Download The study of cell cycle control is entering a new and exciting phase

J. Cell Sci. Suppl. 4, 155-170 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
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CELL CYCLE REGULATION IN YEAST
JA C Q U E L IN E HAYLES a n d PAUL NURSE
Imperial Cancer Research Fund, PO Box 123, Lincoln’s Inn Fields,
London WC2A3PX, UK
INTRODUCTION
The study of cell cycle control is entering a new and exciting phase. The classical
physiological and cytological approaches to investigating the cell cycle have been
supplemented with the powerful techniques of molecular biology and genetics. This
is particularly the case for the budding yeast Saccharomyces cerevisiae and the
fission yeast Schizosaccharomycespombe. As yeasts are simple eukaryotes, cell cycle
regulation is less likely to be confused with the controls involved in growth and
differentiation, which are required in multicellular organisms. Thus they provide
excellent systems in which to study the basic mechanism of cell cycle regulation and
represent good models for understanding eukaryotic cell cycle control.
The ease of classical genetic analysis in the two yeasts has resulted in the isolation
of mutants in many genes that are required for the cell cycle. Study of such mutants
has been useful for formulating hypotheses about how the cell cycle might be
controlled and for identifying genes that have a role to play in those controls. These
abstract studies have then been followed up using the tools of molecular biology to
determine the biochemical basis of the controls involved. In this paper we shall
concentrate on one particular gene function, that of cdc28 from S. cerevisiae and the
analogous gene cdc2 from Schiz. pombe. This gene function has a pivotal position in
cell cycle control. We shall review work that establishes that the cdc2/cdc28 gene
function is required in G\ to commit cells to S phase and the rest of the mitotic cell
cycle, and then again in Gz, where it determines the cell cycle timing of mitosis.
Isolation and characterization of the cdc2 and cdc28 genes have shown that they
encode protein kinases that may be regulated by phosphorylation. This has led to the
hypothesis that phosphorylation of a specific set of proteins may control the
transition from G\ into 5 phase and from Gz into mitosis. The hypothesis has obvious
similarities to several models being developed for control of the cell cycle in other
eukaryotic cells. However, before considering these matters we want to discuss
briefly what we mean by the cell cycle and its control, in order to define more
precisely the problems that we think are important to investigate.
TH E C ELL CYCLE A N D ITS CONTROL
The cell cycle is made up of the events and processes that take place between the
birth of the cell and its subsequent division into two daughters. In steady-state
growth the mass of the cell will also double during the cell cycle. Therefore, taken
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literally, cell cycle events and processes will include all those that are required for
cellular growth as well as those for direct reproduction of the cell. Such a literal
interpretation is rather unfocused since it will include processes such as protein and
RNA synthesis, which are only indirectly relevant to cell reproduction. So we shall
confine our attention to those cell cycle events and processes that are specifically
required for cell reproduction and not for cellular growth. This distinction has not
always been fully appreciated. For example, the changes that occur when quiescent
cells are stimulated are often assumed to be cell-cycle-specific. But, as well as being
stimulated to enter into the cell cycle, such cells are also being stimulated to grow.
Consequently, many of the changes observed will be associated with the growth of
the cell rather than with the cell cycle itself. The two major processes that are
specifically required for cell reproduction are S phase and mitosis. These ensure the
replication and segregation of the chromosomes and are found in all eukaryotic cell
cycles.
When considering cell cycle control we run into the problem that the term ‘control’
can mean very different things to different people. It may be used in the sense of
‘required for’. In this sense, any event that is necessary for successful completion of
the cell cycle can be considered part of its control. We do not think that this is a very
useful way to look at the problem. For us, implicit in the term ‘control’ is the idea of
regulation, and we want to identify events that are important for regulation. Three
useful ways of identifying regulatory events are to find those acting at: (1) initiation
steps, (2) rate-limiting steps and (3) commitment steps. We shall consider each of
these in turn.
An initiation event is simply the first event that is specifically required for the
process under consideration. The difficulty here is knowing when the first event has
been found, as there may always be an earlier event that has not been identified. In
cell cycle terms we are interested in the initiation events of the whole cell cycle, of S
phase and of mitosis.
A rate-limiting step is an event that makes a major contribution to the overall rate
at which a process is completed. Any event necessary for the cell cycle will become
rate-limiting if it is inhibited, but that does not mean that it is normally rate-limiting.
