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Control of the Cell Cycle in Early Embryos
J. RUDERMAN,* F. LUCA,* E. SHIBUYA,* K. GAVIN,*
T. BOULTON,* AND M. COBBt
*Department of Anatomy and Cell Biology, Harvard Medical School, Boston, Massachusetts 02115;
tDepartment of Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas 75235
At the end of oogenesis, oocytes exit from the cell
cycle and arrest at the G2/M border of meiosis I. In
clams, fertilization provides the extracellular signal that
breaks this cell cycle arrest and initiates a series of rapid
cell division cycles. In this paper, we consider two
aspects of this process: the rapid, one-time activation of
a member of the ERK/MAP kinase family that appears
to be a key player in breaking cell cycle arrest, and the
repetitive role of cyclin accumulation and destruction in
driving cycles of cdc2/28 activation and inactivation.
Oocytes of marine invertebrates provide excellent
material for identifying some of the molecules involved
in regulating the cell cycle. The full-grown oocytes
contain large stockpiles of virtually all the enzymes and
structural proteins needed to sustain the rapid, postfertilization cleavage division cycles, yet they remain
arrested. Their responses to fertilization are abrupt,
synchronous and, most important, dramatically exaggerated. This makes it easier to spot important regulatory components, ones that are often barely detectable
in tissue culture cells. Indeed, the cyclins were first seen
in clam embryos, where their synthesis is turned on
strongly within minutes of fertilization (Rosenthal et al.
1980). The discovery that their levels oscillated across
the cell cycle (Evans et al. 1983) focused attention on
potential cell cycle roles for these proteins. Early work
established that the rise and fall in cyclin levels in the
embryonic cell cycles are due to continuous synthesis
followed by a very brief interval of selective proteolysis
(Swenson et al. 1986; Standart et al. 1987; Minshull et
al. 1989a; Hunt et al. 1992). The levels of cyclin A and
cyclin B show offset kinetics of cycling, with cyclin-A
levels dropping in mid-mitosis and cyclin-B levels falling several minutes later, just at the metaphase/anaphase transition (Westendorf et al. 1989).
It is now well established that, during the abbreviated cell cycles of early embryos, it is the rise in
cyclin that drives cells from interphase into mitosis. The
first direct evidence for this role came from the demonstration that the introduction of cyclin A into frog
oocytes, which are arrested at the G2/M border of
meiosis, caused those cells to enter M phase and resume the meiotic cell cycle (Swenson et al. 1986).
B-type cyclin also had the same effect (Westendorf et
al. 1989). Further support for an M-phase-promoting
role came from the demonstration that the depletion of
cyclin mRNA (via antisense constructs) prevented cellfree extracts from entering mitosis (Minshull et al.
1989a,b). The development of an mRNA-dependent
cell-free system from frog embryos allowed Murray and
Kirschner (1989) to establish definitively that the synthesis of a single protein, cyclin B, is sufficient to drive
multiple cycles of certain cell cycle events, including
nuclear envelope breakdown and reformation, chromosome condensation and decondensation, and the rise
and fall in histone H1 kinase activity. Finally, genetic
studies in yeast revealed a gene, cdc13 +, required for
entry into mitosis whose sequence placed it firmly in the
cyclin-B family (Booher and Beach 1988; Goebl and
Byers 1988; Solomon et al. 1988).
Cyclins exert their effect by binding to and activating
the protein kinase cdc2/28. Originally identified in genetic studies of yeast (Hartwell et al. 1974; Nurse and
Bissett 1981), cdc2/28 encodes a 34-kD protein kinase
(Reed et al. 1985) that is required at least twice in each
cell cycle, once in G 1 for commitment to the cell division cycle and again in G 2 for entry into mitosis (for
review, see Ghiara et al. 1991; Surana et al. 1991; see
also other papers in this volume).
The M-phase role of cdc2/28 is best understood: It is
the catalytic subunit of a multiprotein complex called
MPF (for maturation- or M-phase-promoting factor).
