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GREAT EXPERIMENTS
The discovery of cyclins
Tim Hunt
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Background
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Cell division was first described in
modern terms in the late nineteenth century, when people started using
microscopes to look at living cells, especially the fertilized eggs of
marine invertebrates. As the only visible manifestation of
proliferation, as well as a complicated and intriguing process, mitosis
was perhaps the first truly cellular process to be studied. By the turn
of the twentieth century, mitosis was described in great detail, and
its importance for the distribution of chromosomes to the two daughter
cells had been established by Sutton and Boveri (3919) (see 3920
for an interesting discussion of this era). Very little information
about cell division or its control was added for another 50 years,
however, when radioactive phosphate became readily available and was
used to ask when DNA synthesis occurred in relation to mitosis in broad
bean roots. Alma Howard and Steve Pelc, working in a radiation biology
clinic in 1953, discovered that DNA synthesis occurred during a
discrete period after the end of the previous mitosis, with a clear
"gap" before the next mitosis occurred (3921). At the time,
the role of DNA in heredity was only just becoming clear, and Howard
and Pelc's pioneering studies revealed unsuspected complexities to the
cell cycle: it apparently proceeded in discrete phases, implying that
transitions must exist between them. They also discovered that ionizing
radiation delayed entry into S phase or mitosis, depending on when in
the cell cycle cells were irradiated, which showed that cells could
control such transitions (what we would now call "checkpoints").
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But the cell cycle was still just there,
clearly a central problem in biology and of surpassing interest, but
unknown and seemingly unknowable with respect to how it worked. As
Arthur Hughes wrote in 1952, "The factors which determine the onset of
the process of cell division are completely unknown" (3922).
One could read this sentence in a modern way, in terms of specific
genes and proteins, but it is doubtful if Hughes was thinking quite
this way. At the time, biochemists were still unraveling the pathways
of metabolism, and the available concepts simply didn't allow cell
cycle control to be analyzed from a biochemical standpoint. The
functions then known for proteins were mainly structural, like actin
and myosin; respiratory, such as hemoglobin and the cytochromes; or
enzymatic, such as the many enzymes that converted one small soluble
molecule into another in metabolism. In this context there was no way
to formulate the question of control of the cell cycle in molecular
terms. What would be the assay? Largely because of the lack of a
conceivable molecular focus, this condition persisted until well after
complex processes such as DNA replication and protein synthesis had
been reproduced in vitro and were understood in considerable detail.
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My introduction to the possibility of a
biochemical approach to the control of the cell cycle came one
afternoon in a seminar room at the Marine Biological Laboratory (MBL),
Woods Hole, when John Gerhart came to tell the Embryology class of 1979
about maturation promoting factor (MPF) (3923). His approach
to understanding cell cycle transitions took advantage of frog oocytes.
These cells are arrested in a G2-like state before the first meiotic
division, and are normally stimulated to resume meiosis by the steroid
hormone progesterone, which is secreted by surrounding follicle cells.
The oocytes respond to the hormone by undergoing the first meiotic
division and beginning the second, where they arrest in metaphase as
mature eggs. Fertilization releases this arrest. The progression from
oocyte to mature egg is called "maturation." It was known at the time I
heard Gerhart speak that protein synthesis played a significant role in
maturation, since protein synthesis inhibitors blocked the process
whereas inhibition of RNA synthesis did not. The need for new protein
synthesis was not understood, but was apparently related to an
important observation made in 1971 by Yoshio Masui and Clement Markert,
who had found that sucking cytoplasm out of a mature egg and
microinjecting it into an immature oocyte caused the oocyte to mature
without the need for either progesterone or protein synthesis (3924).
They called the unidentified cytoplasmic component responsible maturation promoting factor, or MPF.
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Since its discovery, evidence had
accumulated that MPF was a factor, a biochemical entity, which
catalyzed entry into mitosis. A variety of experiments conducted in the
1970s made it clear that the activity was due to a protein, that it
arose by conversion of an inactive precursor into active MPF, and that
MPF itself was what activated this precursor, acting by some sort of
positive feedback loop: as Gerhart wrote (3923), "the
autoactivation of MPF would seem to resemble the autoactivation of
self-phosphorylating protein kinases or of proteases that activate
their own zymogens by proteolysis." During attempts to purify MPF, Wu
and Gerhart had found that its stability required ATP (3925),
and since they and others had shown that maturation of an oocyte into
an egg was accompanied by significant increases in protein kinase
activity (3926), it was reasonable to surmise that MPF, if not
a protein kinase itself, was closely connected with them somehow. It is
significant, or so it seems to me, that very little, if any, attention
was paid at the time to the problem of how MPF was turned off. If
pressed for a suggestion, people would probably have guessed that MPF
was turned off by dephosphorylation, assuming that its activation
involved phosphorylation. In retrospect, this was a classic case where
it was senseless to speculate, to waste clean thoughts on dirty
enzymes. But speculation was tremendously tempting, as well as great
fun.