It is more informative to speed up an event than inhibit it, since in these
circumstances only genuinely rate-limiting steps will advance the whole process.
Various treatments such as the use of mutants, medium shifts and addition of cell
extracts have been used to advance cells through the cell cycle into 5 phase and
mitosis in order to identify major rate-limiting steps.
A commitment step is more difficult to identify. It can be defined as a point of no
return beyond which no alternative developmental pathways can be initiated until
the cell cycle in progress has finished. Thus practical identification of a commitment
step requires developmental fates as alternatives to the mitotic cell cycle; then, as
cells progress through the cell cycle their ability to undergo these alternatives can be
assessed. Problems arise because the various alternatives may become excluded at
different times during the cycle, making it difficult to define the true point of no
return.
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From our discussion so far it would seem that we are dealing with quite different
controls. However, this need not be the case. For example, a point of commitment to
the cell cycle in G\ might also be a major rate-limiting step of the cell cycle and an
initiation event for 5 phase. Indeed if this were the case we would feel encouraged
that, imperfect as our individual views of control are, together they are leading us
generally in the right direction. As we shall show, this does seem to be the case from
studies with the yeasts.
Cell cycle regulation in yeast
MITOT IC C E L L CYCLES OF THE YEASTS
The first parts of the cell cycle of both yeasts are similar to each other and to those
of other eukaryotic cells. There is a G\ period that becomes much expanded at very
low growth rates. After G\ there is a discrete S phase during which DNA replication
takes place using multiple replicons (Newlon, Petes, Hereford & Fangman, 1974).
However, the second part of the cell cycle differs between the two yeasts (Nurse,
1985a). Fission yeast is more typically eukaryotic, with a G2 period and mitosis.
During mitosis a microtubular spindle forms, chromosomes visibly condense and the
cell divides by medial fission. The condensation of Schiz. pombe chromosomes has
been dramatically demonstrated by Yanagida and co-workers using a mutant strain
defective in tubulin (Umesono, Hiraoka, Toda & Yanigida, 1983). In budding yeast
there is no real Gz period. A short mitotic spindle is formed during 5 phase and the
cell becomes arrested in mid-mitosis for the rest of the cycle. The spindle then
elongates, mitosis is completed and the cell divides by budding. No visible
chromosome condensation takes place. Both yeasts differ from most eukaryotes in
that the nuclear membrane does not break down during mitosis. As we shall see later
the difference in the organization of the two cell cycles is also reflected in their mitotic
controls.
CE L L CYCLE G E N E S OF T H E YEASTS
A large number of conditionally lethal cell cycle mutants have been isolated in the
two yeasts that are unable to complete the cell cycle (Hartwell, Mortimer, Culotti &
Culotti, 1973; Nurse, Thuriaux & Nasmyth, 1976; Reed, 1980; Nasmyth & Nurse,
1981). Most of these mutants are heat-sensitive although some are cold-sensitive
(Moir, Stewart, Osmond & Botstein, 1982; Hiraoka, Toda & Yanigida, 1984). The
mutants fall into two classes: those that continue growth and macromolecular
synthesis at their restrictive temperature and those that do not. Following our earlier
discussion, we consider only those mutants that can continue growth and macromolecular synthesis as being specifically defective in cell cycle progress. By this
criterion, about 50 cdc genes have been identified in budding yeast and about 40
genes in fission yeast. These genes define functions that are specifically required for
successful completion of the cell division cycle, but only a few can be expected to be
involved in cell cycle control.
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J . Hayles and P. Nurse
control
The major cell cycle control in budding yeast is located in G\ at start. ‘Start’ was
originally identified by the pioneering work of Hartwell, Pringle and co-workers. It is
the earliest gene-controlled event of the cell cycle identified to date and, therefore,
may be the point of initiation (Pringle & Hartwell, 1981). Traverse of start results in
commitment to the cell cycle with respect to the alternative developmental pathway
of conjugation (Hartwell, 1974). After start is passed conjugation is not possible until
the cell cycle in progress has been completed. The start event does not take place
until cells have attained a critical size: at low growth rates daughter cells are smaller
at division and so cells must grow for a longer period before passing start (Hartwell &
Unger, 1977; Carter & Jagadish, 1978: Lord & Wheals, 1980). Therefore, under
these conditions traverse of start is a major rate-limiting step of the cell cycle. We can
see that all three ways of identifying regulatory control events can be applied to start.