MPF was discovered as an activity in metaphase-arrested frog eggs that, when transferred by microinjection
into interphase-arrested oocytes, would drive those
cells into M phase (Ecker and Smith 1971; Masui and
Markert 1971). MPF activity was subsequently found to
cycle during each meiotic and mitotic cell cycle; new
protein synthesis was required in each cell cycle to
generate the rise in MPF activity and entry into mitosis
(Wasserman and Smith 1978; Wagenaar 1983; Gerhart
et al. 1984). Partially purified preparations of MPF
from frog and starfish eggs, which displayed a high
kinase activity with strong preference for the substrate
histone H1 (Lohka et al. 1988), were discovered to
contain a 34-kD protein kinase representing the
homolog of cdc2/28 (Arion et al. 1988; Dunphy et al.
1988; Gautier et al. 1988; Labb6 et al. 1988). The
connection with cyclins was fueled by the observation
that monomeric cdc2/28 by itself is inactive, whereas
the active form associates with other proteins (Reed et
al. 1985; Draetta and Beach 1988). The nature of the
cdc2/28-associated proteins was first identified by
Draetta et al. (1989), who found that newly synthesized
cyclin in clam embryos binds to and activates cdc2/28,
forming independent complexes of cyclin A-cdc2/28
Cold Spring Harbor Symposia on Quantitative Biology, VolumeLVI.9 1991 Cold Spring Harbor LaboratoryPress 0-87969-061-5/91$3.00
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RUDERMAN ET AL.
and cyclin B-cdc2/28, each of which displays high H1
kinase activity. This discovery led to the proposal that
the cyclins act as positive regulatory subunits for cdc2/
28, the catalytic subunit of MPF, and that the periodic
rise and fall of MPF activity is due to the periodic
accumulation and destruction of the cyclins across each
cell cycle (Draetta et al. 1989). This view has now been
amply confirmed (see, e.g., Labb6 et al. 1989; Murray
and Kirschner 1989; Pines and Hunter 1989; Gautier et
al. 1990; Solomon et al. 1990).
It is thought that cyclin A and cyclin B, as well as the
more recently discovered cyclin types, confer some sort
of specificity on the timing of cdc2 activation, its substrate specificity, or its localization. Uncovering these
differences has been hampered by the fact that cyclins
A and B can substitute for each other in virtually all
assays tried so far. For example, both can induce
meiotic maturation (Westendorf et al. 1989), phosphorylate the same sites on histone H1 (Minshull et al.
1990), and induce DNA synthesis in G~ lysates
(D'Urso et al. 1990). However, a good case can be
made for the idea that cyclin B targets cdc2/28 for its
functions in mitosis: Cyclin B is high in M-phase cells
(A is low or absent), it is present in partially purified
MPF (A is not), and loss-of-function cyclin-B mutants
in yeast arrest just before they enter M phase (Booher
and Beach 1988; Minshull et al. 1989b; Westendorf et
ai. 1989; Kobayashi et al. 1991). The role of cyclin A is
more mysterious. The fact that it peaks slightly ahead
of cyclin B (Pines and Hunter 1989; Westendorf et al.
1989) and enters the nucleus well in advance of cyclin B
(Pines and Hunter, this volume) suggests that cyclin A
plays a role earlier in the cell cycle, with initiation of S
phase as an obvious candidate. The results of antisense
experiments further support this view (C. Brechot,
pers. comm.). However, the role of cyclin A may be
more complicated, as suggested by the finding that, in
Drosophila, a cyclin-A mutation arrests in G2, not in
G I (Lehner and O'Farrell 1989).
