CHAPTER 40
Introduction to the Cell Cycle
The cell cycle is the series of events that leads to the
duplication and division of a cell. Research on the molec-
ular events of cell-cycle control revealed that variations
of similar mechanisms operate the cell cycles of all
eukaryotes from yeasts to humans. Furthermore, the
components that regulate cell growth and division also
play key roles in the cessation of cell division that is
required for cells to differentiate. Control of the cell
cycle is of major importance to human health because
cancer is usually caused by perturbations of cell-cycle
regulation. Based on 2010 to 2012 data, 40% of Ameri-
cans will develop cancer during their lifetime.
Although animal cells have a wide variety of special-
ized cell cycles, the cells in the stratified epithelium
that forms skin illustrate the most common types of cell
cycles (Fig. 40.1). The basal layer of the epithelium is
composed of stem cells that divide only occasionally
(see Box 41.2). They can activate the cell cycle on
demand and then return to a nondividing state. Stem cell
populations can replenish themselves by symmetrical
division, but when specific signals induce them to pro-
liferate, usually one daughter cell remains a stem cell and
the other enters a pool of rapidly dividing cells. The
dividing cells populate the upper layers of the epithe-
lium, stop dividing, and gradually differentiate into the
specialized cells that cover the surface.
Like stem cells, fibroblasts of the connective tissue
(see Fig. 28.2) typically are in a nondividing state, but
they can be stimulated to enter the cell cycle following
wounding or other stimuli (see Fig. 32.11). In the most
extreme case, the nervous system contains a few stem
cells and a few dividing glial cells, but most neurons,
once differentiated, can live for more than 100 years
without dividing again.
Principles of Cell-Cycle Regulation
The goal of the cell cycle in most cases is to produce
two daughter cells that are accurate copies of the parent
(Fig. 40.2). The cell cycle integrates a continuous growth
cycle (an increase in cell mass) with a discontinuous
division or chromosome cycle (the replication and
partitioning of the genome into two daughter cells). The
chromosome cycle is driven by a sequence of enzy-
matic cascades that produce a sequence of discrete
biochemical states of the cytoplasm. Progress through
the cell cycle is ratchet-like and irreversible because each
new state arises not only by expression or activation of
a new cohort of activities, but also by destruction or
inactivation of key activities characteristic of the pre-
ceding state. Later sections of this chapter explain
these mechanisms.
Phases of the Cell Cycle
In describing the cell cycle, it is convenient to divide the
process into several phases. Recognition of these phases
began in 1882, when Flemming named the process of
nuclear division mitosis (from the Greek mito, or
thread) after he first observed the condensed chromo-
somes. Mitosis was a clear cell cycle landmark, and the
rest of the cell cycle between mitoses was called inter-
phase (Box 40.1).
Once DNA was recognized as the agent of heredity in
the 1940s, it was deduced that it must be duplicated
at some time during interphase so that daughter cells
can each receive a full complement of genetic material.
In 1953, a key experiment identified the relationship
between the timing of DNA synthesis and the mitotic
cycle (Fig. 40.3). This defined the four cell-cycle phases
as they are known today (see Fig. 40.2).
Each cell is born at the completion of the M phase,
which includes mitosis, the partitioning of the chromo-
somes and other cellular components, and cytokinesis,
the division of the cytoplasm. The chromosomal DNA is
replicated during S phase (synthetic phase). The remain-
ing two phases are gaps between mitosis and the S
phase. The G1 phase (first gap phase) is the interval
697
698 SECTION X n Cell Cycle

Death Final stage of

differentiation
in skin

Cessation
of cycling
Terminal
differentiation

Rapid
cell cycles
Expansion of
population

Infrequent
cell cycles
Renewal of
Activation stem cell
population

A B Stem cell

FIGURE 40.1 CELL CYCLES IN A STRATIFIED EPITHELIUM. A, Light micrograph of a section of skin, a stratified squamous epithelium,
stained with hematoxylin and eosin (H&E). B, Diagram showing the different types of cell cycles at the various levels of this epithelium.

A. Cell-cycle details Mitosis

B. Cycle phases in cultured cell
(not to scale)
M Cell-cycle phase Length (hours)
G1 10
Check for Check for
damaged or chromosome S 7.5
unduplicated attachment to Cytokinesis
G2 3.5
DNA mitotic spindle
M 1.0
Generation time 22
DNA
ENLARGED VIEW G0
OF CHROMOSOME

C. Time-scaled diagram
G2 (times in hours)
Growth G
1
in mass 22 0
21
Cohesion M
established
in S phase Check for G2
DNA damage
17.5
G1
Check for DNA Restriction point:
S
damage or stalled pass only if environmental
replication forks conditions favorable
S
Chromosome Centrosome
duplication duplication starts 10

FIGURE 40.2 INTRODUCTION TO THE CELL-CYCLE PHASES. A, Diagrams of cellular morphology and chromosome structure across
the cell cycle. B, Length of cell-cycle phases in cultured cells. C, Time scale of cell-cycle phases.
 CHAPTER 40 n Introduction to the Cell Cycle 699

BOX 40.1 Selected Key Terms

M phase: Cell division, comprising mitosis, when a fully

grown cell segregates the replicated chromosomes to
opposite ends of a molecular scaffold, termed the Nucleus Cell synthesizing DNA
spindle, and cytokinesis, when the cell cleaves between
the separated chromosomes to produce two daughter
cells. In general, each daughter cell receives a comple-
Add 32P, incubate briefly,
ment of genetic material and organelles identical to then wash out free 32P
that of the parent cell.
Interphase: The portion of the cell cycle when cells grow
and replicate their DNA. Interphase has three sections.
The G1 (first gap) phase is the interval between mitosis
and the onset of DNA replication. The S (synthetic)
phase is the time when DNA is replicated. The G2 Photographic
(second gap) phase is the interval between the termina- emulsion
tion of DNA replication and the onset of mitosis. In
multicellular organisms, many differentiated cells no
longer actively divide. These nondividing cells (which
Cell exposes
may physiologically be extremely active) are in the G0 photographic
phase, a branch of the G1 phase. emulsion
Checkpoints: Biochemical circuits that regulate cell-cycle
transitions in response to the physiological condition
of the cell and signals from its environment. Check- VIEW IN
MICROSCOPE
points detect damage to the DNA due to external
agents or problems that arise during DNA replication
and trigger the DNA damage response. Other check-
points detect problems that arise during attachment of
chromosomes to the spindle.