Start gene functions have been investigated by Reed and his co-workers. Four
gene functions, cdc28, -36, -37 and -39, are required to traverse start (Reed, 1980). It
has been claimed that mutants in other genes also block at or before start but in these
cases it has not been firmly established that the mutants can still undergo cellular
growth. For this reason they may not be defective specifically in cell cycle progress.
The mating pheromones oc and a factor also arrest cells at start to prepare them for
conjugation (Bucking-Throm, Duntze, Hartwell & Manney, 1973). Mutants in
cdc36 and -39 appear to be constitutive for mating pheromone response. In cells that
are unable to mate, e.g. a/ar diploids, the cdc36 and -39 mutants do not show cell
cycle arrest. cdc36 and -39 mutants also allow certain ste (sterile) mutants to mate
that are normally unable to do so (Shuster, 1982). This evidence suggests that these
two genes are involved in the mating pheromone response pathway that arrests cells
at start. Mutants in the other two start genes cdc28 and -37 are not influenced by the
mating system and so appear to be more central to the start control system.
A start control has also been identified in the fission yeast. Mutants in two genes,
cdc2 and cdclO, block at a point in the cell cycle during Gi where cells are still able to
conjugate if challenged to do so (Nurse & Bissett, 1981; Aves, Durkacz, Carr &
Nurse, 1985). But once past this point cells are committed to the mitotic cycle and
are unable to conjugate. Traverse of start in fission yeast also appears to be a major
rate-limiting step (Fantes, 1977; Nurse & Thuriaux, 1977; Nasmyth, Nurse &
Fraser, 1979). In mutants that divide at a reduced cell size, the Gi period before
start becomes expanded. A similar expansion is seen when growth rate is reduced
by growing cells in chemostat cultures (Nasmyth, 1979). As with budding yeast,
passage of start also requires growth to a critical cell size.
The cdc2 start gene in fission yeast is functionally similar to the cdc28 start gene in
budding yeast (Beach, Durkacz & Nurse, 1982). When the cdc28 gene is introduced
into mutants of cdc2 these cells are able to divide and grow at the restrictive
temperature. This suggests that the cdc2 and cdc28 genes are functionally inter­
changeable and therefore perform essentially similar functions within the two
organisms. Both genes have been sequenced (Lorincz & Reed, 1984; Hindley &
Phear, 1984) and a comparison of their predicted amino acid sequences shows that
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they are identical at 62% of their amino acid positions. This suggests that the two
genes are related by common descent. The two yeasts are not closely related:
sequence comparisons suggest that they may have diverged as much as 1000 million
years ago, close to the origin of eukaryotic life (Huysmans, Dams, Vandenberghe &
de Wachter, 1983). Therefore, any function that is conserved in both yeasts is likely
to be found in other organisms and it is possible that a similar control mechanism
exists in all eukaryotic cells.
Cell cycle regulation in yeast
g2
m ito tic c o n tr o l
A second major cell cycle control has been identified in the fission yeast (Nurse,
1975). This acts during the Gz period and determines the cell cycle timing of mitosis.
The control was identified as the major rate-limiting step in G2 by isolating ‘wee’
mutants, which are advanced through Gz and undergo mitosis and cell division
prematurely at a reduced size. The mutants define two genes weel and cdc2 (Nurse
& Thuriaux, 1980). The cdc2 gene is required twice during the cell cycle, in G\
at start, as discussed earlier, and then in Gz at the mitotic control. Recessive
temperature-sensitive lethal cdc2 mutants become blocked in Gi or Gz, depending on
where they are in the cell cycle when shifted to the restrictive temperature (Nurse &
Bissett, 1981). In contrast, dominant wee cdc2 and partially recessive weel~
mutations advance cells through Gz- The weel gene codes for an inhibitor and the
cdc2 gene for an activator in the mitotic control (Nurse & Thuriaux, 1980).
Changes in the medium or growth rate of the cells modulate the mitotic control.
Shifting cells into poor medium transiently advances cells through Gz as do wee
mutants (Fantes & Nurse, 1977). This can be explained if a critical cell size is
required before the mitotic control can be passed, which is modulated downwards in
poor growth medium. As a consequence, cells undergo cell division at a small size
and are too small to pass start in the subsequent cell cycle. The G\ period therefore
becomes expanded before the start point of commitment. With further nutrient
deprivation these uncommitted cells will undergo conjugation and sporulation or
enter stationary phase.
