhibition, phosphorylation, or proteolysis due to ubiquitination.
Activation (indicated by an arrow) is presumably achieved by phosphorylation in some
normally requires at least one Cln cyclin (Fig. cases and by dephosphorylation in others. See recent reviews and papers for more
1). However, such mutations did not bypass information (Cross, 1995; Deshaies, 1997; Nasmyth, 1996; Rudner et al., 2000; Rudner
cyclin requirements later in the cell cycle and Murray, 2000; Zachariae and Nasmyth, 1999; Zachariae et al., 1998). (B) The
(Levine et al., 1999).
minimal cell cycle as a Clb activity cycle, in which transitions between the low Clb
Even in cases in which a specific cyclin activity state and high Clbp activity state are regulated by Cln kinase, Cdc14p and
requirement
is
demonstrated,
cyclin Cdc20p.
A
B
1814
JOURNAL OF CELL SCIENCE 114 (10)
budding yeast Cln cyclins to reverse the low Clb activity state
discussed above (Fig. 2A) is probably due to immunity of the
Cln-Cdc28p kinases to Clb-inhibitory mechanisms, rather than
to different abilities of active Cln-Cdc28p complexes vs ClbCdc28p complexes to carry out some functions. This is
evidenced by the ability of Clb-Cdc28p complexes to maintain
the high-Clb activity state once it is established (see above), as
well as by the bypass of the Cln requirement by CLB5, or
certain CDC28 mutations (Epstein and Cross, 1994; Levine et
al., 1999).
Similarly to the situation with Sic1 and Cln cyclins in
budding yeast, the Cig1p and Puc1p cyclins of fission yeast are
immune to the Rum1p inhibitor, but phosphorylate Rum1p and
trigger its degradation (Benito et al., 1998). It is not clear,
though, whether the Cdc13p or Cig2p complexes are able to
overcome Rum1p inhibition if the cyclin-Cdk complexes are
present at a high enough level.
Another interesting example of input specificity could be the
case of the viral cyclins that can activate Cdk6. The viral
cyclin-Cdk6 complexes are resistant to the INK4 and Cip/Kip
families of inhibitors and so allow Rb phosphorylation to occur
independently of the presence of these inhibitors (Swanton et
al., 1997). These viral cyclins may also alter output specificity
in this system, since the substrate specificity of the viral
cyclin-Cdk6 complexes is thought to be higher than that of
endogenous cyclin-Cdk6 complexes (Chang et al., 1996;
Godden-Kent et al., 1997; Jung et al., 1994; Swanton et al.,
1997).
Evidence in favor of intrinsic cyclin functional
specialization
In some cases, cyclin-dependent events are not rescued by
ectopic expression of different cyclins under conditions in
which differences in regulatory input appear to have been
eliminated. Repression of the mating factor response pathway
by CLN2 overexpression was not duplicated by constitutive
CLB5 or CLN3 overexpression (Oehlen and Cross, 1994). The
Clb2p mitotic B-type cyclin is unable to trigger transcription
of genes normally activated by Cln3p and in fact inhibits their
expression (Amon et al., 1993), whereas the Clb5p B-type
cyclin might activate expression of these genes (Li and Cai,
1999; Schwob and Nasmyth, 1993). Overexpression of the
CLN genes or of CLB5, but not of CLB3 or CLB4, eliminates
heat-shock-induced G1 arrest, owing to a specialized ability of
CLB5p to activate CLN1 and CLN2 transcription (Li and Cai,
1999). The function of CLB5 in spindle morphogenesis
or premeiotic DNA replication cannot be rescued by
overexpression of CLB2 (Segal et al., 1998; Stuart and
Wittenberg, 1998). CLN and mitotic cyclins have essentially
opposing effects on budding yeast morphogenesis, and these
effects are exacerbated by overexpression (Lew and Reed,
1993). CLB5 is able to promote the activation of early and late
origins of replication, whereas CLB6 is able to activate only
early origins (Donaldson et al., 1998; in this case, however, the
pattern of expression of Clb6p has not been shown to be the
same as that of Clb5). Spindle pole body (SPB) duplication is
a multistep process both positively and negatively regulated by
the cyclin-Cdc28p complexes. The Cln-Cdc28p complexes are
able to trigger SPB duplication during G1 phase, whereas Btype cyclin-Cdc28p complexes carry out maturation of SPBs.