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The purification of MPF was challenging,
however. This was partly because of problems related to the assay,
which involved microinjection into living oocytes (which vary
significantly from batch to batch, and even from one to another), and
partly because MPF was extremely labile. A considerable amount of
effort was expended trying to purify it, but from its discovery in 1971
until late in the 1980s it resisted all attempts. At the time I heard
Gerhart's talk it remained a "factor" — "innocent of chemistry" as Dan
Mazia liked to say, yet you could feel that it was almost within reach
and clearly a vital key to understanding cell cycle transitions.
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A further aspect of the excitement
surrounding MPF was the gathering sense that it was not a peculiarity
of frog oocytes or of meiosis, but was widespread and was also present
in mitotic cells. Thus, starfish oocytes were found to contain MPF (3927),
and they responded when injected with vertebrate MPF. And in 1979, Sunkara et al. (3928)
found that extracts of late G2 and mitotic human tissue culture cells
also contained MPF (as defined by the frog oocyte assay), which
suggested "that the factors that regulate the breakdown of the nuclear
membrane and chromosome condensation during mitosis and meiosis…appear
to be very similar, if not identical, throughout the animal kingdom."
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Hearing about MPF from Gerhart was
exciting and inspiring. Apparently, getting cells to enter mitosis
could be seen as something quite simple: turning on an enzyme. It ought
to be possible for a biochemist to identify that enzyme, and find out
how it acted and how it was controlled. But this was far from my field
of endeavor at the time and had no apparent connection with it.
Although I probably started to think more seriously about cell cycles
and their control somewhere in the back of my mind, the seminar had no
immediate influence on my own work.
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Protein synthesis and its control
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I had been working on the control of
hemoglobin synthesis in rabbit reticulocytes at the Department of
Biochemistry in Cambridge, England, since 1965. I first went to the
Marine Biological Laboratory (MBL) at Woods Hole in the summer of 1977,
as an Instructor in the Embryology Course, planning to explore a new,
but related problem: how protein synthesis was turned on when sea
urchin eggs were fertilized. This turned out to be difficult. In
reticulocytes, phosphorylation of a protein synthesis initiation factor
was regulated by a variety of things, including heme, oxidizing agents,
and double-stranded RNA, and this phosphorylation turned off protein
synthesis. Naturally, I suspected that phosphorylation controlled sea
urchin protein synthesis, too. Indeed, we soon discovered that
ribosomes of the sea urchin Arbacia punctulata became phosphorylated
at about the same time as protein synthesis increased (3930),
which looked promising, but sadly turned out to be irrelevant to the activation
of protein synthesis (3931). We also knew that this early burst of protein
synthesis was necessary for the eggs to divide (3932), although it was not
required for DNA replication during the first S phase (3933).
That summer was fun and highly educational but not terribly productive,
and I went home without having even begun to understand the problem.
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When I returned to the MBL two years
later in June 1979, I met Joan Ruderman and Eric Rosenthal, who had
discovered that clam oocytes started to make a new set of three very
prominent proteins (for convenience labeled A, B, and C) after
fertilization. I helped with some experiments that demonstrated that
the mRNAs for these proteins were present before fertilization and were
only translated afterwards (one of the first, if not the first,
clear examples of translational control of protein synthesis). We wrote up the results (3934),
ending the Discussion with the sentence: "Bands on gels are all very
well, but what is the role of these proteins in development?" — an
unanswerable question at the time. At some point, probably in 1980 or
1981, Dennis Ballinger and I wondered if we could try a kind of MPF
assay by inhibiting protein synthesis in fertilized sea urchin eggs and
then injecting them with homogenates of later stage sea urchin embryos.
We never reached the point of doing the experiment, but already it must
have occurred to us that new protein synthesis must provide a factor or
factors that were required for cells to enter mitosis. Our ideas were
not very focused, however, and seeing as we never did the experiment we
clearly didn't take the idea very seriously.