In budding yeast there does not appear to be an equivalent of this control because
as explained earlier there is no G2 period. The cdc28 gene is required for mitosis
(Piggott, Rai & Carter, 1982) but its mitotic function is completed very soon after
start, consistent with the early initiation of mitosis. There is an approximately
constant time from S phase to the completion of mitosis and thus a certain period of
time may be required to complete all the events of mitosis (Carter & Jagadish, 1978).
It is this that determines when mitosis is completed.
cdc2/cdc28 g e n e f u n c t i o n
The cdc2/cdc28 gene has a remarkable role in cell cycle control of the two yeasts.
It is involved in the G \/S phase transition at start and then again at the G2/M
transition of the mitotic control in fission yeast. Several powerful molecular
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J . Hayles and P. Nurse
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biological techniques have been used to investigate this gene and we shall now
summarize what these recent studies have shown concerning the molecular role of
this gene function in the cell.
Plasmids containing the cdc2 and cdc28 genes have been isolated from gene banks
by virtue of their ability to allow mutants of cdc2 and cdc28 to grow at their
restrictive temperatures (Nasmyth & Reed, 1980; Beach et al. 1982). The sequence
of the cdc28 gene reveals that it contains a single open reading frame encoding a
protein of 298 amino acids (Lorincz & Reed, 1984). A similar protein of 297 residues
is predicted from the sequence of the cdc2 gene, although in this case four introns
have to be postulated (Hindley & Phear, 1984). The two proteins have a 20%
identity with the src family of oncogene products and with cyclic-AMP-dependent
protein kinase. Most of this similarity can be accounted for by the presence of two
active sites within the protein: an ATP-binding site of the sort found in protein
kinases and a phosphorylation site (Nurse, 1985a). Therefore, it is likely that the
cdc2/28 gene function is a protein kinase that can become phosphorylated. Because
of its similarity to the src family of oncogenes the cdc2/28 gene could be considered
a primitive oncogene. We do not think that this is likely since most of the similarity is
due to the presence of the two active sites, and once these sites are removed from the
comparison there is very little further similarity.
Antibodies have been raised against a afc25-/3-galactosidase fusion, which
identifies a budding yeast protein of the correct relative molecular mass (M r) of
36000 (Reed, 1984). This protein has been shown to be the product of the cdc28
gene, since in strains containing the cdc28 gene on a multicopy number plasmid the
protein is increased in amount (Reed, Hadwiger & Lorincz, 1985). The cdc28 gene
product is phosphorylated and also has protein kinase activity. Exponentially
growing cells labelled with 32PC incorporate the isotope into the p36 protein. The
protein kinase activity has been assayed by following the transfer of phosphate
to a protein of Mr = 40000, which is co-immunoprecipitated with the cdc28
gene product. The 40000 Mr protein may be the natural substrate for the cdc28
protein kinase. Extracts made from temperature-sensitive cdc28 mutants also have
temperature-sensitive activity in vitro, proving that the protein kinase activity is
definitely associated with the cdc28 gene product. The protein kinase activity is
magnesium-dependent and apparently zinc-activated.
Antibodies have also been raised against the cdc2 gene product by using synthetic
peptides (V. Simanis & P. Nurse, unpublished data). These identify a protein from
fission yeast with a relative molecular mass of 34 000. This protein is overproduced in
cells with an increased level of cdc2 transcripts produced by fusion of the cdc2 gene
with the strong alcohol dehydrogenase (adh) promoter. The protein is phosphory­
lated like the cdc28 gene product and also has protein kinase activity. The kinase
activity is divalent-cation-dependent but in contrast with cdc28 is not activated by
zinc. These data confirm that the cdc2lcdc28 gene product is indeed a protein kinase
that becomes phosphorylated.
The cdc2 gene is not regulated at the level of transcription (Durkacz, Carr &
Nurse, 1986). No changes in steady-state transcript level are seen during the cell
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cycle or when cells undergo the transition from rapid growth into quiescence.
Nor are there any changes at the level of RNA processing; all four introns appear to
be used all the time. Therefore, changes in cdc2 transcript level or its processing do
not appear to be responsible for regulating either progress through the cell cycle
or the transition between rapid proliferation and quiescence. Even when cdc2
transcription is increased 30-fold over normal there is no disturbance of normal
regulation of the mitotic cycle.