B-type cyclin-Cdc28p complexes are also able to inhibit
reduplication of SPBs, ensuring that SPB duplication occurs
only once per cell division cycle (which is similar to models
for control of DNA replication; Haase et al., 2001). With
respect to cyclin specificity, it was shown that even
overexpressed CLN2 or CLB5 could not block SPB
reduplication, which suggests that this function is intrinsically
specific to CLB1-CLB4 (Haase et al., 2001). In fission yeast,
the G1-type cyclin-CDK complex Puc1p-Cdc2p cannot induce
S phase, unlike the B-type cyclin-Cdc2p complexes (Fisher and
Nurse, 1996). In higher eukaryotic cells, cyclin D2 and cyclin
D3, but not cyclin D1, prevent differentiation of 32D myeloid
cells in response to granulocyte colony-stimulating factor
(Kato and Sherr, 1993). These data suggest that some CDKdependent events require specific cyclin-CDK complexes or at
the least that specific cyclins strongly potentiate some CDKdependent events.
To demonstrate that cyclin proteins confer functional
specificity to CDKs, cyclin-coding sequences have been placed
under control of a distinct cyclin promoter. In this way, a
direct functional comparison of the two cyclins can be made
independently of differences in expression patterns. This
approach was first used to compare the functional specificities
of the G1-type cyclins Cln2p (representative of the highly
homologous CLN1/CLN2 gene pair) and Cln3p (Levine et al.,
1996; Valdivieso et al., 1993). Previously, functional analysis
of Cln2p and Cln3p had revealed that they support cell cycle
entry by distinct mechanisms; Cln3p is responsible for
transcriptional activation of G1 transcripts, including CLN1
and CLN2 (Dirick and Nasmyth, 1991; Koch and Nasmyth,
1994; Stuart and Wittenberg, 1995). Cln2p is required for
additional G1 events, including bud emergence and activation
of Clb-CDK complexes (Benton et al., 1993; Cvrcková and
Nasmyth, 1993; Lew and Reed, 1993). The intrinsic functional
specificity of Cln2p, compared with that of Cln3p, is
demonstrated by the fact that Cln2p and Cln3p differ in their
support of viability in certain genetic backgrounds, even when
both are expressed from the CLN3 promoter (which produces
relatively constitutive, low levels of expression; Levine et al.,
1996). These data indicate that differences in the timing or the
levels of expression of CLN2 and CLN3 do not account entirely
for their functional specificity. More recent studies reveal that
Cln2p and Cln3p are primarily localized to distinct subcellular
compartments, and that their localization contributes to their
functional specificity (Miller and Cross, 2000; see below).
Despite the clear differences in functional specificity between
Cln3p and Cln2p, both are independently able to support cell
cycle initiation. This is probably due to the existence of parallel
pathways backing up certain aspects of G1 cyclin function.
Thus Cln3p is ‘aided’ by proteins that might mimic some
aspects of Cln2p function (Levine et al., 1996). In the case of
Cln2p, transcripts normally activated by Cln3p are delayed but
eventually are produced at reasonably high levels, possibly
with the aid of Bck2p (Levine et al., 1996). Bck2p works in
parallel with (although independently of) Cln3p to activate
transcription (Di Como et al., 1995; Epstein and Cross, 1994).
