Cyclin from sea urchins to HeLas:
making the human cell cycle
-
Jonathon Pines
WellcomeKRC Institute and Department of Zoology, Tennis Court Road, Cambridge CB2 I QR, U. K.
This lecture will primarily be concerned with
Cyclins, a family of proteins intimately concerned with cell-cycle regulation, but originally a
band on a gel that disappeared every time a sea
urchin egg divided. I will spend just a little time
on some ancient history, a time when cyclin was
‘Cyclin’ - now sadly demoted to simply sea
urchin cyclin B. I will go on to outline our current view of cell-cycle regulation by cyclins and
their partner kinases, and finish with my recent
work, which has focused on cyclins as molecules
that target their partners to discrete parts of the
cell.
Cotworth Medal Lecture
Delivered at University College Dublin
on I2 September 1995
Pre history (or Before Cyclin)
Cyclins were originally identified (by Tim Hunt
on 23rd July 1982) by one-dimensional SDS/
PAGE as proteins in sea urchin eggs that were
rapidly synthesized after fertilization [ 13. This
discovery had its genesis in studies on translational control, because there was a striking
change in the pattern of proteins synthesized
before and after fertilization of marine invertebrate eggs [Z]. Most of these proteins, such as
the small subunit of ribonucleotide reductase
[ 31, simply accumulated through the succeeding
cell cycles, but cyclin was remarkable, being destroyed once per cell cycle, at each mitosis. There
appeared to be one cyclin in sea urchins, but two
in clams, which were designated A and B. Clam
cyclin A was the first to be cloned and
sequenced, by Swenson et al. [4], and its connection to the cell cycle was strengthened by their
observation that cyclin A mRNA was able to
JONATHONPINES
induce meiotic maturation when injected into
frog oocytes. This was the classic assay for MPF
(originally meiosis promoting factor [S], now
referred to as M-phase promoting factor), and it
suggested that cyclin could be a component of
MPF, or at least involved in the initiation of
mitosis. A grand synthesis of cell-cycle studies
soon followed, with the discovery that the human
homologue of the fission yeast cdc2 kinase and
budding yeast CDC28 kinase was able to function in place of the yeast genes [6] and that
cdcZ/CDC28 was a component of MPF [7].
Cyclin finally joined cdcZ/CDC28 at the heart of
the cell cycle when it was shown to be part of
MPF, where it binds and activates cdcZ/CDC28
~ ~ 9 1 .
Abbreviations used: APC, anaphase promoting complex; CDK, cyclin-dependent kinase; CAK, CDK-activating kinase; CDI, CDK-inhibitor protein; MAP
kinase, mitogen-activated protein kinase; MPF, Mphase promoting factor; PKA, protein kinase A; Rb,
retinoblastoma; TGFfl, transforming growth factor fl;
UBC, ubiquitin-conjugating enzyme.
The age of innocence: when cyclin was
cyclin
For a brief moment in cell-cycle studies cyclin
took centre stage, when Andrew Murray showed
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I6
that cyclin (sea urchin cyclin B) synthesis and
destruction was all that was required to drive a
frog egg extract through cycles of both DNA synthesis and mitosis [ 10,111. This emphasizes two
fundamental properties of a cyclin: (i) it activates
a kinase (in this case cdc2 as part of MPF) to
promote a change in the cell cycle; and (ii) the
succeeding phase in the cell cycle is triggered by
cyclin destruction. Indeed, Murray showed that
an N-terminal truncation inhibited cyclin
destruction, and this prevented frog egg extracts
from exiting mitosis [ l l ] . At this point the cell
cycle looked quite straightforward. Cells in interphase synthesized cyclin. When cyclin reached a
threshold level it activated cdc2 kinase to cause
mitosis. Then cyclin destruction released cells
back into interphase, and cyclin synthesis started
all over again.
Things are not quite so simple now; human
cells have at least 14 cyclins with 8 partner
kinases [cyclin-dependent kinases (CDKs)] and
thus far 7 specific inhibitor proteins. Many
cyclins and their partner CDKs play important
roles in the regulation of the eukaryotic cell
cycle, but the cyclin-CDK motif is also used by
the cell to control processes separate from the
cell cycle, such as the response to phosphate
starvation in yeast, DNA repairhranscription in
human cells, and neurofilament phosphorylation
in post-mitotic neurons. This may be because
the cyclin-CDK motif offers a remarkable
degree of flexibility in response to variations in
the environment. Such flexibility is conferred by
the ability to alter the activity of the cyclin-CDK
complex by phosphorylation, or by binding
specific inhibitor proteins, or by varying the level
of the cyclin itself. T h e rest of this review will try
to put some of the cyclins into context, but for
more extensive reviews of the cell cycle and of
cyclin-CDK regulation see [ 12-21].
The cyclin-CDK motif
In essence cyclins are activators of a family of
protein serinehhreonine kinases, the cyclindependent kinases (CDK), which themselves are
defined as protein kinases that need to bind to a
cyclin to be active [22]. As I have explained,
cyclins were originally defined as proteins that
were specifically degraded at every mitosis. However, once several cyclin cDNAs had been cloned
and sequenced (Figure I), the definition became
that of a protein containing a 100-amino-acid
region of similarity to a consensus sequence, the
‘cyclin box’ [23]. T h e cyclin box has since been
Figure I
Schematic of the cyclin family
Cyclins A to H are grouped according to similarities in their primary structures and thelr roles in the cell cycle G I , G2, CAKjtranscription
or unknown Illustrated are the destruction boxes and PEST regions responsible for their destruction The budding yeast Cln (GI cyclins) are
also shown Darker hatching indicates regions that are more closely related
Cyclln
-
CVCLINBOX
-
Sln,
Functlon
48 kDa
S 8 G2-M
48 kDa
Mitosis
66kDa
G1 (START)
D
32 kDa
G1 (R)
E
44
F
87 kDa
G2?
29 kDa
?
n
37 kDa
CAK 8 TFllH
C (mcs2,ccll)
33 kDa
Transcription?
A2
PEST
Ch
G
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H-
kDa
G1-S
Colworth Medal Lecture
demonstrated to be involved in binding a CDK
[24,25]. Cyclin nomenclature has carried on the
alphabetical classification stemming from the
original clam A- and B-type cyclins.
T h e CDKs also share certain structural
similarities (Figure 2), and are numbered
according to the order in which they were shown
to bind a cyclin. CDKs are just large enough to
encompass all the conserved protein kinase
domains [26], and in domain 111 they all have a
sequence related to the canonical EGVPSTAIRISLLKE motif found in the first CDKs to be
isolated: fission yeast ~ 3 4 ' ~ 'and
'
budding yeast
p34"')"2x. This region is involved in binding to
cyclins (see below) and confers some degree of
specificity with which cyclins bind which CDKs
(Figure 3 ) . T h e other region that interacts with
the cyclin includes the threonine residue (T-161
in human cdc2, T-160 in CDK2) in domain VIII
that is phosphorylated in all active protein
kinases. This residue is phosphorylated by a
specific protein kinase, CDK-activating kinase
(CAW.