The cdc2 antibody has been used to monitor changes in protein level and
phosphorylation state of the cdc2 gene product (V. Simanis & P. Nurse, unpublished
data). Surprisingly, no changes in either steady-state protein level or overall
phosphorylation state are found during the cell cycle. However, a dramatic change is
observed when cells become quiescent upon shift into nitrogen starvation conditions.
The level of cdc2 protein does not change, but it becomes rapidly dephosphorylated
and there is a dramatic reduction in the level of its protein kinase activity. Therefore,
exit of cells from the mitotic cell cycle into a quiescent state may well be regulated by
modulating the cdc2 protein kinase activity by changes in phosphorylation. The
reduction in protein kinase activity would then prevent the cell from initiating any
further mitotic cell cycles. The regulation of the phosphorylation state of the cdc2
gene product may be the key to understanding how the cell cycle is initiated. Also,
identification of substrates should be revealing about what the cdc2/28 gene protein
does and how it mediates the G \/S phase transition at start and the Gz/M transition
at the mitotic control (Dudani & Prasad, 1984).
Cell cycle regulation in yeast
cdc2(28 G EN E F U N C T IO N
Gene products that interact with the cdc2/28 gene function are potential
regulators and substrates. Two gene functions that interact closely with the cdc2
function at the mitotic control point are those of weel and cdc25 (Fantes, 1979;
Nurse & Thuriaux, 1980; Fantes, 1983). The weel gene codes for an inhibitor in the
mitotic control as discussed earlier. The cdc25 gene has been implicated in the
control because mutants in weel and wee mutants of cdc2 are able to suppress a
defective cdc25 gene function (Fantes, 1979). Normally, mutants of cdc25 are
unable to undergo mitosis but in a wee mutant background they are able to do so.
The suppressor effects on cdc25 suggest a close relationship between cdc25, weel
and cdc2, and several models have been proposed to account for their close
interaction. Direct evidence in favour of cdc25 being involved in the mitotic control
has recently been obtained after cloning the gene (Russell & Nurse, 1986).
When the cdc25 gene is fused behind the strong adh promoter, thus increasing its
transcription, cells are advanced through Gz into mitosis and cell division at a
reduced size. This establishes that the cdc25 gene function can be rate-limiting for
the timing of mitosis. The cdc25 gene function is also completely dispensable in a
weel mutant background. Deletion of the chromosomal copy of the cdc25 gene using
recombinant DNA procedures is lethal in a weel+ strain whereas a weel~ strain is
still viable. The similarity of the phenotype of cdc2 wee mutants, weel mutants and
G E N E S IN T E R A C T I N G W I T H THE
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of overproducing cdc25 suggests that the weel and cdc25 gene products are
regulators of the cdc2 protein kinase. The cdc25 gene codes for an inducer and the
weel gene for an inhibitor. If cdc25 is overproduced in a weel mutant then the
mitotic control is grossly disturbed, leading to various mitotic catastrophes and cell
death. These data are all consistent with the following model. The cdc2 gene product
is considered to be in two states, an active one and an inactive one. The cdc25 gene
product converts the inactive form into the active form and the weel gene product
converts the active form into the inactive form. The balance of these two gene
product activities determines when the cdc2 gene product becomes sufficiently active
to initiate mitosis.
Another gene product appears to interact directly with the cdc2 protein at both its
G\ and its Gz points of action. This gene, called sucl (for swppressor of cdc2) was
identified by mutants that allow temperature-sensitive lethal mutants of cdc2 to grow
at their restrictive temperature (Hayles, Beach, Durkacz & Nurse, 1986). The gene
has been cloned by exploiting the fact that overproduction of sucl+ transcripts from
a high copy-number plasmid enables certain cdc2 mutants to divide. The sup­
pression is also seen when the sucl+ gene is fused to the strong adh promoter.
Overproduction of the sucl+ transcript, and therefore presumably of the sucl+ gene
product, in a cdc2+ strain causes a delay of mitosis. This suggests that the sucl+ gene
product has a direct effect in its own right upon cell cycle progress and may
physically interact with the cdc2 protein. Further evidence in favour of this
hypothesis is that sucl suppression of cdc2 mutants is allele-specific. The role that
the sucl gene product may play is as yet unclear but it is an obvious candidate for a
protein that directly interacts with the cdc2 protein.