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The summer of 1982 found me back in
Woods Hole once again, still struggling to understand protein synthesis
in fertilized sea urchin eggs. By then, most of the testable hypotheses
had been disposed of, and I was wondering more about why (and how) clam
eggs made new proteins after fertilization, whereas sea urchin eggs
apparently did not. It was in late July, after the formal lab courses
(I was teaching "Physiology and Molecular Biology" by now) were over,
that I had time and space to do some experiments myself. For reasons
that were not particularly clear to me even three weeks later (when I
wrote an excited and detailed letter home), I decided to compare the
patterns of protein synthesis between parthenogenetically activated
eggs and fertilized eggs. The experimental design was simple. Eggs were
suspended in 5-10 ml of Millipore-filtered sea water contained in a 20
ml glass scintillation vial, were either fertilized with sperm or
activated artificially, and [35S]methionine to label newly
synthesized proteins was added as soon as the appearance of
fertilization membranes indicated that development was proceeding
normally. Samples of 50 microliters were pipetted directly into 25%
trichloracetic acid to stop the labeling reaction, and the eggs were
collected, washed twice with acetone and once with ether, air-dried and
finally dissolved in SDS-polyacrylamide gel sample buffer prior to
running them on a gel, which was dried and exposed to X-ray film. It
was about the simplest experiment imaginable. I had long been in the
habit of taking samples at frequent intervals, which gave the results
robustness as well as a dynamic dimension. The autoradiogram of the
very first gel showed a most unexpected and surprising result: one of
the most prominent labeled bands faded away at about the time the cells
started to divide. All the other labeled bands made in the fertilized
eggs went on getting stronger, as I had expected of all the bands. I
was very intrigued by the behavior of this one band, and that very
evening happened to meet John Gerhart at the wine and cheese party that
traditionally follows Friday Evening Lectures at the MBL. I explained
the disappearance of the band, and he told me about recent experiments
that he and Marc Kirschner had been doing in which they had found that
MPF activity disappears between the two meiotic divisions in frog
oocytes (3935). Crucially, they found that inhibiting protein
synthesis both prevented the reappearance of MPF and prevented the
oocytes from entering the second meiotic division.
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This information was electrifying. It
suggested that a protein (or proteins) was (somehow) consumed at the
end of the first division and needed to be resynthesized in order for
the cell to enter the next division. This, of course, was exactly the
sort of behavior shown by the protein that I had seen that morning,
even though I hadn't yet seen "Cyclin," as we soon called it, come back
again. Obviously we couldn't even begin to conclude that cyclin was
part of MPF, or even connected with MPF, but for me, even at that early
time, that conversation strongly connected programmed proteolysis with
the cell cycle.
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Figure 1
Sea urchin eggs were suspended in filtered sea water and
fertilized with a dilute suspension of sea urchin sperm. Radioactive
methionine was added as soon as the fertilization envelope formed so
that all subsequently synthesized proteins would be labeled. Samples
were removed into trichloracetic acid at 10-minute intervals to "fix"
them. The precipitated proteins were analyzed on an acrylamide gel, and
the radiolabeled proteins detected by exposing the gel to X-ray film.
The lower panel shows the intensity of the cyclin band. The second peak
is lower than the first because of dilution of the radioactive label.
Notice how the intensity of the cyclin band increases steadily, and
crashes very abruptly, whereas all the other bands increase steadily in
intensity.
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Figure 2
The red line denotes the levels of cyclin as measured by
an experiment similar to that shown in 1, but at nearly the same time
in parallel, samples of the eggs were taken into formaldehyde to
preserve them for later analysis under the microscope. The numbers of
eggs in the process of dividing into two or four are plotted in blue.
Notice how cyclin levels "crash" well before the cells divide.
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Figure 3
The top panel shows an experiment exactly like that
shown in 1. In the experiment shown in the bottom panel, however,
radioactive methionine was added at intervals to aliquots of a
suspension of fertilized eggs that were left for 10 minutes before
fixing and processing as usual. Each lane thus measures the amount of
cyclin (and other proteins) synthesized in 10 minute "pulses" spaced at
10 minute intervals. To a good approximation, the rate of synthesis of
cyclin is the same as that of the other proteins, even during the
period when it is being degraded (at about 60 minutes).
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Figure 4
Sea urchin eggs were fertilized and labeled with [35S]methionine
as before, and the suspension divided in two. Colchicine was added to
one portion in order to arrest the eggs at metaphase of the first
cleavage division. As the lower panel shows, this prevented the abrupt
disappearance of the cyclin band.