Monomeric CDKs are completely inactive as
protein kinases; they absolutely depend on binding a cyclin for activation, and now we have a
molecular mechanism to explain this depend-
ence. T h e crystal structures of both an inactive
monomeric CDK and an active cyclin-CDK
complex have been solved [27,28]. From a comparison with the active protein kinase A (PKA)
structure [29], two reasons for the inactivity of
monomeric CDK2 were immediately apparent.
Firstly, ATP was bound in a conformation that
would preclude nucleophilic attack on the scissile
1j-v phosphate bond by the substrate hydroxy
group. Secondly, part of the C-terminal lobe of
the enzyme - the 'T loop' domain - blocked the
catalytic cleft. T h e main reason for the differences in structure between CDK2 and PKA was
an a-helical region (stL12) unique to CDK2 that
constrained both the ATP-binding site and the T
loop domain. Thus cyclin binding was predicted
to melt the aL12 helix to allow the ATP-binding
site to reconfigure to resemble that of PKA and
allow the T loop to move away from the catalytic
cleft [27].
T h e structure of human CDK2 bound to an
N-terminal truncated form of cyclin A has now
been solved [28], and this confirms the predictions of De Bondt et al. [27]. In the cyclin ACDK2 complex, ATP is bound in the same
manner as in PKA and is therefore susceptible to
attack from a bound substrate. T h e T loop
17
Figure 2
Schematic of the CDK family
The identified CDKs are shown with their cyclin partners and partial sequences in the PSTAIRE region Two closely related sub-families
(PICTAIRE and PITAIRE). which have not as yet been shown t o be activated by a cyclin, are also shown
4
Kinase Domain
b
Cyclin Partner
PSTARE
A80
cdc2 (CDK1)
PSTARE
CDK2
65%
CDK3
66%
CDK4 (PSKJJ)
44%
E 8 A (8D)
PSTARE
E?
PVfiSTVRE
D
PSSALRE
CDKS (PSSALRE) 57%
D (8 ~ 3 5 )
PLSTIRE
CDKG (PLSTIRE)
47%
CDK7 (p4Omo'7
40%
PlCTAlRE (1-3)
51 -55%
D
NRTALRE
H
PlCTAlRE
?
PITAIRE
PITAIRE
(CHED)
42%
I
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Figure 3
Cyclin-CDK specificity
18
Illustrated in bold type are the known human cyclin-CDK complexes, with the putative budding yeast homologues in plain type.
Also shown are the PhoPcl family of cyclin/CDK complexes
involved in phosphate metabolism in budding yeast
Cyclin B
cdcf (CDKl)
(CLB 14)
(CDc28)
CDK2
Cyclin E
Cyclin D
CDK4, CDKQ
(CLN 1-31
(CDC28)
P35
CDKS
Cyclin H
CDK7
Cyclin C
CDKS
CCLl
KIN28
SRBll
SRBIO
PCLI-4
PH085
PHOBO
domain no longer blocks the catalytic cleft, and
the cleft itself has opened up to allow substrates
to bind.
Cyclin achieves these changes in CDK conformation by binding to the PSTAIRE region and
around the T loop, regions previously shown to
be important for cyclin binding by mutation and
modelling studies [30,31]. Two cyclin a helices
clamp to the middle of the PSTAIRE helix by
hydrophobic interactions, and to either end of
the PSTAIRE helix by hydrogen bonds. This
rotates the PSTAIRE helix
90”, moving it into
the catalytic site, changes the packing of the Nterminal lobe, and melts the aL12 helix. The reorientation of the PSTAIRE helix brings the side
chain of the catalytic residue glutamate-51 into
the active site, reconfiguring the way in which
ATP is bound such that the fl-y phosphate bond
is moved into a position favourable for nucleophilic attack from a bound substrate [28].
Cyclin also strongly interacts with the Nterminal portion of the T loop, which, because
the constraining crL12 helix has melted, moves
-
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the T loop away from the active site [28]. Threonine-160 in the T loop is phosphorylated in the
fully active kinase, which would probably stabilize the T loop in its new position through ionic
interactions with a basic patch of residues in the
C-terminal lobe of the protein. T-160 phosphorylation also appears to stabilize the cyclinCDK complex itself, so there may also be ionic
interactions with the cyclin. Furthermore, T-160
in the cyclin-CDK crystal is much more accessible to phosphorylation by CAK, explaining why
CAK only phosphorylates CDKs complexed with
a cyclin [32].
The crystal structure of the cyclin-CDK
complex shows that the region of cyclin involved
in binding a CDK is an N-terminal helix
followed by a repeat of two sets of five a helices
[28]. Each repeat consists of a three-helix bundle
with the other two helices packed against the
side, and the two sets of a helices can be superimposed on one another. One repeat of a helices
is formed by the cyclin box. The second repeat
extends from the end of the cyclin box to about
40 amino acids from the C-terminus of the protein, but is only 12% identical to the cyclin box.
This repeated structure was noted by Gibson et
al., who also pointed out a similar repeat in
transcription factor (TF)IIB and the retinoblastoma (Rb) family of proteins [33], suggesting
that this may be a conserved structural motif.
There are five very conserved residues in
the cyclin box [24,34] that have also been shown
to be important for CDK binding. Of these, R211 and D-240 form a buried salt bridge that
stabilizes the interaction between the a1 and a2
helices. Two others, K-266 and E-295, hydrogen
bond with the PSTAIRE region of CDKZ and
with each other. Deletions extending into the
helix N-terminal to the cyclin box prevent CDK
binding [24,25], and in the crystal structure this
helix wraps around both cr-helical repeats,
stabilizing the R-211-D-240 salt bridge and
interacting with the C-terminal lobe of CDK2.
The extensive inter-digitation between cyclin A
and CDKZ also makes it feasible that cyclins
could contribute to the substrate specificity of
the cyclin-CDK complex.
T loop phosphorylation
A cyclin-CDK complex is only fully active after
the T loop threonine has been phosphorylated by
CAK [32]. Furthermore, CAK is itself a cyclinCDK complex, composed of cyclin H [35,36],
CDK7 (originally called p40M”” [37-391) and a
Colworth Medal Lecture
third protein of -36 kDa [35,40]. There is
potential for CAK activity to be regulated
through the synthesis and degradation of cyclin
H, but as yet CAK activity has not been found to
vary through the cell cycle. Whether one CAK is
able to phosphorylate another on the domain VIII
threonine, T-176, or whether a different kinase is
required, has not been determined, but this is
not an auto-phosphorylation event [35,36].