Various classes of mutants appear to act at a similar time to the start genes already
mentioned. They include cdc37 in budding yeast and cdclO in fission yeast. The
whil and whi2 (Sudbery, Goodey & Carter, 1980) mutants in budding yeast are
advanced into start at a reduced cell size so these may be important for regulating
cdc28 gene function. Mutants of the rani/pat 1 gene (Nurse, 19856; lino &
Yamamoto, 1985a,b) in fission yeast may also function close to the cdc2 point of
action at start. These mutants undergo conjugation and sporulation in conditions
under which cells should be progressing through the mitotic cell cycle. They appear
to be defective in the monitoring process that determines whether a cell should
become committed to conjugation and sporulation or mitosis. Study of all these
various mutants may turn out to be revealing about how cdc2/28 gene function is
regulated.
A N A L O G O U S CO NT RO L S IN OTHER E UKARYOTES
The work we have described indicates that there are two major cell cycle controls
in the yeasts that both involve the cdc2/28 protein kinase function: a commitment
control acting at the G \/S boundary and, at least in fission yeast, a mitotic control
acting at the Gz/M boundary. We will now consider whether there are any analogous
controls present in other eukaryotic cells.
Cell cycle regulation in yeast
163
Many workers studying animal cells have proposed that the major point of cell
cycle regulation occurs in late G\ (Pardee, Dunbrow, Hamlin & Kletzien, 1978).
These proposals have been based mainly on the observations that arresting cell
proliferation by using a variety of conditions such as serum starvation, nutrient
depletion or high cell density leads to G\ arrest. A good example of this type of
analysis is provided by the work of Pardee and his co-workers (Pardee, 1974), who
put forward the hypothesis that there is a restriction point, R, in late G\, which can
only be passed in conditions favourable for cell proliferation. When conditions are
not favourable cells become arrested at the R point and cannot enter the mitotic
cycle. Transformed cells may be defective in R point control, failing to arrest
correctly in unfavourable conditions.
Such an R point in G\ has obvious similarities to the start commitment control in
the yeasts. There are other features about G\ arrest points in animal cells that also
show similarities. It has been shown that in certain cases of differentiation cells leave
the mitotic cycle from a specific point in Gy (Scott, Florine, Willie & Yun, 1982).
In an in vitro adipocyte differentiation system, cells become committed to differ­
entiation from a point in G\ called Gd- Once past this point they are unable to
differentiate until they have completed the mitotic cycle in progress. However, it is
unlikely that a general argument of this sort can be made, since many cell types
undergoing differentiation still continue to proliferate. Traverse of a G\ arrest point
is also rate-limiting for entry into S phase in certain growth conditions; the G\ period
usually becomes much extended at low growth rates (Darzynkiewicz, Traganos &
Melamed, 1980). The major rate-limiting step may be the attainment of a critical cell
size as in the yeasts, but the evidence for mammalian cells is conflicting and if there is
a relationship between cell size and entry into the mitotic cycle it must be of a much
looser kind and easily dissociated (Shields et al. 1978). What is clear from cell fusion
experiments, though, is that factors accumulate during the G\ period that determine
the rate at which cells initiate S phase (Rao, Sunkara & Wilson, 1977; Rao &
Johnson,1974).
Mitotic controls have received much less attention in mammalian cells, although
some cell types do become arrested at some point in G2 (Gelfant, 1962; Pederson &
Gelfant, 1970). These cells are only a small proportion of the total population, but
when stimulated to divide they undergo mitosis without first entering 5 phase.
Mammalian cell fusion experiments also suggest that there are inhibitory factors that
can block cells in G2 before mitosis (Rao & Johnson, 1974). There have been more
extensive studies of mitotic control in the slime mould Physarum plasmodium.
Fusion of naturally synchronous plasmodia at different times of G2 have indi­
cated that mitotic inducing factors accumulate during the G2 period (Rusch,
Sachsenmaier, Behrens & Gruter, 1966). Such factors have been purified and found
to advance nuclei into mitosis (Loidl & Grobner, 1982). The factors can be
mimicked by Ehrlich ascites cell extracts containing protein kinase activity able to
phosphorylate histone HI in vitro (Inglis et al. 1976), suggesting a role for protein
phosphorylation in the mitotic control. A formally similar approach has been used
recently by Kirschner and his co-workers to investigate mitotic control in Xenopus
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eggs. They have investigated the role of maturation promoting factor (MPF) in
mitosis and meiosis. MPF was first identified as a factor required for maturation of
Xenopus oocytes; the maturation process involves the transition of G -arrested
oocytes into meiotic nuclear division. MPF also varies cyclically during the mitotic
cycle in early Xenopus embryos, peaking at mitosis (Gerhart, Wu & Kirschner, 1984;
Newport & Kirschner, 1984). This suggests that MPF may be a component of a
regulatory system controlling the initiation of mitosis. Preparations containing MPF
activity have been isolated from a variety of organisms including mitotically arrested
yeast and mammalian cells (Weintraub et al. 1982; Sunkara, Wright & Rao, 1979),
and so this regulatory system may be widespread amongst eukaryotic cells. It is also
possible that MPF may involve phosphorylation, since its activity is stabilized by
phosphatase inhibitors, and a fraction that is 20-fold enriched in MPF has protein
kinase activity (Wu & Gerhart, 1980).