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We needed to make sure that what I had
seen was real, and to check the comings and goings of cyclin in
relation to the cell cycle. Tom Evans and I repeated the basic
experiment, this time looking at the eggs under the microscope so as to
get a better idea when in the cell cycle cyclin disappeared. We quickly
discovered that if we extended the labeling over two division cycles,
we could clearly see cyclin come up again and then fade a second time,
as Figure 1 shows. It was also immediately clear that cyclin disappeared
before the cells actually divided (cleaved) in two, as shown in Figure 2.
Even several hours after fertilization we could show, by adding emetine
to inhibit protein synthesis, that cyclin was still being made, and
still going away, although by now the synchrony of division that
attends the first three divisions of sea urchin eggs had been lost. The
experiments shown in Figure 3 made it clear that the disappearance of cyclin was caused by sudden
proteolysis, because its rate of synthesis was more or less constant
throughout the cell cycle, as measured with short pulses of labeled
methionine. We next tested if the oscillations in cyclin could be
altered by compounds that blocked cell division. As shown in Figure 4,
we found that colchicine stabilized cyclin as well as causing a
"metaphase arrest." We would have liked to find out what would happen
if one could inhibit the proteolysis of cyclin: would the cell cycle
stop, and if so, where? But this was unanswerable at the time, for we
had no idea what was responsible for the disappearance of cyclin, and
there was no handy protease inhibitor we could use (although we did
try).
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These observations raised a host of
questions. What was cyclin?; why and how did it behave in this
extremely suggestive manner in relation to the cell cycle? But the
immediate question for the summer of 1982 was: how widespread are
cyclins? We decided to take a look in clam eggs, which were still
available in August. We were surprised and pleased to find that two of
the translationally regulated bands which I had worked on with Eric and
Joan in 1979, previously labeled simply "A" and "B," both behaved like
cyclins (which is how cyclins A and B got their names). Clams and sea
urchins are evolutionarily very distant, so this was encouraging, but
on the other hand, they were both marine invertebrates, so at the time
we scarcely dared imagine that frogs or humans might have cyclins, too.
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One might wonder why the oscillations in
cyclin levels had gone unnoticed in dividing clam embryos, and the
answer is that multiple, closely spaced timed samples were never taken
until Dan Distel (the student who actually performed the experiment)
did it in August 1982. In fact, Eric Rosenthal had noticed before that
the intensity of bands A and B were somewhat variable, and never seemed
to get as strong as band C. Being focused on translational control,
however, he had not considered it worth pursuing this apparently minor
point.
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Figure 5
To discover the precise timing of cyclin disappearance
in relation to the events of the chromosome cycle, fertilized eggs were
labeled as before, but samples were taken for fixation and staining of
chromosomes to determine the timing of the metaphase-to-anaphase
transition. The shaded bar represents the period of mitosis, and the
timing of cytokinesis is indicated by the blue curve.
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Back in Cambridge for the school year, I
was very excited by all this but powerless to do anything except write
a paper. One of the first reviewers said that we engaged in "wild
speculation based on faulty logic" and we had to submit a heavily
revised version, which was accepted (825). By the following
summer, I wondered if it had all been a dream, and it was comforting
when cyclin showed exactly the same behavior in our very first time
course once we were back at the MBL the following June, when Richard
Cornall came as my assistant. We still needed to pin down when in the
cell cycle cyclin disappeared, so Yoshio Masui and Ryoko Kuriyama
showed us how to "see" sea urchin chromosomes, and as shown in Figure 5,
we found that cyclin went away shortly before the chromatids separated
at the metaphase-to-anaphase transition. All this time, of course, I
was trying to learn as much as possible about the control of the cell
cycle, and trying out the idea that protein synthesis followed by
proteolysis was the basis, somehow or another, of the control of MPF
activity. Nobody I talked to seemed to be able to find holes in the
"faulty logic," but equally, nobody had seriously suggested such a
thing before. Periodic enzymes had been considered (3936), and
indeed, actively sought, but as far as I could tell the literature was
silent on the possibility of periodic, programmed proteolysis as the
means of inactivating them. The very idea that it might be possible to
specifically degrade one protein inside a cell at exactly the right
time was challenging, to say the least. At the time, proteases were
known as digestive enzymes whether they operated inside or outside the
cell. The ubiquitin system had recently been discovered, but a
denatured protein was used as the substrate, and in those early days
ubiquitination was seen in the context of ridding the cell of misfolded
or otherwise abnormal proteins.