Up to now, most molecular work has concentrated
on establishing how the cyclin binding activates cdc2/28
and promotes entry into M phase (Draetta et al. 1989;
Murray et al. 1989; Solomon et al. 1990; Kumagai and
Dunphy 1991; Parker et al. 1991). Less effort has gone
into studying how cells exit from mitosis and enter into
the next interphase. The importance of programmed
cyclin destruction in driving this cell cycle transition is
suggested by two kinds of observations. First, when
mitotic exit is prevented by colchicine-mediated dispersion of microtubules, cyclin A drops on schedule, but
cyclin B remains high (Hunt et al. 1992). Second,
synthesis of a truncated, stable version of cyclin B in a
cell-free system keeps cdc2/28 hyperactivated and nuclei arrested in M phase (Murray et al. 1989; Glotzer et
al. 1991). To learn more about the regulatory processes
that control transitions from one cell cycle stage to
another, we have used a cell-free system (Luca and
Ruderman 1989) that reproduces many cell cycle
changes occurring in intact cells. These include the
activation of ERK/MAP kinase within minutes of fer-
tilization, the subsequent activation of cdc2/28, and the
programmed destruction of the cyclins at the end of
each cell cycle.
METHODS
The materials and methods used in these experiments have been described previously in the following
papers: Swenson et al. (1986), Shibuya and Masui
(1989), Draetta et al. (1989), Luca and Ruderman
(1989), and Boulton et al. (1991).
RESULTS AND DISCUSSION
Fertilization Leads to the Rapid Tyrosine
Phosphorylation of pp42 ERI</MApki....
and Its Activation
Clam oocytes are arrested at the G2/M border of
meiosis I. Like other oocytes, they contain a maternal
stockpile of cyclin B and cdc2/28 (Arion et al. 1988;
Draetta et al. 1989; Dunphy and Newport 1989; Westendorf et al. 1989) associated as an inactive complex
that can be isolated using p13 ~uc~beads (Fig. 1). Curiously, in clam oocytes, only a very small portion of the
inactive cdc2/28 is phosphorylated on tyrosine. Entry
into M phase is accompanied by shifts in the mobility of
cdc2/28 and changes in its pattern of tyrosine phosphorylation (Fig. 1). With the aim of following the
kinetics of this switch more carefully, we fertilized
oocytes, took 4-minute time points and followed
tyrosine dephosphorylation of cdc2/28 by blotting total
cell proteins with an antiphosphotyrosine antibody. As
shown in Figure 2, its dephosphorylation occurred
gradually rather than showing an abrupt switch preceding entry into first meiotic M phase (GVBD, or germinal vesicle breakdown) at 11 minutes after fertilization.
In contrast to this leisurely decline in cdc2/28 phosphotyrosine levels, a strong phosphotyrosine signal appeared on a 42-kD protein at 3-4 minutes, stayed high
for the duration of this experiment (20 min), and disappeared at anaphase of meiosis I (about 30 rain),
never reappearing during subsequent meiotic or mitotic
divisions (not shown). The speed of this response and
the fact that many cell-surface receptors contain
tyrosine kinase activities or can activate tyrosine kinases (for review, see Boulton et al. 1991; Ferrell et al.
199t) suggested that this might be a very early step in
the signal transduction pathway turned on by the binding of sperm to its receptor. The size of this protein and
its pattern of phosphorylation suggested that it might
be a member of the ERK (extracellular signal regulated
kinase) family identified in mammalian tissue culture
cells (Cooper et al. 1984; Boulton et al. 1990) and a
homolog of the 42-kD MAP kinase (mitogen-activated
protein kinase) activated during frog oocyte maturation
(Ferrell et al. 1991). In support of this idea, antisera
raised against rat ERK-2 recognized a 42-kD protein in
Downloaded from symposium.cshlp.org on May 12, 2016 - Published by Cold Spring Harbor Laboratory Press
CELL CYCLE CONTROL IN EMBRYOS
Figure 1. Oocytes contain a preformed cyclin
B-cdc2/28 complex that is modified after fertilization, pl3SUCl-bound material from lysates of
unfertilized oocytes (GV) or 15-min postfertilization embryos (GVBD) was electrophoresed
on an SDS-polyacrylamide gel, blotted to nitrocellulose, and reacted with a mix of cyclin B and
cdc2 antibodies (left) or antiphosphotyrosine
antibodies, which were generously provided by
Brian Drucker and Tom Roberts (Harvard
Medical School, Boston) (middle). The positions of cyclin B and cdc2/28 are indicated.