Other evidence also indicates that protein phosphorylation may play an important
role in regulating progress through the eukaryotic cell cycle. A number of pro­
teins have been identified that only become phosphorylated during mitosis
(Sahasrabuddhe, Adlakha & Rao, 1984; Davis, Tsao, Fowler & Rao, 1983; Vandre,
Davis, Rao & Borisy, 1984). Antibodies have been raised against these proteins and a
subset of these have been shown to be associated with microtubular organizing
centres, which are responsible for organizing the mitotic spindle. A variety of other
proteins including histones also undergo changes in phosphorylation during the
cell cycle (Ottaviano & Gerace, 1985; Piras & Piras, 1975; Bradbury, Inglis &
Matthews, 1974; Gurley, Walters, Barham & Deaven, 1978: Yamashita, Nishimoto
& Sekiguchi, 1984).
Taken together these data establish that there are G\ and Gz control points in other
eukaryotic cells, including mammalian cells, which may be analogous to the two
controls identified in the two yeasts. Furthermore, phosphorylation has been
implicated in certain cases, suggesting that protein kinases that are possibly similar to
that encoded by cdc2l28 may have an important role to play in the controls.
2
cdc2/28 F U N C T IO N
We conclude this review by speculating on the molecular basis of the cdc2/28
function and its regulation. The best clue for regulation obtained so far is the
observation that phosphorylation and protein kinase activity of the cdc2 protein is
much reduced when cells become quiescent and accumulate before start. Therefore
phosphorylation and activation of the cdc2 protein kinase may regulate entry into the
mitotic cycle and the transition from G\ into S phase. This raises the question of how
the phosphorylation of the cdc2/28 protein is controlled. One exciting possibility is
provided by recent studies of the ras oncogene homologues and cyclic-AMPdependent protein phosphorylation in budding yeast. Mutants with reduced levels
of cyclic-AMP-dependent protein phosphorylation accumulate in G\ before start
(Matsumoto, Uno, Oshima & Ishikawa, 1982). These observations have led to
the model that cyclic-AMP-dependent protein phosphorylation may regulate the
R E G U L A T I O N OF T H E
165
initiation of the mitotic cell cycle. Furthermore, the control of phosphorylation may
be determined by the ras oncogene homologues modulating adenylate cyclase
activity and thus cyclic AMP levels in the cell (Toda et al. 1985). An obvious
candidate for activation by this phosphorylation system is the cdc2/28 function,
which could then proceed to initiate the cell cycle. This is an intriguing possibility
since it would provide a direct link between the ras oncogene and the control of cell
proliferation.
However, the situation may be more complex than this. The mutants defective in
cyclic-AMP-dependent protein phosphorylation that accumulate in G\ are also
defective in overall growth of the cell (Matsumoto et al. 1982). As explained earlier,
cells that grow at a reduced rate often accumulate in G\. Therefore, the G\ block
point of these mutants may simply be a pleiotropic effect very distant from the initial
biochemical defect. The critical experiment in these studies concerns the bcyl gene,
mutants of which result in a deficiency of the regulatory subunit of cyclic-AMPdependent protein kinase. These mutants have a cyclic-AMP-dependent protein
kinase, which is now cyclic-AMP-independent, and consequently have a constitutive
kinase activity. If the model is correct these mutants should undergo cell pro­
liferation even when conditions are not appropriate, for example under nutrient
deprivation. Mutants that behave in this way do exist, for example whi2 mutants,
continue to proliferate even when they have run out of nutrients. They continue to
bud and divide and produce very small cells, bcyl mutants do not seem to have this
phenotype. It has been claimed that on starvation bcyl mutants continue budding
and eventually arrest in a budded state (Matsumoto, Uno & Ishikawa, 1983).