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>In the fall of 1983, Jon Pines joined me as a graduate student,
with the mission of making cDNA libraries from Arbacia
eggs and obtaining a clone of the original cyclin. Since neither of us
had worked with DNA before in our lives, it was a long and sometimes
frustrating business, ultimately successful just before Christmas, 1986
(3937).
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The link with MPF
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The big question at this stage was: Did
cyclin drive the cell cycle as a central component of what we would now
call the "cell cycle engine," or was it simply a follower, a reflection
of some other underlying and hidden central oscillator? We needed to
come up with approaches to test this, but without a clone that encoded
cyclin, this was impossible. Fortunately, Eric Rosenthal had already
cloned clam cyclin A among other translationally regulated mRNAs, and
as soon as it became feasible to make functional mRNA from plasmid DNA
(through the use of viral RNA polymerases), Katherine Swenson injected
mRNA encoding cyclin A into Xenopus oocytes and found that they matured (2043).
Since frogs were where most of the work on MPF had been done, this
demonstration put cyclins right into the thick of things and strongly
reinforced the idea of a close connection with MPF. Here was a protein
that made the oocytes mature; this was the very definition of MPF. In
some ways, however, it was also a puzzling observation. Taken at face
value, it suggested, paradoxically, that cyclin could not be
a component of MPF itself, because the oocyte maturation assay measured
factors that converted the already-present pre-MPF into active MPF.
Taken literally, the experiment indicated that cyclin must be an
activator of MPF and not MPF itself or a part of MPF. In the end, the
purification of MPF was required to resolve this point (1587),
and in retrospect it was fairly pointless thinking or speculating or
trying to make sense of the known data in terms of an accurate model at
the time. Despite the difficulties in its interpretation, however, this
experiment was very exciting and important, because it demonstrated
that a newly made cyclin could actually bring about a cell cycle
transition.
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By this time, I was heavily involved with the authors of
Molecular Biology of the Cell, the textbook by Alberts et al. (see 3938).
We spent the summer of 1986 in Berkeley in a house near the UC campus,
and I often worked in a library in the same building as John Gerhart's
laboratory. One day, Mike Wu asked if I would like to see an MPF assay,
which of course I did. Inspired by Joan and Katherine's result, I
suggested that we try injecting some maternal mRNA from Urechis caupo,
a large mud-living worm that Eric Rosenthal and Fred Wilt were studying
in the Zoology Department nearby. Mike was very surprised when the frog
oocytes injected with this RNA underwent maturation: he had been
operating an mRNA-injecting service for clients in the Bay Area for
some time, and had never seen that happen before. Encouraged by this
result, we next made mRNA from frog eggs and injected that back into
the oocytes: they too matured, as we discovered creeping back into the
lab late one evening to take a look. This was immensely satisfying, for
it implied (though by no means proved) that frogs contained cyclins;
even if they did not, the experiment suggested that they must contain
mRNA for MPF. We never published these experiments, but they strongly
encouraged us to look for cyclins in Xenopus. Andrew Murray
and Marc Kirschner visited our "safe house," and we discussed the way
forward. It became apparent that to test the role of cyclin in cell
cycle transitions, we would need to develop a way of selectively
removing cyclin mRNA from a cell.
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We thought an antisense approach would
be promising as a way to target individual mRNAs. I suggested the
project to Jeremy Minshull, who had joined my laboratory as a graduate
student the year after Jon Pines. After a great deal of effort and much
cloning and DNA sequencing, now from Xenopus, we were able to
specifically degrade cyclin mRNA in Xenopus
oocytes by injecting antisense oligonucleotides along with RNAse H
(which digests the RNA strand of a DNA-RNA hybrid). With help from
Julian Blow in Ron Laskey's laboratory, we were able to demonstrate
that ablating cyclin mRNA in egg extracts stopped them entering mitosis
(3939). But the antisense experiments with the oocytes didn't
look or feel quite right, and sorting this out took many years (3940).
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While Jon, Jeremy and I were
experimenting in Cambridge, Andrew Murray (who had worked with me as an
undergraduate in Cambridge and who later helped me teach courses in
Woods Hole) had been taking a different and dramatically bold approach
as a postdoc in Marc Kirschner's lab at UCSF. Andrew developed an
extract derived from frog eggs that would perform the remarkable feat
of cell cycles in the absence of cells. He could demonstrate this by
adding purified nuclei to the extract, which would then undergo
repeated cycles of nuclear envelope breakdown, chromosome condensation,
and DNA replication, provided that protein synthesis was allowed. After
many painstaking trials he succeeded in finding a way to destroy all
the endogenous mRNA without compromising the ability of the system to
translate added synthetic mRNA, enabling him to test the ability of Jon
Pines's Arbacia cyclin B clone to "drive" these cell cycles.