Three different cdc2 bands (1, 2, and 3) can be
distinguished by slight differences in their electrophoretic mobilities and reactivity with antiphosphotyrosine antibodies, pl3SUCl-bound
material from embryos (GVBD) and oocytes
homogenized at the prefertilization pH (6.8) or
postfertilization pH (7.2) was assayed for histone H1 kinase activity (right).
Immunoblot
anti-cyclin B
anti-cdc2
Cyclin B ~
GVBD
4
8 T12
I
I
I
I
GV
GVBD
i
~
~
16
20
I
I
p4z
c d c 2 r-
Figure 2. Oocyte activation is accompanied by the tyrosine
phosphorylation of a 42-kD protein. Oocytes were activated
by the addition of additional 40 mM KCI to the seawater, a
treatment that mimics fertilization. Aliquots were removed at
4-min intervals, electophoresed on an SDS-polyacrylamide
gel, and blotted with antiphosphotyrosine antibodies.
anti-p-tyr
H1K Activity
GV
6.8
GV G V B D
i
i
GV
G V B D 7.2
I
~
cdc2 [" *'"~ ~
clam oocytes that became tyrosine phosphorylated
after fertilization (not shown). These and other experiments have now established that this clam pp42 which
becomes phosphorylated on tyrosine within 3-4 minutes of fertilization is a member of the E R K family.
Recent work from other laboratories has highlighted
the potential importance of ERKs in early signal transduction pathways (for review, see Boulton et al. 1991).
In particular, Anderson et al. (1990) argued that, because the activity of these kinases requires phosphorylation on both serine/threonine and tyrosine residues,
they are excellent candidates for coordinating input
signals and activating downstream targets. Experiments
from our laboratory confirm this view. The addition of
molybdate to oocytes at any time between 0 and 3
minutes after fertilization blocks the tyrosine phosphorylation of clam pp42 ERK (which would normally appear
about 4 min postfertilization) and arrests cells in inter-
0
497
I
I
~
9_/1
--2
phase; addition of molybdate just 1 minute later, at 4
minutes, allows the appearance of phosphotyrosine
pp42 ERK and subsequent entry into M phase (not
shown). This result indicates that the activation of
cdc2/28 kinase activity is a late event and strongly
suggests that it depends on the prior activation of
pp42 ERK. It should be noted that during meiotic maturation of frog oocytes, Ferrell et al. (1991) and Gotoh
et al. (1991a) have described a related pp42 MAPki
that is activated after cdc2/28 activation. Unlike the
clam pp42 ERK, which undergoes a one-time activation
during release from cell cycle arrest, that enzyme is
activated during each cell cycle. It probably represents
a different member of the E R K / M A P kinase family
that has a true mitotic function (Gotoh et al. 1991b).
The development of cell-free systems that can go
through one or more rounds of cell cycle events in vitro
and the availability of active, purified recombinant cell
cycle proteins have greatly facilitated investigations of
the molecular mechanisms leading to cycles of cdc2/28
activation and repression during mitosis (Lohka and
Maller 1985; Newport and Spann 1987; Luca and
Ruderman 1989; Murray and Kirschner 1989; Solomon
et al. 1990). In hopes of learning more about the
molecular mechanisms of oocyte activation, we developed a cell-free system from quiescent oocytes that
reproduces several of the events occurring after fertilization, including activation of pp42 ERK and cdc2/28.
Oocyte lysates, which contain inactive cyclin B cdc2/28 complexes, do not develop active cdc2/28
when maintained at the prefertilization pH of 6.8.