Similar conclusions have been drawn about cyclin D1 and
cyclin E in mice. Geng et al. found that phenotypes associated
with the cyclin D1 knockout were rescued by replacement of
the coding sequence of cyclin D1 with that of cyclin E (Geng
et al., 1999). Although this result suggested that cyclin E and
cyclin D1 are functionally redundant, a closer examination of
Cyclin specificity: how many wheels do you need on a unicycle?
specific cyclin-D1-dependent events that occur during normal
cell cycle progression showed that cyclin E did not rescue these
events but instead bypassed the requirement for cyclin D1
during cell cycle progression. These results are reminiscent of
the relationship between Cln3p and Cln2p in which either
cyclin can support entry into the cell cycle, but do so by
different mechanisms.
Cyclin specificity that is independent of differences in
expression patterns has also been demonstrated for B-type
cyclins in budding yeast. CLB5 and CLB6 are expressed
earliest and implicated most directly in regulation of DNA
replication, especially of late-acting origins in the case of
Clb5p (Donaldson et al., 1998; Epstein and Cross, 1992;
Schwob and Nasmyth, 1993). CLB1 and CLB2 are expressed
later and are involved in regulation of mitosis (Surana et al.,
1993). Replacement of the coding sequence of CLB5 with that
of CLB2 (so that CLB2 is expressed from the CLB5 promoter)
can rescue inviability due to deletion of CLB1 and CLB2 but
poorly promotes the onset of DNA replication. This effect
appears to be largely independent of differences in Clb protein
expression (Cross et al., 1999).
Overexpression of a Clb2p mutant lacking its destruction
box (which is therefore immune to APC-mediated
ubiquitination and proteolysis) results in inhibition of mitotic
exit (Ghiara et al., 1991; Surana et al., 1993). Overexpression
of destruction-box-deleted Clb5p to similar levels failed to
inhibit mitotic exit, although it did appear to block DNA
replication in the next cell cycle (Jacobson et al., 2000). This
suggests that Clb2p is much more efficient at blocking mitotic
exit, whereas both Clb2p and Clb5p may block reloading of
origins of replication (see above and Fig. 2A).
A special ability of Clb5p to prevent Clb2p-Cdc28p
inactivation late in the cell cycle was inferred from the result
that, in the absence of Cdc20p and Pds1p, Clb5p plays a unique
role in blocking mitotic exit (Shirayama et al., 1999). It was
concluded that Clb5p is especially active at phosphorylating
Sic1p and Hct1p (Fig. 2A) and keeping Clb2p active.
Thus, we conclude that, although cyclin targeting of CDK
activity is unlikely to be essential, it strongly influences the
biological activity of the complex. Certain activities of specific
cyclin-Cdc28p complexes are simply undetectable in other
complexes; other activities are facilitated by specific cyclins,
even if activity can be detected with inappropriate cyclins at
higher expression levels. There are sure to be truly non-cyclinspecific CDK activities, although none has yet been clearly
characterized.
Cyclin-dependent targeting of CDK activity
How does a cyclin confer functional specificity on a CDK?
Although the expression patterns of most cyclins correspond
to the timing of their functions, the information reviewed above
indicates that this is not the only aspect of cyclins that is
important for functional specificity. Cyclin proteins may
target CDK activity, either through cyclin-specific protein
interactions or by influencing subcellular localization.
Obviously, these two contributing factors may be linked, the
subcellular localization limiting the accessibility of cyclin to a
subset of proteins.