CDK7 is almost exclusively nuclear [40],
both cyclin H and CDK7 have consensus nuclear
localization signals, and a mutant CDK7 without
a nuclear localization signal remains inactive in
Xenopus oocytes. Most cyclin-CDK complexes
are nuclear, and are therefore probable substrates for CAK. However, the primary mitotic
cyclin-CDK complexes, composed of B-type
cyclins and cdc2, are cytoplasmic throughout
interphase (see below), so there may be a cytoplasmic form of CAK, which may or may not be
composed of cyclin H and CDK7.
Cyclin H-CDK7 has also been identified as
a component of TFIIH, which contains the RNA
polymerase I1 C-terminal domain kinase [41]. It
is still unclear whether cyclin H-CDK7 is both
CAK and the RNA polymerase I1 kinase, but its
role in transcription seems to be conserved
through evolution. In budding yeast, the RNA
polymerase C-terminal domain kinase has been
identified as a cyclin-CDK complex of Ccl and
Kin28 [42], and cyclin H and Ccll are more
similar to each other, and to cyclin C, than to any
other cyclin.
Regulating cyclin- CDK complexes by
phosphorylation
. ..
After phosphorylation by CAK, the cyclin-CDK
complex can be regulated in two different ways:
through phosphorylation or through binding an
inhibitor. When bound to a cyclin, the CDK subunit is a substrate for the weel and mikl kinases
[43-471. These kinases very specifically phosphorylate a tyrosine (Y-15 in cdc2) in the ATPbinding region of the CDK, which inactivates the
kinase by interfering with phosphate transfer to a
bound substrate [48]. In animal cells the threonine residue in cdc2 adjacent to Y-15 is also
phosphorylated by a membrane-bound kinase
activity separate from the tyrosine kinase [49].
Modelling studies suggest that T-14 phosphorylation may inhibit the enzyme in a different
manner to Y-15 phosphorylation, by interfering
with ATP binding [31]. Inactivation via Y-15
phosphorylation is especially important in the
control of the cyclin B-cdc2 complexes in fission
yeast and in animal cells. Mutating tyrosine-15 to
phenylalanine causes fission yeast to enter
prematurely into mitosis [50,51], even if the cell
has not yet completed DNA replication. This
mutation has a slightly less severe phenotype in
animal cells, but if threonine-14 is mutated to
alanine the double mutation completely deregulates cdc2 activation [52,53]. In budding yeast,
the homologous tyrosine (Y-18) is not important
in the regulation of mitosis [54,55], but is essential to the proper control of budding [56]. It is
less clear whether Y-15 phosphorylation is
important in the regulation of other CDKs at
other points in the cell cycle, although there are
data indicating that CDK4 is tyrosine phosphorylated and inactivated after UV damage [57].
At mitosis, weel/mikl kinase activity is
down-regulated by at least two separate kinase
activities [58,59]. One of these kinases is the
product of the fission yeast niml gene [58,60],
and it phosphorylates residues in the C-terminus
of weel. Another as yet unidentified kinase(s)
phosphorylates the N-terminus of weel [59].
.. .and dephosphorylation
Threonine-14 and tyrosine-15 are dephosphorylated by a specific family of phosphatases. First
identified as the product of the cdc25 gene in
fission yeast [61], at least three Cdc25 family
members (A, B and C) have been isolated from
animal cells [62]. T h e Cdc25 phosphatases are
dual-specificity phosphatases whose closest relatives are the protein tyrosine phosphatases of
Vaccinia virus and of the plague pathogen, Yersinia pestis, and are highly specific for phosphorylated T-14 and Y-15 in CDKs [63-671.
In animal cells, Cdc25A is phosphorylated
and activated by cyclin E-CDK2 at the initiation
of DNA synthesis [68,69]. It is not clear whether
Cdc25A in turn activates more cyclin E-CDK2
in a positve feedback loop, although this seems
likely. There are also reports that Cdc25A interacts with Rafl, and that both Cdc25A and
Cdc25B are able to co-operate with Ras in cell
transformation [70]. T h e point of action of
Cdc25B in the cell cycle is not yet known.
At mitosis, Cdc25C activates cyclin B-cdc2
complexes, after cdc25 is itself activated by phosphorylation. Cdc25 is a cyclin B-cdc2 substrate,
so the phosphatase and kinase form a positive
feedback loop that ensures rapid activation of a
pool of cyclin B-cdc2 at mitosis [71] (reviewed
in [72]). However, the identity of the kinase that
I996
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20
initially activates Cdc25 at the beginning of mitosis is not known. Cdc25C is recognized by the
MPM-2 monoclonal antibody [73], which recognizes a mitotic phosphorylation epitope [74].
Therefore Cdc25C may be activated by one of
the two partially characterized MPM-2 kinases,
neither of which is a cyclin-CDK kinase [75].
T h e weaker kinase appears to belong to the
mitogen-activated protein (MAP) kinase family,
which ties in with the regulation of the metaphase-anaphase checkpoint by MAP kinase.
CDK inhibitors
T h e most topical means of regulating cyclinCDK activity is through the CDK-inhibitor proteins (CDI or CKI). An in-depth review of the
CDIs is beyond the scope of this lecture, so for a
more detailed treatment see [76,77].
CDIs are usually small (15-27 kDa) proteins that bind and inactivate specific cyclinCDK complexes, or in some cases monomeric
CDKs. All of the inhibitors isolated from animal
cells appear to be primarily concerned with
signal transduction and the decision to senesce,
quiesce or differentiate. In contrast, the rum1
and SIC1 inhibitors in fission and budding yeast,
respectively, are concerned with the proper coordination of DNA replication and mitosis. No
doubt homologues of these yeast proteins will
soon be identified in multicellular organisms.
One candidate protein is Roughex in Drosophila
cells, which is required to establish a G1 phase
as well as being required to prevent an extra M
phase after meiosis I1 [78,79].
Mammalian CDK inhibitors
Two families of CDK inhibitors have been isolated from animal cells so far. T h e members of
the Ink4 family, p16'"K4a [80], ~ 1 5 ' " " [81],
~~
~ 1 8 ' " ~[82,83]
~ " and p19"K4d [83], are each composed of four ankyrin repeats and are closely
related in sequence. T h e genes encoding p16
and p15 are adjacent on the 9p12 locus [84].