When the cytoplasmic pH was raised, cdc2/28 kinase
activity appeared (as assayed by activity of p13 suc~bound material toward histone H1), suggesting that
postfertilization rise in pH has a role in the activation of
the preexisting complexes (Fig. 3) (see also Westendorf
et al. 1989). Lysates were also activated by the addition
of purified recombinant cyclin A or cyclin B protein
(not shown), reproducing in vitro the original observations that the addition of cyclin to intact, G2-arrested
....
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498
RUDERMAN ET AL.
p H 6.8
I
I
'0! 60'
!
pH8.0
I0
I
10' 20' 30' 60' 211
60 I
!
m
Figure 3. In vitro activation of the stored pre-MPF complex
by elevated pH. Oocytes were lysed in buffer held at the
prefertilization pH of 6.8, and a 12,000g supernatant was
prepared. Aliquots of the lysate were mixed with an equal
volume of pH 6.8 or pH 8.0 buffer, and histone H1 kinase
activity was assayed at 0 and 60 min later.
oocytes (Swenson et al. 1986) induces entry into M
phase.
We also used these lysates to look for activators of
pp42 ERK. Purified recombinant rat ERK-2 protein by
itself has very low kinase activity toward the substrate
myelin basic protein and remains inactive when added
to the prefertilization oocyte lysate. In contrast, rat
ERK-2 protein added to lysates made from oocytes
taken at 15 minutes after fertilization became active
(not shown), indicating that the activators are present
and maintained in an active state in lysates from postfertilization cells. This assay system provides an excellent opportunity to identify and isolate activators of
pp42 ERK,including its own tyrosine kinase, and to work
backward through the signal transduction pathway.
Control of Programmed Cyclin Destruction
in a Cell-free System
To ask what controls the periodic accumulation and
destruction of the cyclins across the cells, we developed
a cell-free system that reproduces several aspects of
cyclin destruction in vitro (Luca and Ruderman 1989).
The onset of cyclin destruction and the appropriately
staggered disappearance of cyclins A and B are correctly regulated (Fig. 4). Just as in the intact embryo,
lysates made from early interphase require further protein synthesis to reach the cyclin destruction point,
whereas lysates made from cells past the mitotic commitment point do not. By combining lysates from different cell cycle stages, we found that the timing of
cyclin destruction is set by the cell cycle stage of the
cytoplasm and not the cell cycle stage of the substrate
cyclins themselves (Luca and Ruderman 1989). One
feature of cyclin destruction that was reproduced too
well in vitro, however, was the brief and transient
window of cyclin destruction, considerably less than 5
minutes, making it hard to prepare an extract containing the destruction machinery in its active form. Thus,
biochemical analysis of the proteolytic pathway and its
regulation remained difficult.
Murray et al. (1989) provided an insight into this
problem when, in the course of adding cloned cyclin
mRNAs into their frog embryo cell-free system, they
~'~j.:g~ 9 9
RRe
Figure 4. Cyclin destruction in vitro. Embryos were labeled
with [35S]methionine during the first mitotic cell cycle, and a
lysate was prepared from early M-phase cells and incubated at
18~ After the start of the incubation in vitro (t = 0 min),
samples were taken at the indicated times and analyzed by gel
electrophoresis followed by autoradiography. The positions of
cyclin A, cyclin B, and ribonucleotide reductase (RR) are
indicated. The dashes on the right denote the positions of
molecular-weight markers, from top to bottom, 116, 94, 56,
and 40 kD. (Reprinted, with copyright permission of the
Rockefeller University Press, from Luca and Ruderman
1989.)
found that a sea urchin B-type cyclin missing the first
amino-terminal 90 amino acids remained stable, whereas the full-length version was destroyed on schedule.