between mitosis and the start of DNA replication. The

G2 phase (second gap phase) is the interval between the
completion of DNA replication and mitosis. All cycling
cells have an M phase and an S phase. The G1 and G2
phases vary in length and are very short in some early
embryos. The following sections describe the stages of
the cell cycle, starting just after the birth of the cell.
G1 Phase
G1 is typically the longest and most variable cell-cycle
phase. When cells are born at cytokinesis, they are
roughly half the size they were before mitosis, and
during G1, they grow back toward an optimal size.
During this time, many activities involved in cell-cycle
progression are repressed so that the cell cannot initiate
a new round of proliferation. This repressive control
system is called the restriction point. If the supply of
nutrients is poor or if cells receive an antiproliferative
stimulus such as a signal to embark on terminal differen-
tiation, they delay their progress through the cell cycle
in G1 or exit the cycle to enter G0 (see G0 and Growth
Control below). However, if appropriate positive stimuli
are received, cells overcome the restriction point block
and trigger a program of gene expression that commits
them to a new cycle of DNA replication and cell division.
Cancer cells often have defects in restriction point
20% of cells turn the emulsion black
FIGURE 40.3 DISCOVERY OF CELL-CYCLE STAGES. To deter-
mine whether cells synthesize DNA during a defined portion of the cell
cycle or constantly throughout the entire cycle (as is the case in bac-
teria, for example), Howard and Pelc fed a radioactive component of
DNA (32P) to onion root tip cells, spread the cells in a thin layer on a
microscope slide, washed away the 32P that had not become incor-
porated into DNA, and overlayered the slide with photographic emul-
sion. After incubation in the dark, the emulsion was developed like film
and examined with a light microscope. The nuclei of cells active in
replicating their DNA incorporated the radioactive 32P into DNA and
exposed the photographic emulsion above them. Two possible out-
comes were predicted. If cells synthesized DNA constantly during
interphase, then all cells would incorporate the radioactive label. Con-
versely, if each cell synthesized DNA only during a discrete portion of
the cell cycle, then only cells engaged in active replication during the
period of exposure to 32P would expose the photographic emulsion.
When the slides were examined, 20% of the interphase cell nuclei were
labeled, proving that cells synthesize DNA only during a discrete
portion of interphase. Mitotic cells were unlabeled. Assuming that the
cells traverse the cycle at a more or less constant rate, it was possible
to calculate the length of the synthetic phase. Overall, the time between
successive divisionsthe generation timewas approximately 30
hours in the root tip cells. If approximately 20% of the cells were
labeled, then approximately 20% of the 30-hour generation time must
be spent in DNA synthesis. Thus, 0.2 30, or 6 hours, was spent in
replication. (Data from Pelc HA Sr. Synthesis of DNA in normal and
irradiated cells and its relation to chromosome breakage. Heredity
Suppl. 1953;6:261273.)
700 SECTION X n Cell Cycle
control and continue to grow and attempt to divide even
in the absence of appropriate environmental signals.
G0 and Growth Control
Most cells of multicellular organisms differentiate to
carry out specialized functions and no longer divide.
Such cells are considered to be in the G0 phase. Cells
often enter G0 directly as they exit their last mitosis. G0
cells are not dormant; indeed, they are often actively
engaged in protein synthesis and secretion, and they may
be highly motile. Many G0 cells have a nonmotile primary
cilium, which is an important sensory organelle (see Fig.
38.19). The G0 phase is not necessarily permanent. In
some specialized cases, G0 cells may be recruited to
reenter the cell cycle in response to specific stimuli.
Cell-cycle reentry involves changes in gene expression
and protein stability and disassembly of the primary
cilium, if present. This process must be highly regulated,
as the uncontrolled proliferation of cells in a multicel-
lular organism can lead to cancer.
S Phase
Chromosomes of higher eukaryotes are so large that
replication of the DNA must be initiated at many differ-
ent sites, termed origins of replication. In budding
yeast, the approximately 400 origins are spaced an
average of 30,000 base pairs apart. An average human
chromosome contains about 150 106 base pairs of
DNA, approximately 10 times the size of the entire
budding yeast genome, so many more origins are
required. Each region of the chromosome that is repli-
cated from a single origin is referred to as a replicon.
Groups of neighboring replicons cluster in topologically
associating domains (TADs) (see Chapter 8).
Proliferating diploid cells replicate their DNA once,
and only once, each cell cycle. Each origin of replication
is prepared for replication by the formation of a prerep-
lication complex during G1 (a process that is referred to
as licensing). As each origin fires during S phase, the
prereplication complex is dismantled and cannot be
reassembled until the next G1 phase. This ensures that
each origin fires only once per cell cycle. The cyclic
nature of origin licensing is driven at least in part by
fluctuations in the activity of cyclin-dependent kinases
and protein destruction machinery (discussed later).
During replication, the duplicated DNA molecules,
called sister chromatids, become linked to each other
by a protein complex called cohesin (see Fig. 8.18). This
pairing of sister chromatids is important for their orderly
segregation later in mitosis (see Fig. 44.16).
G2 Phase
In most cells of metazoans, G2 is a relatively brief period
during which key enzymatic activities that will trigger
the entry into mitosis gradually accumulate and are con-
verted to active forms. When these activities reach a
critical threshold level, the cell enters mitosis. Along the
way, the chromatin and cytoskeleton are prepared for
the dramatic structural changes that will occur during
mitosis. If damaged DNA is detected during G2, a check-
point activates the DNA damage response and delays
entry of the cell into mitosis.
M Phase
During M phase (mitosis and the subsequent cytokine-
sis), chromosomes and cytoplasm are partitioned into
two daughter cells. Mitosis is normally divided into five
discrete phases.
Prophase is defined by the onset of chromosome
condensation and is actually the final part of G2 phase.
TADs disassemble inside the intact nucleus and mitotic
chromosomes begin to form their characteristic array
of loops (see Chapter 8). In the cytoplasm, a dramatic
change in the dynamic properties of the microtubules
decreases their half-lives from approximately 10 minutes
to approximately 30 seconds. The duplicated centro-
somes (centrioles and associated pericentriolar material
in animal cells; see Fig. 34.14) separate and form the two
poles of the mitotic spindle.
Prometaphase begins when the nuclear envelope
breaks down (in higher eukaryotes) and chromosomes
begin to attach randomly to microtubules emanating
from the two poles of the forming mitotic spindle. Other
microtubules originate on chromosomes and within the
mitotic spindle. As both kinetochores on a pair of sister
chromatids attach to microtubules from opposite spindle
poles, the pair of chromatids slowly moves to a point
midway between the poles. When all chromosomes are
properly attached, the cell is said to be in metaphase.
The exit from mitosis begins at anaphase with abrupt
separation of the two sister chromatids from one
another. Most cohesion molecules linking sister chroma-
tids are removed without cleavage during prophase in a
process initiated by mitosis-specific phosphorylation.
Proteolytic cleavage of the remaining cohesin molecules
triggers the metaphase-anaphase transition. During ana-
phase, the separated sister chromatids move to the two
spindle poles (anaphase A), which themselves move
apart (anaphase B). As the chromatids approach the
spindle poles, the nuclear envelope reforms on the
surface of the chromatin. At this point, the cell is said to
be in telophase.
Finally, during telophase, a contractile ring of actin
and myosin assembles as a circumferential belt at the
cortex midway between spindle poles and constricts the
equator of the cell. The separation of the two daughter
cells from one another is called cytokinesis.
Control of Cell-Cycle Progression
Control networks and checkpoints regulate progres-
sion of the cell cycle. Checkpoints are biochemical