Several laboratories have found that phosphorylation of
some potential Cdk substrates is dependent on the identity of
1815
the cyclin. This work has been carried out primarily in higher
eukaryotic cell lines, in which a limited number of CDK
substrates are known. Sherr et al. have found that the LxCxE
motif in cyclin D is required for interaction of the cyclinD–CDK complex with and efficient phosphorylation of Rb
(Sherr, 1993). In fact, specificity exists among different types
of cyclin D with respect to their ability to activate CDK2 and
phosphorylate Rb (Ewen et al., 1993). Mutation of a similar
sequence in cyclin E prevents phosphorylation of Rb by cyclinE–CDK2 (Kelly et al., 1998) and has interesting biological
consequences. Peeper et al. found that cyclin-A–CDK2 and
cyclin-A–CDC2, but not cyclin-B–CDK2 or cyclin-B–CDC2
complex can phosphorylate the Rb-like protein p107 (Peeper
et al., 1993). Cyclin-A–CDK2 and cyclin-B–CDK2, but
not cyclin-D–CDK2 can phosphorylate the E2F-1–DP-1
transcription factor complex. Interestingly, phosphorylation of
E2F-1–DP-1 by cyclin-A–CDK2 inhibits the DNA-binding
activity of the transcription complex, whereas cyclin-B–CDK2
phosphorylation does not (Dynlacht et al., 1997). CyclinA–CDK2, but not cyclin-E–CDK2, phosphorylates the 34 kDa
subunit of the human single-stranded-DNA-binding protein
complex (SSBP) and lamin B (Gibbs et al., 1996; Horton and
Templeton, 1997). The human papillomavirus DNA replication
initiation factor E1 binds to cyclin E, cyclin A, cyclin B and
cyclin F, but not cyclin D1(Ma et al., 1999). Studies of T470
breast cancer cell nuclear lysates find that cyclin-D1–CDK4
phosphorylates a 24 kDa and 34 kDa protein, whereas
cyclin-D3–CDK4 phosphorylates 24-kDa, 42-kDa, 102-kDa,
and 105-kDa proteins. The cyclin-D3–CDK6 complex
phosphorylates 102-kDa and 105-kDa proteins, but not a 24
kDa protein. These data suggest that there are substrates
specific to the cyclin (the 102-kDa and 105-kDa proteins
phosphorylated in the presence of cyclin D3 but not cyclin D1)
and substrates specific to the CDK (the 24 kDa protein
phosphorylated in the presence of CDK4, but not CDK6;
Sarcevic et al., 1997).
Cyclin-specific protein interactions are not limited to
potential substrates. In budding yeast, specific inhibitors
recognize specific cyclin-CDK complexes. As described in the
previous section, the Far1p inhibitor is able to bind to and
inhibit Cln-Cdc28p complexes but not Clb-Cdc28p complexes
(Jeoung et al., 1998; Peter and Herskowitz, 1994). Conversely,
the Sic1p and Swe1p inhibitors are able to inhibit Clb-Cdc28p
complexes but not Cln-Cdc28p complexes (Schwob et al.,
1994). Cks1p is required for Cln2-Cdc28p complex formation
but not Clb-Cdc28p complex formation (Reynard et al., 2000).
The fission yeast Rum1p protein inhibits the Cdc13p and Cig2p
cyclins, but not Puc1p or Cig1p cyclin in complex with Cdc2p
(Benito et al., 1998). In vitro, the higher eukaryotic CDK
inhibitors p21 and p27 are able to bind cyclins directly. This
binding exhibits some specificity, since p21 is able to bind to
cyclin E more efficiently than to cyclin A and cyclin B, and
does not bind to D-type cyclins (Chen et al., 1996). Specific
inhibitors are also known to bind preferentially to different
CDK components of cyclin-CDK complexes in higher
eukaryotes (Morgan, 1995; Sherr and Roberts, 1999).
Viral cyclin D homologs have been identified in three types
of γ-herpesviridae (Cesarman et al., 1996; Nicholas et al.,
1992; Virgin et al., 1997). Two have been shown to bind and
activate eukaryotic CDK6 and CDK4 in higher eukaryotic
cells. When bound to CDK4 and CDK6, viral cyclins are able
1816
JOURNAL OF CELL SCIENCE 114 (10)
to phosphorylate substrates normally phosphorylated by
cyclin-D–CDK complexes, such as Rb, yet are resistant to
inhibition by the INK4 and Cip/Kip families of CDK
inhibitors. The viral cyclin-CDK6 complexes also demonstrate
a broader substrate range than that observed for endogenous
cyclin-D–CDK6 complexes (Chang et al., 1996; Godden-Kent
et al., 1997; Jung et al., 1994; Li et al., 1997b; Swanton et al.,
1997).