In the critical experiments he found that addition of the mRNA for sea
urchin cyclin B was sufficient to restore cell cycles to the
mRNA-depleted extracts, allowing a bold title for his paper: "Cyclin
drives the cell cycle" (1573). And when he expressed a form of
cyclin that could not be degraded (a version of the sea urchin cyclin
missing its N-terminus) the extracts still entered mitosis, but were
then unable to leave it (832). Almost as satisfying was news
from Christian Lehner and Pat O'Farrell that an oligonucleotide whose
sequence we had suggested revealed the presence of cyclins in Drosophila
(3941), and from Steve Reed and Bruce Futcher that budding yeast contained
cyclins too (3942; 3943).
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Figure 6
Cyclin accumulates continuously and associates with Cdc2
protein during interphase. A regulatory mechanism prevents the kinase
activity of the Cdc2-cyclin dimer (MPF) from being activated until
various conditions are fulfilled, such as completion of DNA replication
(or, in the case of Xenopus oocytes, the presence of
progesterone). The abrupt activation of MPF is facilitated by a
positive feedback look. Once activated, MPF drives the cell into
mitosis by phosphorylating a large number of other proteins. The
proteolysis of cyclin inactivates MPF, allowing the cell to leave
mitosis and start the whole process over again. A variety of regulatory
inputs can delay the activation or inactivation of MPF.
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The grand unifying concept (321)
came soon afterwards when MPF was finally purified and its components identified (1587).
It proved to be a complex of cyclin and Cdc2, a protein that had
emerged from yeast genetics as a central regulator of entry into
mitosis (322). Cdc2 was shown to be a protein kinase that was
completely inactive unless combined with a cyclin partner. The basic
picture that emerged was that cyclin's appearance and disappearance
caused the kinase to be periodically activated, driving the cell in and
out of mitosis, as shown in Figure 6.
Much subsequent work has embellished this basic mechanism with
significant complexities of regulation. Cdc2 is phosphorylated at no
less than three different positions after it binds cyclin, the
modifications being performed by at least two different kinases, one
inhibitory and the other activating. The activation of Cdc2 as a
protein kinase required the removal of the inhibitory phosphates by an
enzyme (Cdc25 phosphatase) that is itself regulated by phosphorylation.
Such a complicated sequence of activation not only accounts for the
abrupt activation of the kinase despite the gradual accumulation of
cyclin, but also permits many inputs to control progression into
mitosis, including the progesterone activation of frog oocytes, the
fertilization activation of clam eggs, and the DNA damage checkpoints
seen in virtually all cell types. In retrospect, it is hard to
understand why so many very clever people took so long to consider the
idea that a protein kinase (which the sequence of Cdc2 had clearly
indicated long before) might require an activating partner (3944).
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As for the periodic degradation of
cyclins, it did not take Michael Glotzer and the Kirschner laboratory
long to discover that cyclin was modified by the covalent attachment of
ubiquitin before it was degraded, and to identify the short cis-acting
sequence that was recognized by the destruction machinery (845).
It took much longer to identify the complicated, highly regulated
control machinery that regulates the addition of ubiquitin (847).
It's worth noting that we still do not know how the little "destruction
box" is recognized, or quite how the activity of the "anaphase
promoting complex/cyclosome" that ubiquitinates cyclin is regulated,
despite intense effort.
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After the initial simplification of the
grand unifying model, it was not long before things became much more
complicated again. More cyclins were discovered, and it became clear
they controlled the G1-to- S phase transition as well as entry into
mitosis. From there the complications only continued. A cell as simple
as budding yeast proved to have nine different cyclins devoted to
various stages of the cell cycle, and the human genome contains even
more of them, perhaps reflecting the greater complexity of human cells.
By the end of 2002, 20 years after their accidental discovery, there
were roughly 15,000 papers containing the word "cyclin," of which
10,000 also contain the word "human."