circuits that detect external or internal stimuli and send
appropriate signals to the cell-cycle system. The restric-
tion point in G1 phase is a control network that
integrates the physiological state of the cell with its
environment, including input from other cells and
interactions with the surrounding extracellular matrix.
Cells must receive appropriate growth stimuli from
their environment to progress past this point in the
G1 phase; if not they may live on without dying or
commit suicide by apoptosis (see Chapter 46).
DNA damage checkpoints operate throughout
interphase. If damage is detected, the DNA damage
response initiates a cascade of events that blocks cell-
cycle progression and can also trigger cell death by apop-
tosis. Problems with DNA replication generally produce
single-stranded DNA and activate the DNA damage
response. This response stabilizes stalled replication
forks so that they can be repaired. During mitosis, the
spindle assembly checkpoint delays the onset of
chromosome segregation until all chromosomes are
attached properly to the mitotic spindle.
The DNA damage response regulates cell-cycle pro-
gression in a three-tier pathway (Fig. 40.4). First sensors
detect DNA damage. These sensors activate transduc-
ers, which include both protein kinases and transcrip-
tional activators. The transducers act on effectors that
ultimately block cell-cycle progression and may also
fulfill other functions. Two key protein kinases, ataxia-
telangiectasia mutated (ATM) and ataxia-telangiectasia
and Rad9 related (ATR), lie at the head of the pathway
and may also act as sensors of DNA damage. They acti-
vate two transducer kinases, Chk1 and Chk2, as well as
a transcription factor called p53 that induces the expres-
sion of a cohort of genes that halt cell-cycle progression
by inhibiting cyclin-dependent kinases as well as genes
Problems at
DNA breaks replication forks
Genotoxic
stress
ATM ATM ATR + cofactors Sensors
dimer monomer bind ssDNA
Chk2 p53 Chk1 Transducers
kinase transcription kinase
factor
Cell cycle proteins Effectors
DNA repair proteins
Apoptosis Cell-cycle DNA repair Responses
arrest
FIGURE 40.4 ELEMENTS OF THE DNA DAMAGE CHECK-
POINT AND RESPONSE SYSTEM. ATM, ataxia-telangiectasia
mutated; ATR, ataxia-telangiectasia and Rad9 related; ssDNA, single-
stranded DNA.
CHAPTER 40 n Introduction to the Cell Cycle 701
that trigger cell death by apoptosis. Chapters 41 and 43
discuss these proteins in detail.
Biochemical Basis of Cell-Cycle Transitions
Transitions between cell-cycle phases are triggered by a
network of protein kinases and phosphatases that is
linked to the discontinuous events of the chromosome
cycle by the periodic accumulation, modification, and
destruction of several key components. This section pro-
vides a general introduction to the most important com-
ponents of this network.
Cyclin-Dependent Kinases
Genetic analysis of the cell cycle in the fission yeast
Schizosaccharomyces pombe identified a gene called
cell division cycle2+ (cdc2+) that is essential for cell-
cycle progression during both the G1 S and G2 M
transitions (Box 40.2). The product of this gene, a protein
kinase of 34,000Da originally called p34cdc2, is the pro-
totype for a family of protein kinases that is crucial for
cell-cycle progression in all eukaryotes. This mechanism
of cell-cycle control is so well conserved that a human
homolog of p34cdc2 can replace the yeast protein, restor-
ing a normal cell cycle to a cdc2 mutant yeast. Boxes
40.3 and 40.4 present a number of the key experiments
and experimental systems that led to the identification
of the molecules that drive the cell cycle.
Humans have more than 10 distinct protein kinases
related to p34cdc2, although only a few are involved in
cell-cycle control. To be active, each enzyme must associ-
ate with a regulatory subunit called a cyclin. Thus, they
have been termed cyclin-dependent kinases (Cdks).
p34cdc2, now termed Cdk1, seems to function primarily
in the regulation of the G2 M transition in animal cells.
Cdk2 (plus Cdk4 and Cdk6 in some cell types) is involved
in passage of the restriction point during G1. Cdk2 also
contributes to the G2 M transition, although Cdk1 is
the only Cdk absolutely essential for this step (Appendix
40.1). Cdk7 is important for activation of other Cdks,
and also appears to participate in transcribing RNA
and repairing damaged DNA. Other Cdks participate in
diverse processes ranging from transcriptional regulation
to neuronal differentiation. Surprisingly, fibroblasts from
mice that lack Cdk2, Cdk4, or Cdk6 are viable; other
Cdks can drive the cell cycle if necessary. The mice suffer
developmental difficulties because those genes are
needed for the differentiation of particular cell types.
Cyclins
Cdks require cyclin binding for catalytic activity (Fig.
40.13). Cyclins were discovered in rapidly dividing inver-
tebrate embryos as proteins that accumulate gradually
during interphase and are abruptly destroyed during
mitosis (see Fig. 40.11). This process of cyclic accumula-
tion and destruction inspired their name.
702 SECTION X n Cell Cycle
BOX 40.2 Use of Genetics to Study the Cell Cycle
Studies of the distantly related budding and fission yeasts
Saccharomyces cerevisiae and Schizosaccharomyces pombe
(see Fig. 2.8) were important for understanding the cell
cycle for several reasons. First, the proteins that control the
cell cycle are remarkably conserved between yeasts and
mammals. Second, both yeast genomes are small, simplifying
the discovery of important gene products. Third, genetic
analysis is straightforward, as both yeasts can grow as hap-
loids, and both efficiently incorporate cloned DNA into their
chromosomes by homologous recombination.
These two yeasts evolved very different strategies for cell
division. Budding yeasts divide by assembling a single bud
on the surface of the cell every cell cycle. Fission yeasts
divide by fission across the center of an elongated cell. A
useful feature of using yeast to study the cell cycle is that
the stage of the cell cycle is revealed by the cellular morphol-
ogy in the light microscope. For budding yeast, unbudded
cells are in G1, cells with buds smaller than the mother cell
are in S phase, and cells whose buds are similar in size to
the mother cell are in G2 or M. For fission yeast, cell length
provides a yardstick for estimating cell-cycle position.
The cell cycles of both yeasts differ from those of animal
cells. In budding yeast, much of the 90-minute cell cycle is
spent in G1. Thus, the networks controlling the G1 S transi-
tion are particularly amenable to study. In contrast, a fission
yeast spends most of its 2-hour cell cycle in G2. S phase
follows separation of sister chromatids and occurs prior to
cytokinesis. Thus, the control of the G2 M transition is
readily studied in fission yeast. During mitosis, the nuclear
envelopes of both yeasts remain intact, so chromosomes
segregate on a spindle inside the nucleus.
Genetic studies revealed that the yeast cell cycle is a
dependent pathway whereby events in the cycle occur nor-
mally only after earlier processes are completed. The cell
cycle can be modeled as a line of dominoes, each domino
corresponding to the action of a gene product that is essen-
tial for cell-cycle progression (Fig. 40.5) and the nth domino
Model of the cell cycle as a
simple dependent pathway
Wild type
CDC mutant
FIGURE 40.5 THE CELL CYCLE MAY BE MODELED AS A
SIMPLE DEPENDENT PATHWAY. A cell division cycle (CDC)
mutation can block further progression along the pathway at a
characteristic point in the cell cycle.
falling only when knocked down by the (n-1)th domino.
According to the model, mutations in genes that are essential
for cell-cycle progression cause an entire culture of yeast to
accumulate at a single point in the cell cycle (the point at
which the defective gene product first becomes essential).
This is referred to as the arrest point. Fig. 40.5 shows this
by including a mutant domino that does not fall over when
struck by the upstream domino. Mutants that meet this cri-
terion are called cell division cycle mutants or CDC
mutants. Genetic screens for CDC mutants have identified
many important genes involved in cell-cycle control.
Because CDC genes are essential for cell-cycle progres-
sion, it is impossible to propagate strains of yeast carrying
CDC mutants unless the mutants have a conditional lethal
phenotype. The most commonly used conditional lethal
mutations are temperature sensitive (ts). Many yeast
temperature-sensitive mutants are viable at 23C (the per-
missive temperature), but cease dividing at 36C (the
restrictive temperature). Temperature-sensitive proteins
often have an altered amino acid sequence, but occasionally,
the lack of a gene product can cause a ts phenotype. More
recently, the use of auxin-inducible degrons (see Chapters 6
and 23) has enabled experimenters to study the conse-
quences of depleting an essential protein from yeast in a
matter of minutes.
Fission yeasts with CDC mutants affecting the entry into
mitosis have distinctive morphologies. Cells mutant in Wee1
(a kinase that keeps cyclin-dependent kinase1 [Cdk1] inac-
tive prior to mitosis) enter mitosis prematurely and are
shorter than normal (Fig. 40.6B). In contrast, cells lacking
Cdc25 (a phosphatase that counteracts Wee1 and activates
Cdk1) are unable to undergo mitosis but continue their
growth cycle, therefore becoming greatly elongated (Fig.
40.6C). This simple morphologic assay allowed straightfor-
ward classification of yeast CDC genes into those that stimu-
late progression through mitosis and those that retard entry
into mitosis.
A. Wild type B. Wee1 mutant C. Cdc25 mutant
FIGURE 40.6 FLUORESCENCE MICROGRAPHS OF
FISSION YEAST CELLS ILLUSTRATING PHENOTYPES OF
CELL-CYCLE MUTATIONS. Cell walls and nuclei are stained.
A, Wild-type cells. B, A wee1 mutation that accelerates entry into
mitosis at the restrictive temperature. C, A cdc25 mutation that
delays entry into mitosis at the restrictive temperature. (Courtesy H.
Ohkura, Wellcome Trust Institute for Cell Biology, University of Edin-
burgh, United Kingdom.)