Tyrosine phosphorylation of CDK inhibits its activity, and
CDK can subsequently be activated by Cdc25p-dependent
dephosphorylation. In the case of cyclin-B–Cdc2p complexes,
the inhibitory phosphorylation of Cdc2p occurs after complex
formation, which suggests that the cyclin binding is important
for this regulation (Solomon et al., 1990). Cdc25p is also better
able to bind to cyclin-CDK complexes than to monomeric CDK
in vitro, which suggests that the cyclin brings the CDK into
contact with Cdc25p more efficiently (Morris and Divita, 1999).
The relationship between CDC25 and cyclins is complex, since
cyclin B, but not A- or D-type cyclins, is able to bind to
CDC25A, which results in activation of the phosphatase
(Galaktionov and Beach, 1991; Zheng and Ruderman, 1993).
During early embryogenesis, the cyclin-A–CDC2 complex
appears to be a poor substrate for the kinase that phosphorylates
and inhibits CDC2, whereas cyclin-B–CDC2 is not.
Additionally, cyclin-A–CDC2 complexes do not require the
CDC25 tyrosine phosphatase for activation, whereas cyclinB–CDC2 complexes do (Devault et al., 1992). In these cases
CDC2 is inhibited differentially depending on the identity of its
cognate cyclin. A finding consistent with this is that cyclinB–CDC2, but not cyclin-A–CDC2 complexes, are regulated by
tyrosine phosphorylation during meiosis in starfish oocytes
(Okano-Uchida et al., 1998). Interactions between CDC25 and
cyclin might occur through the highly conserved hydrophobic
patch region (described below), and potential competition might
exist between p21 inhibitor, substrate and CDC25 for
interaction with cyclin-CDK complex through the hydrophobic
patch (Adams et al., 1996; Saha et al., 1997).
The hydrophobic patch region of cyclin A has been
described as a docking site for substrates (Shulman et al.,
1998). Mutation of this region reduces the ability of cyclin A
to bind to specific substrates but does not reduce activation of
CDK2 kinase activity (Shulman et al., 1998). Likewise, stable
association of substrates such as p21, Rb and E2F requires
specific substrate domains that, when mutated, inhibit substrate
interactions (Adams et al., 1999; Adams et al., 1996). Small
peptides designed to mimic substrate domains show specificity
in their ability to block cyclin A activity but not cyclin B
activity (Adams et al., 1996).
Residues within the hydrophobic patch region of cyclin are
highly conserved, and mutation of these residues in yeast
Clb5p strongly impairs Clb5p function but not activity of
Cdc28p towards the nonspecific substrate histone H1, which is
consistent with data for cyclin A hydrophobic-patch mutants
(Cross et al., 1999). The hydrophobic patch in cyclin A
mediates interactions with the cyclin inhibitor p27. In twohybrid analysis, Clb5p also interacts with the higher eukaryotic
cyclin inhibitor p27 through this conserved domain (Cross and
Jacobson, 2000). Although no p27 homolog is thought to exist
in budding yeast, these data indicate that this domain is capable
of mediating cyclin-protein interactions. Confirmation of this
idea requires identification of relevant Clb5p targets. Two-
hybrid analysis has identified proteins that interact specifically
with Clb5p but not Clb2p, and almost all of these targets also
require the Clb5p hydrophobic patch to be intact (M. D.
Jacobson, B. R. Drees and F.R.C., unpublished data); however,
it is unclear whether any of these are authentic Clb5p
interactions in vivo.
The yeast mitotic cyclin Clb2p is unable to bind to p27 in
the two-hybrid assay, although it retains most sequence
signatures of the conserved hydrophobic patch. However,
mutation of the hydrophobic patch domain does alter the
functional profile of Clb2p. The hydrophobic patch mutant of
Clb2p is almost negative in Clb2p-specific genetic assays, but,
intriguingly, inactivation of the hydrophobic patch allows
rescue in contexts normally restricted to Clb5p (Cross and
Jacobson, 2000). These data suggest that the normal activity of
Clb2p may require this targeting domain, and abnormal Clb2p
activity towards normally Clb5p-specific events might be
restricted by the targeting domain.