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The author
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tim Hunt is a Principal Scientist at Cancer Research UK, Clare Hall
Laboratories, 15 miles due north of central London. Born in England on
the Wirral (not far from Liverpool) in 1943, his family moved to Oxford
in 1945 where he was educated at the Oxford High School for Girls, the
Dragon School, and Magdalen College School before going up to Cambridge
University in 1961 to read Natural Sciences. He spent close to 30
years, man and boy, at the Department of Biochemistry in Cambridge,
with intervals working in the USA. The first was a "sabbatical" he took
as a graduate student at the Albert Einstein College of Medicine during
the summer of 1966. He returned there (in the Department of Medicine)
for a postdoctoral fellowship from 1968-70, and from 1977-85 spent most
summers at the Marine Biological Laboratory, Woods Hole, Massachusetts,
teaching practical (i.e. laboratory) courses and doing research on sea
urchin eggs and clam oocytes. In Cambridge, Dr. Hunt began his career
working on the control of protein synthesis in red blood cells. A long
and involved story with many twists and turns but ultimate success, it
turned out that protein synthesis was regulated by protein kinases, one
controlled by the availability of heme, the other by double-stranded
RNA. In 1982, he did the experiment on sea urchin eggs described in
this essay that revealed an important aspect of how cell division is
controlled. He has worked on cell division ever since. His work led to
the Nobel Prize in Physiology or Medicine for 2001, which he shared
with Leland Hartwell and Paul Nurse. Together with Andrew Murray, he
wrote The Cell Cycle: An Introduction, and with John Wilson
composed A Problems Book to accompany Alberts et al.'s
Molecular Biology of the Cell.
Tim Hunt
Cancer Research UK
Clare Hall Laboratories
South Mimms, Herts EN6 3LD
United Kingdom
phone: (44) 0207 269 3981
E-mail: [email protected]
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Last Revised on September 10, 2004
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321 Murray, A. W. and Kirschner, M. W.
(1989).
Dominoes and clocks: the union of two views of the cell cycle.
Science 246, 614-621.
PubMed
-
322 Nurse, P.
(1990).
Universal control mechanism regulating onset of M-phase.
Nature 344, 503-508.
PubMed
-
3920 Martins, L.A.-C.P.
(1999).
Did Sutton and Boveri propose the so-called Sutton-Boveri Chromosome hypthesis?
Genet. Mol. Biol. 22, 261-271.
Journal
-
3922 Hughes, A.
(1952).
The mitotic cycle: The cytoplasm and nucleus during interphase and mitosis. (London: Butterworths Scientific Publications).
-
3923 Gerhart, J. C.
(1980).
Mechanisms regulating pattern formation in the early amphibian egg and early embryo. In Biological Regulation and Development R. F. Goldberger, ed. (New York: Plenum Press), pp. 133-316.
-
3936 Mitchison, J. M.
(1969).
Enzyme synthesis in synchronous cultures.
Science 165, 657-663.
PubMed
-
3938 Wilson, J., and Hunt, T.
(2003).
Molecular Biology of the Cell: A Problems Approach (New York: Garland Science).
-
3944 Dorée, M., and Hunt, T.
(2002).
From Cdc2 to Cdk1: when did the cell cycle kinase join its cyclin partner?
J. Cell Sci. 115, 2461-2464.
PubMed Journal
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825 Evans, T., Rosenthal, E. T., Youngblom, J., Distel, D., and Hunt, T.
(1983).
Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division.
Cell 33, 389-396.
PubMed Journal
-
832 Murray, A. W., Solomon, M. J., and Kirschner, M. W.
(1989).
The role of cyclin synthesis and degradation in the control of maturation promoting factor activity.
Nature 339, 280-286.
PubMed Journal
-
845 Glotzer, M., Murray, A. W., and Kirschner, M. W.
(1991).
Cyclin is degraded by the ubiquitin pathway.
Nature 349, 132-138.
PubMed Journal
-
847 King, R. W., Peters, J. M., Tugendreich, S., Rolfe, M., Hieter, P., and Kirschner, M. W.
(1995).
A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B.
Cell 81, 279-288.
PubMed Journal
-
1573 Murray, A. W. and Kirschner, M. W.
(1989).
Cyclin synthesis drives the early embryonic cell cycle.
Nature 339, 275-280.
PubMed Journal
-
1587 Lohka, M. J., Hayes, M. K., and Maller, J. L.
(1988).
Purification of maturation-promoting factor, an intracellular regulator of early mitotic events.
Proc. Natl. Acad. Sci. USA 85, 3009-3013.
PubMed
-
2043 Swenson, K. I., Farrell, K. M., and Ruderman, J. V.
(1986).