BOX 40.3 Studies of the Cell Cycle in Vitro
Amphibian oocytes and eggs are storehouses of most compo-
nents needed for cell-cycle progression. Oocytes are arrested
in G2 until a surge of the hormone progesterone causes them
to mature into eggs, which are then naturally arrested in
metaphase of the second meiotic division (see Chapter 45).
After fertilization, the embryo of the South African clawed
frog (Xenopus laevis) undergoes a rapid burst of cell divi-
sions. An initial cell cycle 90 minutes long is followed by a
rapid succession of 11 cleavages spaced only 30 minutes
apart to produce an embryo of 4096 cells (Fig. 40.7).
Thirty minutes per cycle is insufficient to transcribe and
translate all the genes needed to make the daughter cells that
are produced at each division. The frog solves this problem
1 Cleavage 11 Cleavages
90 minutes 30 minutes
apart
A B C they are readily accessible to biochemical manipulation. For
Somatic cell enlarged 10
FIGURE 40.7 CLEAVAGES SUBDIVIDE THE EGG DURING
XENOPUS EARLY DEVELOPMENT. A, Fertilized egg. B, Two-cell
stage. C, Multicellular embryo. Compare size of somatic cell and
egg.
A. Tightly B. Eggs crushed C. Added sperm
packed by centrifugation nucleus with
eggs membrane
removed
Lipid
Centrifuge
hard
Extract
Xenopus
eggs
Pellet
FIGURE 40.8 USE OF XENOPUS EGG EXTRACTS TO STUDY THE CELL CYCLE. AB, Making a Xenopus egg extract that is
competent to carry out cell-cycle oscillations in vitro. CF, A cycling Xenopus extract undergoes alternating S and M phases. G1 and G2
phases are minimal (as they are during early development of the frog).
Humans have at least 16 different cyclin proteins that
range in size from 35 to 130kD. The highly conserved
cyclin box domain, which docks with the Cdk partners,
is the defining structural feature of these proteins. Only
a handful of cyclin isoforms are involved in cell-cycle
control. Of those that are, some function during G1
phase, others during G2 phase, and still others during
M phase.
Positive Regulation of Cyclin-Dependent Kinase
Structure and Function
Cdks monomers are intrinsically inactive, so they depend
on activation by cyclins and are regulated by positive and
CHAPTER 40 n Introduction to the Cell Cycle 703
by making oocytes extremely large (~500,000 times the
volume of a typical somatic cell) and storing within them
vast stockpiles of the structural components needed to make
cells. As a result, only DNA and a very few proteins need be
synthesized during early embryonic divisions. In addition to
structural components, many factors that regulate normal
cell-cycle progression are also stockpiled in oocytes. These
features make Xenopus oocytes an excellent source of mate-
rial for biochemical analysis of the cell cycle.
Remarkably, it is possible to make cell-free extracts from
Xenopus eggs that progress through the cell cycle in vitro
(Fig. 40.8). Nuclei from G1 cells or haploid sperm nuclei,
when added to these extracts, efficiently replicate their DNA
and proceed through the cell cycle into mitosis, complete
with chromosome condensation, nuclear envelope break-
down, chromosome alignment on a spindle, and anaphase
segregation of sister chromatids without any additions to
the tube. Because these events occur in a cell-free milieu,
example, antibodies and other proteins can be added to the
extracts, and their effect on the cell cycle can readily be
determined. Thus, the Xenopus extract system offers a pow-
erful tool for testing the role of various proteins in the cell
cycle in higher eukaryotes.
D. Reassembly E. DNA replication F. Mitosis
of nucleus
negative controls. Like other eukaryotic protein kinases
(see Fig. 25.3), Cdks have a bilobed structure with the
active site in a deep cleft between a small N-terminal and
larger C-terminal domain. Monomeric Cdks have a flex-
ible T loop that blocks the mouth of the catalytic pocket.
In addition, a short -helix is oriented such that a glu-
tamic acid required for adenosine triphosphate (ATP)
hydrolysis points away from the catalytic cleft. As a
result, ATP bound by the monomeric kinase cannot
transfer its -phosphate to protein substrates (see
Fig. 40.13A).
Several different mechanisms regulate Cdk activity
(Fig. 40.14). On one hand, cyclin binding and
704 SECTION X n Cell Cycle