In summary, the hydrophobic patch could be a conserved
substrate recognition domain co-opted by p27 to anchor a
kinase-inhibitory region (Russo et al., 1996). We speculate that
differences in residues flanking the core domain could confer
binding specificity. The hydrophobic patch region is important
for cyclin-CDK interactions that are cyclin specific (as
described above). The genetic and two-hybrid studies of Clb5p
and Clb2p summarized above are consistent with this
speculation but do not prove it.
Thus, the ability of cyclin-CDK complexes to interact with
cellular proteins is influenced by the identity of the cyclin
component of the complex. In many cases, there is evidence
that this can occur independently of potential differences in
expression patterns or cyclin subcellular localization. Taken
together, the ability of cyclin proteins to mediate specific
protein interactions provides a likely mechanism for cyclindependent functional specificity of cyclin-CDK activity,
although clearly defined and physiologically relevant cyclinCDK targets will need to be defined and tested for specific
cyclin binding before this idea can be established. There has
been significant progress along these lines in mammalian
systems in the identification of specific cyclin–CDK-complexbinding proteins and substrates. Progress in the yeast field has
been significantly slower on this important front.
Cyclin specificity and subcellular localization
An additional mechanism that contributes to the functional
specificity of cyclin-CDK complexes is the control of their
subcellular localization (reviewed in Pines, 1999). Differential
subcellular localization of complexes could bring specific
complexes into contact with potential substrates, keep
complexes sequestered from improper substrates or expose the
complex to activators or inhibitors that are localized to specific
compartments. In this sense, control of cyclin subcellular
localization is potentially linked to the control of cyclin-protein
interactions.
The subcellular localization patterns of many cyclins have
been characterized and generally correlate well with cyclin
function, such that cyclins are in the right place at the right
time for their predicted functions (Baldin et al., 1993; Cardosa
et al., 1993; Diehl and Sherr, 1997; Hagting et al., 1998; Hood
et al., 2001; Knoblich et al., 1994; Lukas et al., 1994; Maridor
Cyclin specificity: how many wheels do you need on a unicycle?
et al., 1993; Ohtsubo et al., 1995; Pines and Hunter, 1991). In
addition to this correlation, experimental data indicate that
cyclin subcellular localization is important for cyclin function.
The higher eukaryotic G1-type cyclin D1 is localized to the
nucleus during G1 phase and then redistributes to the
cytoplasm once DNA replication begins (Baldin et al., 1993).
A mutant cyclin D1 that is unable to enter the nucleus is unable
to activate DNA replication in fibroblasts (Diehl and Sherr,
1997). Higher eukaryotic cyclin B1 has a complex localization
pattern, accumulating in the nucleus during mitosis just prior
to nuclear envelope breakdown. The ability of cyclin B1 to
promote mitosis in frog eggs is abolished in a mutant that does
not localize to the nucleus (Li et al., 1997a). Interestingly,
cyclin B1 that is targeted to the nucleus by addition of a nuclear
localization signal (NLS) or through disruption of the cyclin
B1 nuclear export signal (NES) shows a defect in DNAdamage-induced G2 arrest (Jin et al., 1998; Toyoshima et al.,
1998). The cyclin-E–CDK2 complex and its substrate p220 (or
NPAT) colocalize to Cajal bodies (specific subnuclear
organelles associated with histone gene clusters). The timing
of p220 phosphorylation correlates with the appearance of
cyclin E in Cajal bodies at the beginning of G1/S boundary and
appears to be important for histone transcription (Ma et al.,
2000; Zhao et al., 2000). Cyclin B1 and cyclin B2 are localized
to microtubules and the Golgi apparatus, respectively; this
suggests that these two cyclins might have distinct functions in
the cell cycle that are independent of their expression patterns
and CDK partner (Draviam et al., 2001; Jackman et al., 1995).