However, if the results are examined more carefully it appears that cells do not
continue to bud, they simply fail to complete the mitotic cycle that is in progress
when shifted into nutrient starvation conditions. This can be seen by the low increase
in cell number observed in these mutants when shifted into nutrient starvation
conditions. They fail to complete the cell cycle in progress and thus arrest as budded
cells. Therefore, an alternative interpretation of the data is that the cells are sick and
are unable to complete the cell cycle in progress in starvation conditions. Until these
points are clarified the roles of cyclic AMP and the ras gene product in cell cycle
regulation, and thus their effects on the cdc2/28 gene function and overall
phosphorylation, will remain uncertain.
Overall phosphorylation of the cdc2/28 protein is unlikely to be responsible for
regulating the mitotic control since phosphorylation levels are unchanged during G2
and mitosis. Of course, it is possible that there may be a more subtle change in
phosphorylation not detected to date, such as different amino acid residues becoming
modified. Alternatively, the cdc2)28 gene function could be regulated by substrate
availability. Changes in compartmentation of the cdc2/28 protein or its substrates
may be responsible for determining the cell cycle timing of mitosis.
Cell cycle regulation in yeast
cdc2/28 F U N C T IO N
What could the single cdc2/28 gene function be doing that facilitates both the
transitions from G\ into S and from Gz into M ? Many different changes occur as cells
M O L E C U L A R BASIS OF T H E
166
J . Hayles and P. Nurse
progress through the cell cycle and it is difficult to identify those processes that may
be important. We suggest that the most important processes that occur are those
central to DNA replication and chromosome segregation. One such process is a
change in the chromatin condensation state. Chromatin must decondense to become
available for replication at 5 phase, and then condense in mitosis to permit
segregation of the chromosomes. The hypothesis has been suggested that there is a
cycle of chromatin condensation and decondensation (Mazia, 1963): when chroma­
tin is maximally decondensed 5 phase occurs and when it is maximally condensed
mitosis takes place. Decondensation of chromatin may make the DNA available to a
constantly present replication apparatus giving rise to a discrete S phase (Newport &
Kirschner, 1984).
A second important process concerns alterations in the cytoskeleton. Changes
occur in the cytoskeleton during the cell cycle, the most dramatic being the
disappearance of cytoplasmic microtubules at the Gz/M transition and the formation
of a mitotic spindle, which is necessary for accurate segregation of the sister
chromatids. A transient disassembly of microtubules has also been suggested as a
necessary requirement for the initiation of 5 phase (Otto et al. 1979; Friedkin, Legg
& Rozengurt, 1979; Crossin & Carney, 1981; Thyberg, 1984). Therefore, there may
well be changes in both chromatin and cytoskeletal organization at the G\/S and
Gz/M transitions, and we suggest that these changes may be brought about by the
cdc2/28 protein kinase phosphorylating specific proteins. Obvious candidates for
these proteins are those associated with chromatin and the cytoskeleton; for example,
both histones and microtubular associated proteins (MAPs) are believed to be
important for organization of chromatin and the cytoskeleton. Several of the histones
undergo substantial changes in phosphorylation during the cell cycle and may be
partly responsible for the state of chromatin condensation (Dolby, Belmont, Borun
& Nicolini, 1981; Gurley et al. 1978; Bradbury et al. 1974; Hanks, Rodriquez &
Rao, 1983). Both MAPs 1 and 2 can become phosphorylated (Herrmann, Dalton &
Wiche, 1985) and their level of phosphorylation may be important for levels of
microtubular disassembly.
The cdc2/28 protein when activated could phosphorylate a series of proteins
within the cell which could bring about changes in chromatin and cytoskeletal
organization. The reason for the different behaviour at G\/S and Gz/M is not clear
but could be due to different availability of substrates. The cdc2/28 protein at the
apex of a protein phosphorylation cascade can potentially control the two major
transitions involved in cell cycle control. We propose that this is how the cell cycle is
regulated in yeast and knowledge gained from these studies may provide a useful
framework for studying cell cycle regulation in higher eukaryotes.
We thank Steve Aves, Tony Carr, Iain Hagan, Pete Magee, Paul Russell and Viesturs Simanis
for helpful discussions and for supplying unpublished data.
Cell cycle regulation in yeast
167
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