The clam embryo protein cyclin A induces entry into M phase and the resumption of meiosis in Xenopus oocytes.
Cell 47, 861-870.
PubMed Journal
-
3919 Boveri, T. (1902). Über mehrpolige
Mitosen als Mittel zur Analzyse des Zellkerns. Verhandlungen der
physicalisch-medizinischen Gesselschaft zu Würzburg. Neu
Folge. 35, 67-90. Journal
-
3921 Howard, A. and Pelc, S. R.
(1953).
Synthesis of desoxyribonucleic acid in normal and irradiated cells and its relation to chromosome breakage.
Heredity 6, 261-273.
Journal
-
3924 Masui, Y., and Markert, C. L.
(1971).
Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes.
J. Exp. Zool. 177, 129-145.
PubMed
-
3925 Wu, M., and Gerhart, J. C.
(1980).
Partial purification and characterization of the maturation-promoting factor from eggs of Xenopus laevis.
Dev. Biol. 79, 465-477.
PubMed
-
3926 Maller, J., Wu, M., and Gerhart, J. C.
(1977).
Changes in protein phosphorylation accompanying maturation of Xenopus laevis oocytes.
Dev. Biol. 58, 295-312.
PubMed
-
3927 Kishimoto, T., and Kanatani, H.
(1976).
Cytoplasmic factor responsible for germinal vesicle breakdown and meiotic maturation in starfish oocyte.
Nature 260, 321-322.
PubMed
-
3928 Sunkara, P. S., Wright, D. A., and Rao, P.
N. (1979). Mitotic factors from mammalian cells induce germinal
vesicle breakdown and chromosome condensation in amphibian oocytes.
Proc. Natl. Acad. Sci. USA 76, 2799-2802. PubMed
-
3930 Ballinger, D. G., and Hunt, T.
(1981).
Fertilization of sea urchin eggs is accompanied by 40 S ribosomal subunit phosphorylation.
Dev. Biol. 87, 277-285.
PubMed
-
3931 Ward, G. E., Vacquier, V. D., and Michel, S.
(1983). The increased phosphorylation of ribosomal protein S6 in
Arbacia punctulata is not a universal event in the activation of sea
urchin eggs. Dev. Biol. 95, 360-371. PubMed
-
3932 Hultin, T.
(1961).
The effect of puromycin on protein metabolism and cell division in fertilized sea urchin eggs.
Experientia 17, 410-411.
PubMed Journal
-
3933 Wagenaar, E. B.
(1983).
The timing of synthesis of proteins required for mitosis in the cell cycle of the sea urchin embryo.
Exp. Cell Res. 144, 393-403.
PubMed
-
3934 Rosenthal, E. T., Hunt, T., and Ruderman, J.
V. (1980). Selective translation of mRNA controls the pattern of
protein synthesis during early development of the surf clam, Spisula
solidissima. Cell 20, 487-494. PubMed Journal
-
3935 Gerhart, J., Wu, M., and Kirschner, M.
(1984).
Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs.
J. Cell Biol. 98, 1247-1255.
PubMed
-
3937 Pines, J., and Hunt, T.
(1987).
Molecular cloning and characterization of the mRNA for cyclin from sea urchin eggs.
EMBO J. 6, 2987-2995.
PubMed
-
3939 Minshull, J., Blow, J. J., and Hunt, T.
(1989).
Translation of cyclin mRNA is necessary for extracts of activated xenopus eggs to enter mitosis.
Cell 56, 947-956.
PubMed Journal
-
3940 Hochegger, H., Klotzbücher, A., Kirk, J., Howell, M., le Guellec, K., Fletcher, K., Duncan, T., Sohail, M., and Hunt, T.
(2001).
New B-type cyclin synthesis is required between meiosis I and II during Xenopus oocyte maturation.
Development 128, 3795-3807.
PubMed Journal
-
3941 Lehner, C. F., and O'Farrell, P. H.
(1989).
Expression and function of Drosophila cyclin A during embryonic cell cycle progression.
Cell 56, 957-968.
PubMed Journal
-
3942 Richardson, H. E., Wittenberg, C., Cross, F., and Reed, S. I.
(1989).
An essential G1 function for cyclin-like proteins in yeast.
Cell 59, 1127-1133.
PubMed Journal
-
3943 Nash, R., Tokiwa, G., Anand, S., Erickson, K., and Futcher, A. B.
(1988).
The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog.
EMBO J. 7, 4335-4346.
PubMed
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©Jones and Bartlett Publishers (2007)
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