Members of this family only bind CDK4 and
CDK6 and appear to compete for binding with
the D-type cyclins. p16 is a potential tumour
suppressor gene; it is rearranged, deleted or
mutated in a large number of tumour cell lines,
and in some primary tumours [84, 851, and p16
is able to counteract Ras-induced cell transformation [86]. T o some extent it appears that the
loss or mutation of either p16 or Rb has a similar
effect in deregulating the D-type cyclins. p15
appears to be on one pathway through which
Volume 24
some cells arrest in response to transforming
growth factor B (TGFB). For example p15
mFWA and protein levels are induced more that
30-fold when HaCaT cells are exposed to TGFB
[81]. Like p16, p18 appears to require Rb to
arrest the cell cycle [82], whereas p19 does not.
T h e levels of p19 appear to vary during the cell
cycle, peaking as cells enter S phase [83].
T h e second family of inhibitors comprises
P21("P'MIAFI, ~ 2 7 ~ "and
' p57K'pZ[77]. ~ 2 7 ~ ' is"
another effector of cell-cycle arrest in response
to TGFB, and seems to be present in proliferating cells in a latent or masked form [87,88].
Several stimuli in addition to TGFB are able to
unmask p27, including cell-cell contact [87] and
cyclic AMP treatment of macrophages [89]. Once
unmasked, p27 binds and inhibits the cyclin ECDK2 complex, although in proliferating cells
most p27 is bound to cyclin D-CDK4/6 complexes [90]. p27 may inhibit cyclin D-CDK4
through preventing the phosphorylation of the T
loop threonine residue in CDK4 (T-172) by CAK
[89]. Increasing the amount of p27 (cyclic AMP
treatment of macrophages) or decreasing the
amount of CDK4 (TGFB treatment) would both
block cells in G1 phase [77]. Conversely, in Tlymphocytes, interleukin 2 treatment depresses
p27 levels, while antigen stimulation increases
cyclin D2 and D3 synthesis, causing T-cells to
proliferate [91]. p27 is closely related to p57 in
its N- and C-terminal sequences, but p57 has in
addition one large proline-rich and one acidicrich domain in the middle of the protein.
Hybridization in situ shows that both p27 and
p57 are most abundant in post-mitotic, differentiated tissues, such as muscle and brain,
although p57 is more restricted in its distribution
[92,93]. p57 also maps to llp15, a chromosomal
locus that is re-arranged in many tumours [93].
Like ~ 2 7 ~ " p57K'1'2
",
is able to inhibit cyclin D-,
cyclin E- and cyclin A-associated kinases, and
does not require Rb or wild-type p53 to induce
G 1 arrest when transfected into tissue-culture
cells.
T h e N-termini of p27 and p57 have strong
similarity to that of p21 [87,90]. p21 forms a
ternary complex with PCNA (a subunit of DNA
polymerase 6) in several cyclin-CDK2 complexes, including cyclins A, D1 and E, and p21
also binds, more weakly, to CDKl and CDK3
[94-961. T h e exact mechanism by which p21
inhibits cyclin-CDK kinase activity is not clear.
Unlike p27, it does not appear to affect the phosphorylation state of the CDK, and more than one
Colworth Medal Lecture
p21 molecule needs to bind to a cyclin-CDK
complex in order to inhibit protein kinase activity
[97]. An analysis of p21 mRNA in growing, quiescent and senescent cells correlates with a role
as a negative regulator of entry into S phase. p21
mRNA is up-regulated as cells become senescent
or quiescent, and after serum stimulation of quiescent cells, and decreases as cells enter S phase
[981.
Overexpressing p21 suppresses growth in
various human tumour cell lines, and inhibits
DNA synthesis in non-transformed cells [94].
T h e negative effect of p21 on DNA replication in
normal cells can be overcome by overexpressing
SV40 T antigen, and in T antigen-transformed
cells p21 is absent from cyclin-CDK complexes
[96]. T h e p53-dependent G1-phase arrest in
response to UV damage appears to be effected
through p21. p21 is induced by wild-type, but not
mutant, p53 [99], and cells irradiated in G1
phase require wild-type p53 in order to arrest the
cell cycle before S phase [loo], although there is
some controversy over whether cells arrest transiently, or indefinitely in a state resembling senescence [ l o l l .
In an SV40 T antigen-dependent replication
system in zlitro, p21 is able to inhibit DNA replication without the mediation of cyclin-CDK
complexes, by binding to PCNA [102]. Furthermore, DNA excision repair, which requires
PCNA, is not sensitive to p21 inhibition [103].
T h e C-terminus of p21 has been shown to be
responsible for inhibiting PCNA in vztro
[104,105], but it remains to be seen whether p21
ever acts through
inhibiting PCNA in vivo. On
the one hand, in Xenopus egg extracts, exogenous
p21 inhibits DNA replication exclusively through
inhibiting cyclin-CDKs [ 104,105], such that
replication can be restored by the addition of
cyclin E or cyclin A kinase [lob]. On the other
hand, tissue-culture cells are arrested when
transfected with either the amino or the carboxyl
half of p21 [lo71 (Figure 4).
As yet a specific inhibitor for the cyclin 0cdc2 complex has not been identified, although
p21 will weakly inhibit this kinase [94,95]. One
indication that there is an inhibitor for the
mitotic form of cdc2 is that purified frog MPF
(essentially, but not exclusively, cyclin B-cdc2)
is inactivated when added to an interphase
extract in a manner independent of T-14B-15
phosphorylation of cdc2 [108].
Thus we have some understanding of how
cyclins activate their partner CDKs, and of how
the cyclin-CDK complex can be further modulated by phosphorylation and by binding inhibitors. Progress is also being made in dissecting
the other defining feature of a cyclin, its rapid
cell-cycle-dependent destruction. Indeed, the
cell-cycle-dependent destruction of specific proteins, including both CDK activators (cyclins)
and inhibitors, is one of the most conserved
Figure 4
Roles of CDK inhibitors in the control of cell fate
The putative roles of the different CDK inhibitors in the decision made at R (restriction point) whether to proceed to DNA replication, arrest
in G I , senesce or differentiate
DNA damage
uv
G1
w S -G2
M
PCNA
Rb
-.\,
Senescence
Dlfferentiation
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Biochemical Society Transactions
features of cell-cycle control, and is central to the
proper regulation of DNA replication and mitosis.
22
Cyclin destruction
Cyclins can be divided into the same two classes
according to where they act in the cell cycle (G1
or G2 phase) or to the character of their destruction. T h e G1 cyclins ( D and E) are constitutively
unstable through the cell cycle, and their level is
therefore determined by the rate of their transcription. By contrast, the G 2 cyclins (A and B)
are unstable in only one phase of the cell cycle,
mitosis.