This truncated, stable cyclin also had the very interesting property of permanently activating the cyclin destruction machinery toward full-length cyclin B. The
importance of the amino-terminal region was further
confirmed by the observation that fusion of that region
to a marker protein led to the temporally regulated
destruction of the fusion protein. In this amino-terminal region, Glotzer et al. (1991) identified a short
stretch of amino acids (RxxLxxlxN) conserved in all
B-type cyclins. When the arginine was changed to cysteine by site-directed mutagenesis, the resulting cyclin
proved resistant to stage-specific mutagenesis. Related
regions noted among the A-type cyclins seemed likely
to be involved in their specific proteolysis but had not
been tested. Here we have directly tested the importance of the amino-terminal region of cyclin A, which
contains the candidate A-type motif RAALGVITN.
Clam cyclin proteins lacking amino-terminal regions
were produced by using convenient restriction sites to
remove regions encoding the first 60 amino acids of
cyclin A and the first 97 amino acids of cyclin B. The
truncated proteins, termed AA60 and BA97, were synthesized in Escherichia coli using the pET5 expression
Downloaded from symposium.cshlp.org on May 12, 2016 - Published by Cold Spring Harbor Laboratory Press
CELL CYCLE CONTROL IN EMBRYOS
a
CaB
499
H1 Kinase Assay
b
0'
M ~A ~B
5' 10' 15' 30' 60' 2tl 4h
;.
+ Buffer
,.,
9
116 ....
96 ,--
H1
68~
+A~60
56
~
H1
+Baa7
31
21
-'-
Figure 5. Bacterially expressed clam cyclin AA60 and BA97 can each induce H1 kinase activity when added to interphase lysates
lacking endogenous cyclins. (a) Purified cyclin AA60 (AA) and BA97 (AB) produced in E. coli were electrophoresed on a 15%
polyacrylamide gel and stained with Coomassie brilliant blue (CBB).(b) A 150,000g supernatant lacking endogenous cyclins was
prepared from two-cell embryos arrested in interphase. Portions of the lysate were incubated with buffer, cydin AA60 or cyclin
BA97, and incubated at 18~ At the indicated times, aliquots were taken and assayed for H1 kinase activity. Autoradiograms
of the 32p-labeled histone H1 from each reaction are shown.
vector (Studier et al. 1990), gel purified, and renatured
by step-wise dialysis (F. Luca et al. in prep.). The
truncated cyclins were active as M-phase inducers in
several different types of assays. For example, both
were capable of inducing G2-arrested frog oocytes to
resume meiosis (not shown). When added to emetinearrested interphase lysates, which contain inactive
cdc2/28 and lack endogenous cyclins, both AA60 and
BA97 induced histone H1 kinase activity, a marker for
activated cdc2/28 (Fig. 5). Further experiments established that the Acyclins induced this activity by binding
to and activating cdc2 (not shown).
Just as in cyclin B (not shown), removal of the amino
terminus from cyclin A converted it into a stable cyclin
but did not inhibit destruction of endogenous, fulllength cyclins (Fig. 6). Not unexpectedly, the presence
of stable AA60 in lysates kept cdc2/28 kinase activity
high for hours (not shown). To compare the effects of
Anti-cyclin A
Anti-cyclin B
+ Buffer
w
cyc A-
30' 45' 60' 90' 2h 3h 5h
0' 3 0 ' 4 5 ' 6 0 ' 9 0 ' 2 h
3h 5h
---
.cyc B
.X
.
9
o ' , 3 o ' 45' ~
go' : ~ ~
sh
m*
9 r
30' ~
-
;
A
" ~:-,--
-
.,-:
~
8o' ~ ~
,-.
:'
,
, - , ! , y ] - 7 o. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
,
:~
-cycB
x
?
i
Figure 6. Cyclin AA60 is stable and does not inhibit the destruction of endogenous full-length cyclin A or B. 150,000g
supernatants from embryos taken at mid-late interphase were incubated with buffer (top) or cyclin AA60 (bottom). At the
indicated times after starting the incubation at 18~ aliquots were taken, electrophoresed on a 15% polyacrylamidegel, blotted to
nitrocellulose, and probed with polyclonal serum raised against cyclin A (left) or cyclin B (right). X indicates a non-cyclin protein
that cross-reacts with this particular cyclin-B antiserum.