BOX 40.4 Discovery of Factors Essential for Cell-Cycle Progression

The best early evidence for the existence of positive induc-
ers of cell-cycle transitions in mammals was obtained in cell
fusion experiments. When cultured cells in S phase were
fused with cells in G1, the G1 nuclei initiated DNA replication
shortly thereafter. In contrast, if S phase cells were fused
with G2 cells, the G2 nuclei did not rereplicate their DNA
until after passing through mitosis. The most dramatic results
were obtained when mitotic cells were fused with inter-
phase cells. This caused the interphase cells to enter into
mitosis abruptly (as judged by nuclear envelope breakdown
and chromosome condensation). The phenomenon was
termed premature chromosome condensation (PCC).
The mitotic inducer could work in any cell-cycle phase (Fig.
40.9). If mitotic cells were fused with cells in G1 phase,
interphase chromosomes condensed into long, single fila-
ments. If the interphase cell was in G2 phase, the duplicated
chromosomes appeared as double filaments. If the inter-
phase cell was in the S phase, the partially replicated chro-
mosomes condensed into a complex pattern of single and
double condensed regions separated by regions of decon-
densed chromatin corresponding to sites where DNA was
actively replicating at the time of fusion.
Working independently, developmental biologists who
were interested in the control of cell division during early
development in frogs also discovered an activity that could
cause interphase cells to enter the M phase. They used a
micropipette to extract a tiny bit of cytoplasm from a mature
egg that was arrested in metaphase of meiosis II and
inject it into oocytes (which are in G2 phase). The oocytes
rapidly entered M phase, with concomitant chromosome
condensation and nuclear envelope disassembly (Fig. 40.10).
This stimulation to enter M phase is called maturation,
and the unknown factor present in the egg cytoplasm
that induced oocyte maturation was termed MPF, or
maturation-promoting factor (now often referred to as
M phasepromoting factor). It was realized early on that
MPF might be related to the inducer of mitosis detected in
the PCC experiments. In fact, extracts from mitotic tissue
culture cells could induce meiotic maturation when injected
into oocytes. Similar extracts from cells in other phases
of the cell cycle did not cause the G2/M phase transition
in oocytes.
Other cell biologists studying protein synthesis in starfish
and sea urchin embryos noticed a curious protein that
seemed to accumulate across the cell cycle but was then
destroyed during mitosis. They were well aware of the work
on MPF, and immediately suspected that their protein,
which they called cyclin, might be somehow involved in
MPF activity (Fig. 40.11).
In a third line of investigation, geneticists working on
yeasts realized that the cell cycle could be dissected through
the isolation of cell division cycle (CDC) mutants (see Box
40.2). The analysis of the cell cycle with these mutants
dominated cell-cycle research to such an extent that many
human genes that are important in cell-cycle control bear
the CDC name if they are related to well-characterized yeast
genes. The best-known genes to emerge from this analysis
were Cdc2 (Cdk1) and Cdc25, both of which were deter-
mined genetically to encode proteins that actively promote
the G2/M transition. Other genes, such as Wee1, were found
to encode activities that act as antagonists that inhibit the
G2/M transition.
When eventually purified from Xenopus eggs (Fig. 40.12),
active MPF consisted primarily of two polypeptides of 32,000
and 45,000Da. The smaller component of MPF is the
Xenopus equivalent of the fission yeast Cdc2 gene product
(now known as Cdk1). The larger component of Xenopus
MPF is a B-type cyclin. Only 15 years later was it recognized
that fully functional MPF also requires Greatwall kinase
and its small substrates to inhibit protein phosphatase PP2A-
B55 and give Cdk activity a chance to take off and trigger
mitotic entry.

A. MG1 fusion B. MS fusion C. MG2 fusion

5 m

FIGURE 40.9 FUSION WITH MITOTIC CELLS CAUSES INTERPHASE CELLS TO ENTER MITOSIS PREMATURELY, NO MATTER
WHERE THEY ARE IN THE CELL CYCLE. The resulting prematurely condensed chromosomes are single threads if the interphase cell
was in G1 phase (A), double threads if the cell was in G2 phase (C) and a complex mixture of both interspersed with uncondensed regions
if the cell was in S phase (B). M, mitosis. (From Hanks SK, Gollin SM, Rao PN, etal. Cell cycle-specific changes in the ultrastructural orga-
nization of prematurely condensed chromosomes. Chromosoma. 1983;88:333342.)
 CHAPTER 40 n Introduction to the Cell Cycle 705

BOX 40.4 Discovery of Factors Essential for Cell-Cycle Progressioncontd

A B C D

Meiotic Nucleus
spindle Nuclear disassembly Meiotic
and chromosome spindle
condensation

Suck out Inject it Oocyte enters

cytoplasm into oocyte M phase
Egg Fully grown oocyte Egg
FIGURE 40.10 THE EXPERIMENT THAT IDENTIFIED MATURATION-PROMOTING FACTOR. A, The box shows the meiotic spindle
in a Xenopus egg arrested in metaphase II of meiosis. B, The box shows the interphase nucleus in a mature oocyte. Following injection of
MPF, the nucleus disassembles, mitotic chromosomes form (C), and the cell assembles a meiotic spindle (D). Disassembly of the oocyte
nucleus and entry into M phase is called maturation, and the factor triggering this event was named maturation-promoting factor (MPF).

A. SDS gel of
proteins in
A purified MPF
75
B 97

Molecular weight (kDa)

67
B
Intensity of bands A and B

50

43
Cyclin B
A

25

30
Cdk 1

Cleavage Induction of M phase (%) 0 0 0 0 50 85 25 0 0 0

index
0 B. H1 kinase
0 1
Time (hours)
FIGURE 40.11 EXPERIMENTAL IDENTIFICATION OF A
CYCLIN. Newly synthesized proteins (labeled with 35S-methionine)
in fertilized sea urchin eggs were separated by sodium dodecylsul-
fate (SDS) polyacrylamide gel electrophoresis. It was noted that the
protein labeled A (which was named cyclin) first accumulated, was
greatly reduced at the metaphase/anaphase transition, and then
began to accumulate again. Protein B, which is not involved in
cell-cycle regulation, accumulated progressively over this time.
Cleavage index refers to the percentage of dividing cells observed
in the microscope at varying times after fertilization. (From Evans T,
Rosenthal ET, Youngblom J, etal. Cyclin: a protein specified by
maternal mRNA in sea urchin eggs that is destroyed at each cleav-
age division. Cell. 1983;33:389396.)
2
activity
Fraction number 6 9 12 15
FIGURE 40.12 PURIFICATION OF MATURATION-PROMOT-
ING FACTOR (MPF). A, Sodium dodecylsulfate (SDS) polyacryl-
amide gel electrophoresis of fractions from the final step of the
purification. The numbers at the bottom show the percentage of
oocytes that entered M phase when a portion of each column frac-
tion was injected (the classical MPF assay). The roughly 32-kD band
is Cdk1 (p34cdc2). The roughly 45-kD band is cyclin B. B, Assay of
the ability of the column fractions to phosphorylate histone Hl. This
is a standard assay for active Cdk enzymes. (Modified from Lohka
M, Hayes MK, Maller JL. Purification of maturation-promoting factor,
an intracellular regulator of early mitotic events. Proc Natl Acad Sci
U S A. 1988;85:30093013.)