Cyclin proteolysis: 'destruction boxes'
and ubiquitin
T h e original cyclins, A and B, are stable throughout interphase and are specifically destroyed at
mitosis. This property is conferred by a partially
conserved destruction box in the N-terminus of
the protein [109]. T h e destruction box almost
certainly marks cyclins for degradation at mitosis
through ubiquitination [ 109,1101. On its own the
destruction box is not necessarily sufficent to
mark the cyclin for destruction, because both
cyclin A and cyclin B2, and in some circumstances, cyclin B1, need to be able to bind to
their CDK partner in order to be degraded
[34,111].
T h e ubiquitin degradation pathway involves
up to three enzyme components to charge and
transfer the ubiquitin moiety (reviewed in [ 1121)
(Figure 5). Ubiquitin is transferred to the substrate protein either by a ubiquitin-conjugating
enzyme (UBC) alone or by a UBC in conjunction
with a ubiquitin ligase. Cyclins are recognized by
particular UBCs in yeast, and a potential cyclinspecific UBC has been isolated from clam
oocytes [ 1131. How specific any one UBC is for a
particular motif is unknown. There could be a
cyclin A-specific and a cyclin B-specific UBC,
because the sequence of the destruction box
varies between the A- and B-type cyclins, and the
A-type cyclins begin to be destroyed in metaphase, whereas the B-types are destroyed when
cells enter anaphase [ 114-1 181. In budding yeast
the degradation of the S-phase-specific Clb5,
perhaps analogous to cyclin A, and the mitosisspecific Clb2 cyclin, analogous to cyclin B, both
require the product of the UBC9 gene [119].
However, in a reconstituted system using Xenopus components, the frog UBC9 homologue did
not cofractionate with the E2 responsible for
cyclin destruction. A homologue of yeast UBC4
did cofractionate, and bacterially expressed
human UBC4 was able to ubiquitinate sea urchin
cyclin B with the other components of the reconstituted system.
T h e other major component involved in the
Figure 5
The control of cyclin destruction by ubiquitination
Shown are the E I , E 2 (UBCS in budding yead, UBC4 in frogs) and E3 (APC/cyclosorne) involved in mitotic cyclin destrudion, and the E 2
(Cdc34) involved in G I regulation in budding yeast. Ubq, ubiquitin, APC, anaphase-promotingcomplex
B
ATP
+@
( T > S "
s
H
(,W
ADP+Pi
\/,
(-q-G&-d(zJ-@
UBC (6g CDC34. UBCS
8 frog UBC4)
pzJ+@
CDC16.23.27
(TPR proteins)
-7-
Cvclin B-CDK
DESTRUCTION
26 S Proteosome
Volume 24
Cyclosome
Colworth Medal Lecture
ubiquitination of cyclin is a specific ubiquitin
ligase that is activated in mitosis, probably by
phosphorylation. This is a very large ( > 1 MDa)
multiprotein complex called the anaphase promoting complex (APC) or the cyclosome
[113,120-1221. B-type cyclins trigger their own
destruction when they activate Cdc2 at mitosis,
whereas A-type cyclins are unable to activate
their own or B-type cyclin destruction [115]. In
agreement with this, in a reconstituted system
from clam oocyte extracts, the APC/cyclosome is
only active in mitotic extracts, but can be stimulated in interphase by cyclin B-cdc2 kinase
[113]. Three conserved components of the APCI
cyclosome have been identified from fractionated
Xenopus extracts, yeast and human cells. They
are tetratricopeptide-repeat proteins that are
encoded in budding yeast by the CDC16, CDC23
and CDC27 proteins [120-1221. Yeast that are
mutant in CDC16 and CDC23, or in a nuclear
import protein (CSEl), are unable to degrade Btype cyclins and therefore arrest in mitosis [121].
T h e human CDC16 and CDC27 proteins have
been shown to associate with the spindle and the
centrosome [ 1201, which is almost certainly connected with the observation that B-type cyclin
destruction is sensitive to the integrity of the
mitotic apparatus at the end of metaphase. If the
spindle is incorrectly assembled, or chromosomes are incorrectly aligned, then B-type cyclin
destruction is inhibited. T h e MAP kinase ERK2
is part of the mechanism that prevents cyclin B1
destruction when the cell arrests at metaphase in
adverse conditions [ 1231. Spindle integrity is
assayed through the tension on the microtubules
created by the attachment of kinetochores to
microtubules [124,125].
Ubiquitinated proteins are degraded by the
2 6 s protease, which is made up of a core 2 0 s
proteosome particle and various ATPase subunits. A yeast with a mutation in one of the
ATPase subunits is known to arrest cells in
mitosis through an inability to degrade B-type
cyclins, because the mutation can be suppressed
by a mutation in the B-type cyclin encoded by
CLB2 [126,127].
Other proteins are also specifically degraded
in mitosis. Some, such as the unidentified components that link sister chromatids together
[127a], are degraded at the same time as the
cyclins, and their degradation can be competitively inhibited by a cyclin substrate. Others,
such as CENP-E [128], are degraded later in
mitosis, suggesting that there are specific waves
of protein destruction as cells move through
mitosis.
In yeast the B-type cyclins remain unstable
through G1 phase until G1 cyclin-CDK (ClnCdc28) activity appears at START [129], suggesting that the G1 cyclin-CDK protein kinases
turn off B-type cyclin destruction. There are
indications that this holds true in the animal cell
cycle too. Ectopic expression of the G1 cyclin,
cyclin E, in Drosophila is sufficent to cause postmitotic G1 cells to undergo another round of
DNA replication and cell division [130]. In these
cells, ectopic cyclin E was sufficent to stimulate
the accumulation of the mitotic cyclins A and B
with no increase in their mRNA levels, suggesting that cyclin E stabilizes cyclin A and B by
shutting off the proteolytic machinery [ 1301.
G I cyclin proteolysis: PEST sequences
and ubiquitin
T h e human D- and E-type cyclins are short-lived
proteins, as are the Cln proteins in budding
yeast. T h e short half-life ( - 20 min) is due to
PEST sequences in the C-terminal regions of
the proteins; removing the PEST sequences
stabilizes the proteins [ 131- 1331. There is some
debate over whether PEST regions confer
instability directly or not, and the biochemical
basis for the degradation of proteins containing
PEST regions has not yet been elucidated. However, recent evidence shows that yeast G1 cyclin
destruction, like that of the G2 cyclins, is mediated by ubiquitin [ 1341.
In yeast the cyclin-dependent kinase inhibitors Sicl and Farl are destroyed once cells pass
START, the point in G1 phase when cells are
committed to the mitotic cell cycle. Farl inhibits
the G1 cyclin-CDK complex (Cln2-Cdc28)
[135-1371 in response to activation of the MAP
kinase pathway, and may therefore be the paradigm for p21 and p27. Sicl inhibits the S-phase
cyclin-CDK complex (Clb5-Cdc28) [ 1381 to
prevent cells from entering prematurely into S
phase, and is therefore involved in co-ordinating
the cell cycle proper. Sicl is recognized by the
UBC product of the CDC34 gene, and there are
indications that both Sicl and Farl are only
degraded in their phosphorylated form.