Downloaded from symposium.cshlp.org on May 12, 2016 - Published by Cold Spring Harbor Laboratory Press
500
RUDERMAN ET AL.
AA60 and BA97 in living cells, each was injected into
one cell of a two-cell frog embryo. Both AA60 and
BA97 were potent inhibitors of cell division: 100% of
the recipients in each set arrested without undergoing
any further cleavage, whereas all the buffer-injected
controls continued to divide normally for hours. All of
the Acyclin-blocked cells contained highly condensed
chromosomes reminiscent of metaphase arrest (not
shown).
of the other (not shown). Thus, regardless of any functional differences between the full-length A- and Btype cyclins when present at normal levels in the intact
cell, the presence of high levels of either truncated
cyclin in lysates had identical effects on the persistence
of cyclin destruction in vitro.
Both AA60 and BA97 Lead to Persistent Activation
of the Cyclin Destruction Machinery by Preventing
Cyclin Destruction from Being Turned Off
To ask whether AA60 or BA97 could turn on cyclin
destruction in an interphase-arrested lysate, an Mphase lysate was incubated with emetine for 4 hours at
18~ to take it well past the cyclin destruction point.
Radioactively labeled proteins were then added and
found to be stable over the next 4 hours, indicating that
the lysate had indeed turned off the cyclin destruction
system. When AA60 was added, it bound to and activated cdc2 kinase to high levels, but the lysate failed to
activate cyciin destruction. In striking contrast, when
BA97 was added, it was able to both activate cdc2
kinase and proceed to activate the cyclin destruction
In normally dividing embryos, cyclin destruction is a
brief and transient process, lasting for less than 5 minutes (Hunt et al. 1992). In contrast, lysates containing
urchin AB displayed constitutive cyclin destruction
(Glotzer et al. 1991). The presence of AA60 led to the
same appearance of constitutive cyclin destruction activity that acted on both A- and B-type cyclins, indicating that one cyclin type is able to influence destruction
a
Cyclin-BA97-activated cdc2 Turns on Cyclin
Destruction but Cyclin-AA60-activated cdc2 Does Not
+ Buffer
+ AA60
o' 5' 10' 15' 30' 60' 2h 5h
0' 5' 10' 15' 30' 60' 2h 5h
116 9668- A - -------------
5640-
b
0' 5' 10' 15' 30' 60' 2h 5h
O' 5' 10' 15' 30' 60' 2h 5h
116-,
96-'
68-
-A-
56
40 -
,"
~
~
~'~'~'-'-
Figure 7. Truncated cyclin A does not turn on or advance the onset of cyclin destruction. Mid-interphase lysates containing
[35S]methionine-labeled cyclin A reticulocyte translation product were mixed with buffer or AA60 on ice and then incubated at
18~ Samples were taken at the indicated times and analyzed by SDS gel electrophoresis followed by autoradiography. In both
cases, cyclin destruction occurred on schedule and was preceded by the formation of a series of higher-molecular-weight bands
that probably represent ubiquinated intermediates in the destruction process. (Upperpanels) Short exposure; (lowerpanels) long
exposure.
Downloaded from symposium.cshlp.org on May 12, 2016 - Published by Cold Spring Harbor Laboratory Press
C E L L CYCLE C O N T R O L I N E M B R Y O S
system (Luca et al. 1991). These results suggest that the
two cyclins, which have been indistinguishable in virtually all other assays tried so far, are functionally different and that it is cyclin B that activates the cyclin
destruction system and, in doing so, promotes the last
event of the cell cycle, anaphase onset and entry into
interphase of the next cell cycle.
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Control of the Cell Cycle in Early Embryos
J. Ruderman, F. Luca, E. Shibuya, et al.
Cold Spring Harb Symp Quant Biol 1991 56: 495-502
Access the most recent version at doi:10.1101/SQB.1991.056.01.056
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