phosphorylation of the T loop stimulate enzyme activity.
On the other, Cdks are inhibited by phosphorylation of
residues adjacent to the ATP-binding site and binding of
inhibitory proteins.
Cyclin binding causes the T loop to retract away from
the mouth of the catalytic pocket (see Fig. 40.13B). In
addition, the secondary structure of the N-terminal
domain is altered, allowing the bound ATP to assume a
conformation suitable for reaction with substrates.
Despite these changes, the Cdkcyclin complex has
low catalytic activity. Full activation of most Cdks
requires a kinase called Cdk-activating kinase (CAK),
which phosphorylates a threonine in the T loop of the
Cdk (this threonine gives the loop its name). In
706 SECTION X n Cell Cycle

A. Cdk2 B. Cdk2cyclin A C. Active Cdk2cyclin A

N lobe Active site

PSTAIR ATP
helix

T loop

C lobe Cyclin A Phosphorylation

site

FIGURE 40.13 ATOMIC STRUCTURES OF CYCLIN-DEPENDENT KINASES. A, Cdk2. The PSTAIR helix, found in most Cdks, is named
after a sequence of six amino acids that binds to cyclins (one letter code). (For reference, see Protein Data Bank [PDB; www.rcsb.org] file 1DM2.)
B, Cdk2cyclin A (kinase at basal activity level). (PDB file 1FIN.) C, Cdk2cyclin (kinase fully active following phosphorylation of threonine160). (PDB
file 1JST.) ATP, adenosine triphosphate.

vertebrates, CAK is composed of Cdk7-cyclin H. The A. Kinase activation B. Inactive forms

phosphorylated threonine fits into a charged pocket on
the surface of the enzyme, flattening the T loop back
even farther from the mouth of the catalytic pocket (see INK4
Cyclin
Figs. 40.13C and 40.14A). This stimulates the catalytic
activity up to 300-fold, in part because the flattened Cdk
T loop forms part of the substrate-binding surface. Threo-
nine phosphorylation also stabilizes the association of Cyclin
Inactive kinase cannot
Cdk2 with cyclin A. bind
In addition to their cyclin partner, Cdk1 and Cdk2 Cyclin
bind an additional small Cdc kinase subunit (Cks) protein
to their C-terminal domain, away from the active site.
Bound Cks enables the kinase to better hold onto its INK4
substrates and increases the efficiency with which Cdks
can phosphorylate substrates at multiple sites (a hall-
mark of Cdk target phosphorylation). Interference
p27 with ATP use

Negative Regulation of Cyclin-Dependent Kinase

Structure and Function
At least two mechanisms slow or stop the cell cycle by
inactivating Cdks (see Fig. 40.14). During G2 phase, the CAK
phosphorylation
protein kinases Myt1 and Wee1 hold Cdk1 in check by
phosphorylating threonine14 and tyrosine15 in the roof of ATP
cannot
the ATP-binding site. These phosphates interfere with bind
ATP binding and hydrolysis. Threonine14 and tyrosine15 Wee1 and Myt1
phosphorylation
are accessible to the regulatory kinases only following
cyclin binding, so this phosphorylation of Cdks depends,
Cdc25
at least in part, on the availability of cyclins. phosphatase
In mammals, three Cdc25 phosphatases (see Fig. dephosphorylation
25.5) reverse these inhibitory phosphorylations. Cdc25A Interference
regulates both the G1 S and G2 M transitions and Active kinase with ATP use
is essential for life of the cell. Ccd25B is dispensable for FIGURE 40.14 POSITIVE AND NEGATIVE REGULATION OF
mitosis, but it is essential for the production of gametes CYCLIN-DEPENDENT KINASES. A, Pathway of activation by cyclin
in meiosis. Cdc25C is a target of the G2 DNA damage binding and phosphorylation. B, Pathways of inactivation by inhibitor
binding and phosphorylation. When INK4 binds, twisting of the Cdk
checkpoint that prevents cells from undergoing mitosis
upper lobe blocks cyclin binding or interferes with ATP hydrolysis.
with damaged DNA (see Fig. 43.11), but cells can survive When p27 binds, a loop insinuates into the upper lobe of the Cdk and
without it. blocks ATP binding. (For reference, see PDB files 1B17 [Cdk2-INK4],
A parallel mechanism for inactivating Cdks involves 1FIN and 1BI7 [Cdk2-INK4-cyclin], and 1JSU [Cdk2-p27-cyclin A].)
binding of subunits from two families of small inhibitory CAK, Cdk-activating kinase.

proteins called the cyclin-dependent kinase inhibitors
(CKIs) and inhibitors of Cdk4 (INK4) (for their names,
see Appendix 40.1). When activated in the DNA damage
response pathway, p53 turns on transcription of the CKI
p21, which inhibits Cdk-cyclin A. CKI p27Kip1 inactivates
complexes of Cdk2 and cyclin A by having a protein loop
invade the N-terminal domain of the Cdk, disrupting its
structure and competing with ATP for binding to the
active site (see Fig. 40.14B).
Members of the INK4 family preferentially inactivate
Cdk4 and Cdk6 in two ways (see Fig. 40.14B). First,
binding to monomeric Cdk distorts the orientation of
the N- and C-terminal lobes, so cyclin D does not bind.
INK4 family inhibitors also inhibit preformed Cdk4/6
cyclin D complexes by binding the Cdk and distorting
the ATP-binding site so that the kinase uses ATP much
less efficiently.
Cdk inhibitors are important for growth regulation
during the G1 and G0 phases of the cell cycle (see Chapter
41). They also play a critical role in the cell-cycle arrest
that occurs in response to DNA damage and to antipro-
liferative signals. Mutations in the INK4 locus are strongly
linked to cancer.
Role of Phosphatases in
Counter-Balancing Cdk Activity
Two important phosphatases counter-balance Cdk activ-
ity in mitosis. Just as Cdks exhibit cyclic behavior and
are activated in mitosis, these counteracting phospha-
tases must also be cyclicbut they are inhibited in
mitosis. Protein phosphatase 1 (PP1), associates with
numerous targets on chromosomes and the mitotic appa-
ratus, and is highly active during mitotic exit. Many
target proteins have a simple loop with the sequence
RVS/TF that inserts into a groove on the enzyme. During
mitosis, Cdk phosphorylation of adjacent sites often
blocks this interaction with substrates. Cdk1-cyclin B
also phosphorylates the PP1 catalytic subunit during
mitosis, thereby inactivating the enzyme.
PP2A is more directly involved in Cdk regulation. It
is a trimeric enzyme with catalytic, scaffolding, and
regulatory subunits. The latter include B55- and
B56- (see Appendix 40.1). PP2A-B55 is largely (15-subunit) complex called the anaphase-promoting
responsible for removing phosphates added by Cdks.
Consequently PP2A-B55 must be inhibited to allow
Cdks to drive the cell into mitosis. The Greatwall (Gwl)
kinase regulates PP2A. Gwl is unusual, because 500
amino acids are inserted into its large lobe roughly adja-
cent to the T loop (see Fig. 40.13). Phosphorylation
by Gwl allows two small proteins, Arpp19 (cyclic
adenosine monophosphate [cAMP]-regulated phospho-
protein 19) and ENSA (-endosulfine), to bind and
inhibit PP2A-B55. Thus, Gwl confers the necessary
cyclic behavior on PP2A. Understanding how Gwl is
turned on and off is important for developing a full
CHAPTER 40 n Introduction to the Cell Cycle 707
picture of mitotic regulation, and this is a subject of
active study.
Role of Protein Destruction in
Cell-Cycle Control
During mitosis active Cdk1cyclin BCks phosphorylates
key substrates leading to dramatic reorganization of the
cell and, ultimately, to separation of sister chromatids on
the mitotic spindle. Once chromatids are separated, the
cell must return to a state with low levels of Cdk activity
so that nuclear envelope reassembly, spindle disassem-
bly, and cytokinesis can occur.
Exit from mitosis requires Cdk inactivation by the
ubiquitin-directed proteolytic machinery. The destruc-
tion of A- and B-type cyclins inactivates Cdk1 and Cdk2.
This allows PP1, PP2A-B55, and other phosphatases to
reverse the action of Cdks and bring mitosis to a close.
Ubiquitylation also results in proteolysis of a protein
called securin, which regulates the onset of sister chro-
matid separation at anaphase.
Ubiquitin-mediated destruction of cyclins involves a
cascade of three enzymes described in Chapter 23 (see
Fig. 23.3). First, an E1 enzyme (ubiquitin-activating
enzyme) activates the small protein ubiquitin by
forming a thioester bond between the ubiquitin
C-terminus and a cysteine on the enzyme. Activated ubiq-
uitin is next transferred to another thioester bond on an
E2 enzyme (ubiquitin-conjugating enzyme). The E2
often cooperates with an E3, which is important for
imparting substrate specificity, to transfer ubiquitin to
the -amino group of a lysine on a target protein. The
resulting polyubiquitinated proteins are usually targets
for destruction by the cylindrical 26S proteasome (see
Fig. 23.8). This large multienzyme complex functions
like a cytoplasmic garbage disposal, grinding target pro-
teins down to short peptides and spitting out intact
ubiquitin monomers for reuse in further rounds of
protein degradation. Its role was originally thought to be
the removal of damaged proteins from the cytoplasm;
however, it is now recognized as a central factor in cell-
cycle control.
The key E3 ligase regulating cyclin proteolysis is a large
complex/cyclosome (APC/C) (Fig. 40.15). The APC/C
is inactive during the S and G2 phases of the cell cycle.
Phosphorylation by Cdk1-cyclin B-Cks1 and binding of
the protein coactivator Cdc20 activate the APC/C in
early mitosis. APC/CCdc20 then triggers the metaphase
anaphase transition.
An important checkpoint, the spindle assembly
checkpoint, regulates APC/CCdc20 during mitosis,
keeping it inactive until all kinetochores are produc-
tively attached to spindle microtubules. The checkpoint
effector is the mitotic checkpoint complex whose for-
mation is triggered by unattached kinetochores. This
708 SECTION X n Cell Cycle