Thus cell-cycle control appears at heart to
be due to the sequential destruction of different
sets of cell-cycle regulators at specific phases of
the cell cycle [129]. Sequential waves of proteolysis could in part be achieved by activating the
I996
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Biochemical Society Transactions
ubiquitin-conjugating machinery only at particular points in the cell cycle, perhaps through
phosphorylation by cyclin-CDK kinases.
24
(Figure 6) are due in part to proteolysis and in
part to transcription.
G I cyclins
The importance of transcription (but
you won’t find the details here)
In addition, there is a complex interplay between
the transcription machinery and the cyclinCDKs. This has been most clearly defined in
budding yeast, where G1 cyclin complexes form a
positive feedback loop with the SBF (Swi4/Swi6
cell cycle box factor) and MBF (MluI cell cycle
box factor) transcription factors to regulate G1phase and S-phase entry. Later in the cell cycle,
the G2 cyclins (Clbs) activate their own transcription and turn off G1 cyclin transcription
([139]; reviewed in [16]). T h e interplay between
transcription factors and the cell-cycle machinery
in human cells is equally important, but much
less exactly defined. T h e E2F family of transcription factors has been strongly implicated in
controlling the induction of S phase (see below),
but the interplay between these factors and
cyclin-CDK complexes is much less clear. Later
in the cell cycle, the transcription of G2-phase
components such as Cdc25C and cdc2 appears to
be under the control of a repressor that binds to
a specific repressor element [ 1401.
Thus the waves of cyclin-CDK activity that
appear sequentially through the cell cycle
So, how do the plethora of cyclins, CDKs and
CDK inhibitors act to regulate the human cell
cycle? Different cyclin-CDK complexes have
essential roles in the transition from one cellcycle state to another, most importantly the initiation of DNA replication or cell division, and also
in the integration of cell progression with other
cell fates such as differentiation, senescence and
quiescence.
T h e D-type cyclins ([141]; reviewed in
[ 1421) are most closely connected with a control
point in mid-late G1 phase called the Restriction
point (R) [143], which is the point at which cells
respond to the presence of serum by commitment to the mitotic cell cycle, or to the absence
of serum by becoming quiescent. This is also
probably the point at which cells exit the cell
cycle when they differentiate or become senescent. T h e D-type cyclins are especially suited to
a role in signal transduction, because their transcription is serum dependent. In the absence of
serum, their short half-life means that the Dtype cyclins rapidly disappear.
There are three D-type cyclins in mammalian cells, which are cell-type specific [141].
Cyclin D1 is the Bcl-l/PRADl proto-oncogene
Figure 6
Periodic cyclin-CDK activity throughout the cell cycle
Illustrated are the kinase profiles for the D-. E-, A- and B-associated CDKs. The inputs from
cyclin transcription and from the Cdc25 family of phosphatases are also shown.
G1
Volume 24
S
G2
n
M
G1
Colworth Medal Lecture
product [ 144,1451, and the overproduction of
cyclin D1 through gene amplification or mRNA
stabilization has been correlated with several
types of cancer (reviewed in [76,146,147]). T h e
exact role of the D-type cyclins in the proliferating cell cycle is not clear, but cyclin D1 only
appears to perform an essential function in cells
that have a functional Rb protein [148].
D-type cyclins bind to several different
CDKs, CDK2, CDK4, CDK5 and CDK6
[149-1521, of which the main partners appear to
be CDK4 [149] and CDK6 [151,152]; in many
cell types CDK2, CDK5 and CDK6 are not
associated with cyclin D [151]. T h e cyclin DCDK4 complex is unusual because it forms for
only a short period in the cell cycle, at R through
to early S phase [149], even though cyclin D and
CDK4 remain at almost constant levels in cycling
cells. Thus part of the control of R is through
regulating the association between cyclin D and
CDK4, for example through the effect of p27 on
the phosphorylation of the T loop threonine in
CDK4. CDK4 synthesis itself is also subject to
regulation by negative growth factors such as
T G F P [153].
Cyclin D-CDK4
complexes have a
restricted substrate specificity. These include the
E2F family of transcription factors [ 1541,
thought to regulate the cell-cycle-dependent synthesis of proteins required for S phase, such as
DNA polymerase a, thymidylate synthetase and
ribonucleotide reductase, as well as cyclins E and
A (reviewed in [155,156]). E2F is a dimer composed of a member of the E2F family [157,158]
(five different cDNAs have been isolated) and a
member of the DP family [159,160] (of which
three cDNAs have been found so far).
T h e second substrate identified for the Dtype cyclin-CDK complexes is Rb [161]. Rb is
under-phosphorylated in most of G1 phase,
phosphorylated at or after R, and remains phosphorylated until late mitosis [162,163]. T h e
hypophosphorylated form of Rb is able to block
cells in G1 phase, and it binds, and potentially
sequesters, a large number of proteins, including
E2F. T h e D-type cyclin-CDK complexes may
phosphorylate and inactivate Rb in mid-late G1
phase. In vztro, the D-type cyclins are able to
bind to Rb through an L-X-C-X-E
motif in
their N-terminus [ 164- 1661.
T h e role of Rb itself in the cell cycle is
contentious. An Rb- mouse undergoes multiple
normal rounds of cell division and considerable
tissue differentiation until day 13, at which point
it dies because of problems in the development
of the liver and blood system [167-1691. This
suggests that Rb is only important for a subset of
cell differentiation, notably haematopoeisis and
neurogenesis, rather than regulating the cell
cycle. Rb probably acts to arrest the cell cycle at
R, by sequestering or inactivating transcription
factors such as E2F that are involved in the synthesis of genes required for DNA replication.
Differentiation and transformation
T h e D-type cyclins and Rb are important in the
switch between proliferation and differentiation,
and there are also some data to suggest that
CDK4 needs to be down-regulated in order to
allow differentiation [ 165,1701. In the 32D myeloid cell line cyclins D2 and D3 are normally
expressed in a growth-factor-dependent manner
[171]. However, if the cells are transfected with
either cyclin D2 or D3 under a constitutive promoter, cells are unable to differentiate. T h e constitutive expression of cyclin D1 has no effect on
their differentiation, nor does expression of
cyclin D2 and D3 mutants that are unable to
bind Rb [171]. In an analogous fashion, ectopic
cyclin D1 inhibits the differentiation of the
C2C 12 myoblast cell line, apparently through
inhibition of MyoD [172], and this can be overcome by ectopic p21. p21 and MyoD could form
a positive feedback loop in muscle differentiation, because p21 is induced by MyoD. Thus, as
muscle cells begin to differentiate, MyoD could
increase p21 levels, inhibiting cyclin Dl-associated kinase activity, and allowing MyoD to
increase transcription of p21 as well as musclespecific genes [172-1741. However, MyoD is not
solely responsible for the induction of p21,
because myoblasts are able to induce p21 synthesis and differentiate in mice lacking both MyoD
and myogenin [ 173,1741. A correlation between
p21 transcription and differentiation has also
been observed in other cell types, such as cartilage and epithelium [174].