Similarly, Drosophila cyclin A and cyclin B both accumulate
in the cytoplasm, whereas cyclin B3 accumulates in the nucleus
(Jacobs et al., 1998).
Strong evidence for an influence of subcellular localization
on cyclin specificity comes from the study of G1 cyclin
localization in budding yeast. The cyclins Cln2p and Cln3p
support cell cycle progression through the same cell cycle step
but through distinct mechanisms (see above). These two
cyclins have distinct subcellular localization patterns. Cln3p is
found primarily in the nucleus, which is consistent with its role
as a transcriptional activator (Miller and Cross, 2000). Cln2p
is found primarily in the cytoplasm, although a pool of Cln2p
also localizes to the nucleus (Miller and Cross, 2000; M.E.M.
and F.R.C., unpublished). Nuclear localization of Cln3p
requires a C-terminal NLS (M.E.M. and F.R.C., unpublished),
and deletion of this sequence results in the cytoplasmic
accumulation of Cln3p. Functional analysis of a Cln3p mutant
that lacks this NLS indicates that it can rescue strains that
Cln2p can rescue, but that wild-type Cln3p cannot. Therefore,
when Cln3p takes on a Cln2p-like subcellular localization
pattern, it also takes on Cln2p-like functions. This is due to the
shift in localization patterns and not to other defects associated
with the Cln3p mutant, because addition of a heterologous
NLS moves the mutant Cln3p back into the nucleus and
abolishes Cln2p-like functions. These data (Miller and Cross,
2000; M.E.M. and F.R.C., unpublished) indicate that the
subcellular localizations of the G1 cyclins Cln2p and Cln3p
contribute to their functional specificity.
The higher eukaryotic cyclins B1 and B2 also exemplify the
importance of subcellular localization. When cytoplasmic,
cyclin B1 is partially localized to microtubules and is capable
of reorganizing nuclear, cytoskeletal and membrane
compartments; whereas cyclin B2 is localized to the Golgi
1817
apparatus and is able to disassemble it. Using chimeric
proteins, Draviam et al. replaced the N-terminus of cyclin B2
with the N-terminus of cyclin B1 and found that this chimera
was redirected to the Golgi (Draviam et al., 2001). The
reciprocal chimera, in which the N-terminus of cyclin B1 was
replaced with the N-terminus of cyclin B2, was liberated from
the Golgi. Directing cyclin B1 to the Golgi in this way
restricted its function to re-organization of the Golgi apparatus,
whereas liberation of cyclin B2 allowed it to re-organize the
cytoskeleton, taking on cyclin B1-like function. The CDKbinding region and potential substrate-binding hydrophobic
patch domain was not switched in these chimeras. In this case,
it appears that localization, and not substrate targeting through
the hydrophobic patch, influenced the activity of these B-type
cyclins (Draviam et al., 2001).
Concluding remarks
In summary, cyclins can influence when and where CDK is
active, and therefore may be more than a conformational on/off
switch for CDK activity. This cyclin-dependent targeting can
occur through temporal regulation of cyclin expression and
spatial regulation of cyclin localization. Additionally, cyclins
influence CDK activity by mediating critical interactions
between cyclin-CDK complexes and cellular proteins
(including substrates, inhibitors and activators). Targeting is
likely to occur through a combination of factors discussed here,
which include temporal expression, protein associations,
subcellular localization and possibly additional mechanisms
not yet elucidated. Regulated oscillations of CDK activity
required for the minimal cell cycle can probably be achieved
through control of the level of a single cyclin protein, and this
is arguably the condition under which the eukaryotic cell cycle
initially evolved (Nasmyth, 1995; Nasmyth, 1996). The
information reviewed above suggests that the situation in most
modern eukaryotes may be more complex and that significant
enhancement of biological efficiency of cyclin-CDK activity
occurs through specific interaction of different cyclins with
regulators and targets.
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