SCFSkp2, SCFTrCP
APC/CCdc20 APC/CCdh1 APC
(active) (active)
Cdc20 Cdh1
E2 Cdh1 Degron
SCFSkp2

Cdk
cyclin

P M A T
P M G1 S G2 P M A T
P M G1 APC/CCdh1 surface and backbone

FIGURE 40.15 TWO FORMS OF THE ANAPHASE-PROMOTING COMPLEX/CYCLOSOME (APC/C) CONTROL THE CELL CYCLE.
At the metaphase-anaphase transition, the APC/C with associated Cdc20 triggers the onset of anaphase by signaling the degradation of securin
and cyclin B. During mitosis Cdh1 phosphorylated by Cdk1cyclin B is unable to bind the APC/C, so APC/CCdh1 activity is low. As Cdk1cyclin
B activity declines in anaphase, Cdh1 binds the APC/C and APC/CCdh1 drives the exit from mitosis into G1. APC/CCdh1 remains active throughout
G1 but is inactivated following synthesis of the specific inhibitor Emi1. After the onset of S phase, SCF (shown here adding a ubiquitin chain to
a docked substrate) directs the degradation of cell-cycle substrates such as p27Kip1, following their phosphorylation by protein kinases.

complex inhibits APC/CCdc20 by acting as a competitive A. Structure of SCFSkp2-E2 complex

substrate. As a result, cyclin B and securin are stable until Skp 2
the checkpoint is satisfied. A few substrates, including ~50 E2
cyclin A, bind directly to the APC/C without requiring
Cdc20, so they are marked for degradation even when
the checkpoint is active. Skp 1 RbX 1
Exiting from mitosis and allowing the G1 cell to
prepare chromatin for DNA replication (see Chapter 42)
requires low Cdk activity and destruction of Cdc20. This
is accomplished late in mitosis by Cdh1, a different Cullin 1
co-activator of the APC/C. Phosphorylation of Cdh1 by
Cdks blocks its binding to the APC/C early in mitosis.
Thus, APC/CCdh1 forms only after cyclin levels (and there- B. Some F-box proteins C. Their target substrates
fore Cdk activity) decline late in mitosis. As cells pass Skp 2
F E2F-1: Cell-cycle regulator
from G1 into S phase, a newly synthesized inhibitory
protein, Emi1, binds to and inactivates APC/CCdh1. This F box
p27Kip1: Cdk2 inhibitor
allows cyclins to accumulate during S and G2. Remark-
Skp 1
ably, APC/CCdh1 is also involved with regulating the
activity of synapses in nondividing neurons. -TrCP1 Cdc25A: Cdk1 activator
After the G1 S transition and throughout the remain- F Wee1: Cdk1 inhibitor
der of interphase members of a different family of E3
activities called SCF regulate the levels of proteins that Emi1: APC/CCdh1 inhibitor
control Cdk and other cell cycle factors. SCF is named -Catenin: Cell-proliferation
Skp 1 regulator
after three of its four subunits: Skp1, cullin, and F-box
protein (Fig. 40.16). SCF is a molecular toolbox built on
FIGURE 40.16 STRUCTURE AND FUNCTION OF SCF.
a bow-shaped scaffold formed by the cullin subunit. The A, Structure of SCFSkp2. Left, SCF recognizes target proteins through
fourth subunit, Rbx1, binds near the C-terminus of cullin its F-box subunit (Skp2 in this case). Right, Ubiquitin is then transferred
and uses a protein motif called a RING finger to dock to from an E2 enzyme. The whole is assembled on a rigid bow-like scaf-
a ubiquitin-linked E2 enzyme. Skp1 binds to the other fold composed of the cullin subunit. Structures of F-box proteins Skp2
end of the cullin, where it provides a docking site for an and -Trcp (B) and a list of several of their known target proteins (C).
F-box protein that recognizes and binds the substrate.
(The F-box got its name because it was first discovered
in cyclin F.) Humans have 78 F-box proteins, giving SCF SCF is fundamentally different from the APC/C,
enormous versatility. Two examples shown in Fig. 40.16 because it is constitutively active. However, it ubiquity-
are Skp2, which targets the Cdk inhibitor p27 helping to lates substrates only after they have been phosphory-
drive the G1 S transition, and -Trcp, which targets lated, often by Cdks. This feature links SCF activity to
Cdc25A, Wee1, and Emi1. the cell cycle.
 CHAPTER 40 n Introduction to the Cell Cycle 709

1 2 3 4 5 1 2 3 4

Increasing Cdk activity

Cdk1-cyclin B
Cdk2-cyclin A
Cdk2-cyclin E

P M A T P M A T
P M G1 S G2 P M G1
Increasing APC activity

APC/CCdh1 APC/CCdc20

Emi1
SCFSkp2, SCFTrCP
PP2AB55, PP1

FIGURE 40.17 DIAGRAM SHOWING THE CHANGING STATES OF THE CYTOPLASM AS CELLS TRAVERSE THE CELL CYCLE.
Between G2 and G1 are shown the various stages of mitosis: P, prophase; PM, prometaphase; M, metaphase; A, anaphase; T, telophase. The
states of the cytoplasm discussed in the text are shown as green arrows across the top. Increased Cdk activity is shown as peaks upwards from
the central bar. Increasing anaphase-promoting complex/cyclosome (APC/C) activity is shown as a mirror image, with peaks going down from
the central bar. APC, anaphase-promoting complex; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A.