These observations may provide an explanation for the ability of D-type cyclins to act as
proto-oncoproteins; if their deregulation signalled the cell to proliferate rather than differentiate, then this is one of the conditions necessary
for cellular transformation [ 1751. Cyclin D
expression would therefore be expected to cooperate with other oncoproteins in cellular transformation, and indeed cyclin D 1 will co-operate
with Myc in transgenic mice [176], and withRas
and a defective E1A protein in cultured cells
I996
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Biochemical Society Transactions
26
[ 1771. For a comprehensive review of the connections between cyclins and oncogenesis see
[76,1781.
In proliferating cells, CDK5 is bound to the
D-type cyclins but has little detectable kinase
activity. However, in post-mitotic neurons, CDK5
takes on the role of a neurofilament kinase [179].
Interestingly, its partner is p35, a protein with
very limited homology to cyclins [180,181]. It will
be interesting to solve the crystal structure of
CDK5, and of the p35-CDK5 complex, to determine whether p35 activates CDK5 in the same
way as a cyclin.
[ 1891. The relationship between E2F and cyclin
E looks to be conserved in evolution because
mammalian cyclin E (and cyclin A) synthesis is
strongly stimulated by ectopic E2F-1 [191].
Further evidence for a requirement for
cyclin E-CDK2 to initiate S phase is that in
Xenopus egg extracts almost all the CDK2 is
bound to cyclin E, and DNA synthesis is blocked
when CDK2 is depleted [192]. Similarly a dominant negative form of CDK2 will inhibit the initiation of DNA replication in mammalian cells,
blocking cells in G1 phase, at which point CDK2
is primarily bound to cyclin E [193].
Cyclin E and the G I to S transition
Cyclin A, the M&S cyclin
Once cells have entered S phase, the predomi-
The other G1 cyclin that has been identified in
human cells is cyclin E. Overproducing either Dtype cyclins or cyclin E moderately shortens G1
phase in mammalian cells [ 182- 1841, whereas
overproducing both D- and E-type cyclins significantly shortens G1 phase [185]. This suggests
that the two types of cyclin regulate different
aspects of G1 phase.
The cyclin E-CDK2 complex appears after
the D-types [186,187], such that there is a burst
of cyclin E transcription only in late G1 and early
S phase. Additionally, cyclin E-CDK2 protein
kinase activity may be modulated by Cdc25A
phosphatase at the end of G1 phase [69]. The
cyclin E-CDK2 complex is essential for the cell
to begin DNA replication. The best evidence for
this comes from studies on developing Drosophila embryos. In Drosophila embryogenesis, the
disappearance of cyclin E transcripts after mitosis 16 causes cells to stop dividing and arrest in
G1 [ 1301. Some cells go on to complete endoreplication (DNA synthesis without cell division)
after cycle 16, and the presence of cyclin E transcripts correlates exactly with cells that are capable of endoreplication [ 130,1881. Furthermore,
cells of a Drosophila mutant in cyclin E are
unable to enter S phase after the maternal store
of cyclin E has been exhausted.
A homologue of the mammalian E2F transcription factor has been isolated from Drosophila,
and one of its target genes is cyclin E [189,190].
Moreover, ectopically expressed cyclin E is able
to overcome the cell-cycle arrest caused by a
defective E2F gene [189]. Thus it appears that
cyclin E is one of the major limiting downstream
targets of E2F in the initiation of S phase. Conversely, E2F is unable to rescue a defect in cyclin
E, so cyclin E-associated kinase activity must be
required for more that just the activation of E2F
Volume 24
nant cyclin-CDK complex that is active through
to the end of G2 phase is cyclin A-CDK2
[194-1961. Cyclin F is also active at this point,
and, although its substrates have not been identified, anti-sense data suggest that cyclin F is
required for entry into M phase [197]. A continuing debate over cyclin A is whether it has a role
in initiating S phase, or whether it only maintains DNA synthesis once S phase has been initiated by cyclin E. If cyclin A is artificially
produced in G1 phase, this has the effect of
advancing S phase [198]. However, the cyclin A
promoter normally begins to be transcribed only
in late G1 or the beginning of S phase, so
whether there is normally enough cyclin A in late
G1 to contribute to the initiation of DNA replication is unclear.
S-phase substrates for cyclin A have been
reported to include E2F-1, which is apparently
unable to bind DNA after phosphorylation
[199,200]. Thus a model has been proposed by
which cyclin E activates E2F-1, and thus initiates
the transcription of genes required for DNA synthesis in late G1 phase, and cyclin A turns E2F
off once cells are in S phase. Cyclins A and E
also bind to the Rb-related proteins p107 and
p130 [201,202], and recent data suggest that this
interaction may be the means by which another
member of the E2F family, E2F-4, is regulated
[203]. In G1 phase a minor proportion of E2F-4
[204,205] and p107 is found in a complex with
cyclin E-CDK2, and in S phase a larger proportion of E2F-4 and p107 associates with cyclin ACDK2 [201,206,207]. Activated cyclin A-CDK2
causes p107 to dissociate from E2F-4, but p107
appears to be able to inhibit cyclin A-CDK2
activity in an analogous fashion to p21 or p27;
furthermore, these three proteins share a small
Colworth Medal Lecture
region of similarity and bind to cyclin-CDKs in a
mutually exclusive fashion [203]. Thus it has
been proposed that p107 may function as an
inhibitor of cyclin-CDK activity as well as to
target cyclin-CDK complexes to E2F [203].
T h e role, if any, of cyclin A in DNA replication itself is also contentious. Cyclin A co-localizes with origins of replication [208,209], and
cyclin A-CDK, as well as cyclin E-CDK2, complexes are able to rescue DNA replication in cellfree extracts from Xenopus eggs that have been
inhibited by ~21"'"' [ 1061. However, neither
Drosophila embryos nor an in vitro SV40 T-antigen-dependent system need cyclin A to replicate
DNA [210-2121. In the latter case, this indicates
that if cyclin A-associated kinase activity is
required, then it is only needed for origin
unwindinglinitiation and not elongation.
T h e G2 phase substrates of cyclin A-CDK2,
and of cyclin A-cdc2, which forms later in G2
phase, also remain obscure. It is clear from an
analysis of Drosophila embryos mutant in cyclin
A that this cyclin plays an essential role in the
initiation of mitosis. Once embryos have
exhausted their supply of maternal cyclin A, all
the cells arrest in G2 phase, having completed
DNA replication [211]. Cyclin A-CDK complexes may be involved in the initiation of spindle
assembly, because in Xenopus cell-free extracts
cyclin A-CDK kinase activity causes centrosomes to nucleate arrays of microtubules
[213,214].
Cyclin B: the mitotic cyclin
T h e cyclin with the clearest cell-cycle role is the
'original cyclin', cyclin B. Cyclin B, in partnership with cdc2, is the primary mitotic kinase, and
a large number of mitotic substrates have been
proposed for it (reviewed in [215]). These
include both downstream mitotic kinases such as
NIMA [216,217] and karyoskeletal and cytoskeletal elements such as the nuclear lamins
[218], actin-binding proteins [219,220], and
microtubule-associated proteins [221]. Thus
cyclin B-cdc2 is both a regulator and a 'workhorse' of mitosis [215]. As outlined above, the
activation of cyclin B-cdc2 is very carefully regulated by phosphorylation and dephosphorylation
of cdc2, through the weel/mikl kinases and
Cdc25C phosphatase. In addition, some of the
cyclin B-cdc2 complexes undergo cell-cycledependent translocation
to the nucleus
[ 116,222,2231, and my own research has focused
on this aspect of the cyclins.
Localization
A third feature of the cyclins, along with their
activation of CDKs and cell-cycle-dependent
destruction, is that cyclins appear to target their
partner kinases to particular sub-cellular compartments and substrates. Localization may be
one way in which the cyclins can confer substrate
specificity on their partner kinase. For example,
the nuclear cyclins A and E bind to p107 [201]
and p130 [202], and this means that cyclin ACDK2 phosphorylates p107 much more efficently than does cyclin B-cdc2 [224]. Also, the
NAP1 protein interacts specifically with mitotic
B-type cyclins in yeast and frogs, and is required
for the proper regulation of mitosis [225,226].
Localization is likely to be especially important
for the mitotic cyclin-CDK complexes that have
almost identical substrate specificities in vitro
[215]. My recent research has concentrated on
the A- and B-type cyclins to define their locations
in the cell, and to determine which parts of the
molecule are responsible for their localization.
Cyclins are mostly nuclear proteins, with the
exception of the metazoan B 1, B2 [ 116,222,2231
and Drosophila A-type cyclins [210]. Mammalian
cyclin B 1 [ 1161 and avian cyclin B2 [222] accumulate in the cytoplasm in G2 phase and translocate into the nucleus at the beginning of
mitosis. Similarly, in starfish oocytes the B-type
cyclin translocates into the germinal vesicle when
the oocytes are fertilized or activated [223]. Subsequently, cyclin B associates with the spindle
apparatus, in particular with the spindle caps
[ 116,222,2231, and this is congruent with the
behaviour of fission yeast cyclin B (cdcl3), which
associates with the spindle poles [227]. T h e
association of cyclin B with the spindle has at
least two implications. Firstly, it means that
cyclin B-cdc2 kinase may be involved in the formation of the spindle through phosphorylating
components of the mitotic apparatus. Secondly,
it would facilitate the feedback mechanism that
links cyclin B1 destruction to the correct assembly of the metaphase mitotic apparatus.
Human cyclin B2-cdc2 has an identical substrate specificity in vitro to cyclin B1-cdc2, and
its associated protein kinase activity is turned on
and off at the same time in the cell cycle. However, cyclin B2 differs strikingly from cyclin B1 in
its localization in human cells, in that it is almost
exclusively associated with the membrane compartment, and in particular the Golgi apparatus
[228]. This immediately suggests that cyclin B2cdc2 is involved in the disassembly of the Golgi
I996
27
Biochemical Society Transactions
28
apparatus when cells enter mitosis [229]. At
mitosis membrane traffic is inhibited, and data
from systems in vitro have shown that cdcZassociated protein kinase activity is able to
inhibit membrane fusion [230,23 11.
B-type cyclins are targeted to the
cytoplasm
T h e ‘default’ localization of a cyclin-CDK complex is the nucleus. Through a series of deletion
mutants and domain swaps, I have shown that
the cyclin box alone is nuclear [232], and Erich
Nigg and colleagues have shown that, in order to
be transported to the nucleus, the cyclin needs to
bind to its CDK [233]. T h e cytoplasmic localization of the B1 and B2 cyclins is determined by a
42-amino-acid region in the N-terminus
between the destruction box and the cyclin box
[232]. This region appears to act as a cytoplasmic anchor, because it is able to re-direct
cyclin A to the cytoplasm when fused to the Nterminus. T h e cytoplasmic anchor region of
cyclins B1 and B2 is partially conserved, and is
both necessary and sufficent to direct cyclin B1
to microtubules and cyclin B2 to the membrane
compartment of the cell. Ookata et al. [221] have
shown that one of the proteins that interacts with
cyclin B1 on microtubules is MAP4, and cyclin
B1 is able to phosphorylate MAP4, which appears
to increase the dynamic instability of microtubules in vitro. This is the behaviour of microtubules in mitosis and has also been shown to
occur when cyclin B1 kinase is added to Xenopus
extracts, where in addition it was shown to
depress microtubule nucleation on centrosomes
[213,214].
-
Future studies: cyclins in real time
Mitosis is the most dynamic part of the cell
cycle; most of the cellular architecture is reorganized and new structures such as the spindle
are formed. Some of the cyclin-CDK complexes,
such as cyclin B 1-cdc2, are themselves dynamic
in mitosis, translocating from the cytoplasm to
the nucleus and associating with the spindle.
Th u s one of our aims is to be able to observe
cyclins in living cells. T o this end I have created
fusion proteins between the cyclins and the naturally fluorescent 26 kDa Green Fluorescent Protein of the jellyfish Aequoriu victoria [234]. Our
studies have only just begun, but we have been
able to detect cyclin fusion proteins, and have
shown that they are localized to the same compartments as the endogenous cyclins [235]. T h u s
Volume 24
we hope to be able to unlock some of the secrets
of cell-cycle regulation by observing cyclins
throughout the cell cycle in living cells. Just one
more transition in the short but eventful life of
the cyclins: from a band on a gel, to a consensus
protein sequence, to a crystal structure, and now,
I hope, to a green light for cell-cycle research!
This lecture is dedicated to Dr. Tim Hunt, who introduced me to cyclin and the cell cycle, and not least to
John Elliot Gardner in a Metro over the Alps. True
inspiration.
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Received 22 August 1995
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