Changing States of the Cytoplasm During
the Cell Cycle
The cell cycle is characterized by five discrete physiolog-
ical states of the cytoplasm (Fig. 40.17). Changing levels
of Cdk activity drive transitions between these states,
sometimes counteracted and sometimes reinforced by
targeted proteolysis.
1. Early mitosis. Cdk2-cyclin A peaks in prophase,
followed by Cdk1-cyclin B. When the nuclear enve-
lope breaks down, the APC/CCdc20 starts degrading
cyclin A, but the spindle checkpoint inhibits destruc
tion of other substrates. When the last chromo-
some has attached correctly to spindle microtubules
(metaphase), the checkpoint is satisfied, and APC/
CCdc20 starts to degrade cyclin B and securin, an inhibi-
tor of a key protease called separase. Their degrada-
tion continues throughout metaphase.
2. Anaphase and mitotic exit. When securin levels fall
below a critical threshold, active separase cleaves a key
component of the cohesin ring (see Fig. 8.18). This
triggers sister chromatid separation. Cyclin destruction
continues throughout anaphase and telophase, and
falling Cdk1 activity allows the formation of APC/CCdh1,
which marks Cdc20 for destruction along with the
remaining B-type cyclins. SCF-Trcp destruction of Emi1
allows APC/CCdh1 to be active when it forms.
3. G1 phase. APC/CCdh1 and Cdk inhibitors of the CKI
and Ink4 families cooperate to inhibit Cdk activity.
Low Cdk activity is required for cytokinesis, spindle
disassembly, chromosome decondensation, nuclear
envelope reassembly, reactivation of transcription,
reassembly of the Golgi apparatus, and assembly of
prereplication complexes on the chromosomes.
4. G1S phase transition. Growth signals from the envi-
ronment promote the transcription of Cyclin E. If
the levels pass a critical threshold, a burst of Cyclin
E-Cdk2 activity allows the cell to pass the restriction
point, leading to synthesis of proteins required for
DNA replication and cell-cycle progression. Cdk phos-
phorylation targets the CKI peptides for destruction
by SCF allowing Cdk2 to become activated. In addi-
tion, APC/CCdh1 is inactivated by newly synthesized
Emi1.
5. SG2 phase. Cdk activity remains high throughout the
remainder of the cell cycle, and SCF continues to
degrade selected proteins tagged by Cdk phosphoryla-
tion. SCF-Trcp destruction of Cdc25A keeps Cdk1 inac-
tive, preventing a premature entry into mitosis. The
APC/C remains inactive, allowing mitotic cyclins to
accumulate. It is not known what ultimately triggers
entry into mitosis, but an important factor may be a
switch in the specificity of SCF-Trcp, which spares
Cdc25A and instead degrades the Cdk-inhibitory
kinase Wee1.
Although this sounds complicated, the underlying
principles are actually quite straightforward. The follow-
ing chapters discuss the cell-cycle transitions in greater
710 SECTION X n Cell Cycle

detail and show how the process is modulated in
response to a changing environment.
ACKNOWLEDGMENTS
We thank Tim Hunt, David Morgan, and Jonathon Pines
for their suggestions on revisions to this chapter.
SELECTED READINGS
Bartek J, Lukas J. DNA damage checkpoints: from initiation to recovery
or adaptation. Curr Opin Cell Biol. 2007;19:238-245.
Brown JS, Jackson SP. Ubiquitylation, neddylation and the DNA damage
response. Open Biol. 2015;5:150018.
Craney A, Rape M. Dynamic regulation of ubiquitin-dependent cell
cycle control. Curr Opin Cell Biol. 2013;25:704-710.
Hartwell LH, Weinert TA. Checkpoints: Controls that ensure the order
of cell cycle events. Science. 1989;246:629-634.
Hunt T. On the regulation of protein phosphatase 2A and its role in
controlling entry into and exit from mitosis. Adv Biol Regul. 2013;
53:173-178.
Lorca T, Castro A. The Greatwall kinase: a new pathway in the control
of the cell cycle. Oncogene. 2013;32:537-543.
Morgan DO. The Cell Cycle: Principles of Control. London: New
Science Press; 2007: 297p.
Nasmyth K. A prize for proliferation. Cell. 2001;107:689-701.
Nurse P. A long twentieth century of the cell cycle and beyond. Cell.
2000;100:71-78.
Primorac I, Musacchio A. Panta rhei: the APC/C at steady state. J Cell
Biol. 2013;201:177-189.
Qian J, Winkler C, Bollen M. 4D-networking by mitotic phosphatases.
Curr Opin Cell Biol. 2013;25:697-703.
Stukenberg PT, Burke DJ. Connecting the microtubule attachment
status of each kinetochore to cell cycle arrest through the spindle
assembly checkpoint. Chromosoma. 2015;124:463-480.
Wieser S, Pines J. The biochemistry of mitosis. Cold Spring Harb Per-
spect Biol. 2015;7:a015776.

APPENDIX 40.1

Inventory of the Enzymes of the Cell-Cycle Engine

Cyclin-Dependent Kinases and Their Cyclin Partners
Kinase Cyclin (+ Other) Partner Function
Cdk1 (p34cdc2) A Mammals: triggers G2 M transition. Yeasts: triggers G1 S and G2
B1, B2 (Xenopus has 5 B-type M transitions. Cyclin A is synthesized in S and destroyed starting at
cyclins) prometaphase. Cyclins B are synthesized in S/G2 and destroyed
Cdk1cyclin B binds Cks1 following the completion of chromosome attachment to the spindle.
(Cdc kinase subunit) Cyclins A1, B3 function preferentially in meiosis.
Cdk2 A, E Triggers G1 S transition. Can be replaced by other Cdks in mouse.
Cdk4, Cdk6 D1D3 Phosphorylation of the retinoblastoma susceptibility protein (pRb) in G1.
Triggers passage of the restriction point and cyclin E synthesis in some
cell types. Extracellular growth factors control synthesis of D cyclins.
Can be replaced by other Cdks in mouse.
Cdk5 CDK5R1 or CDK5R2 Neuronal differentiation, sensory pathways.
Cdk7 (CAK) H; also binds assembly factor Cdk activation by phosphorylation of the T loop. Also in TFIIH, important
MAT1 for regulation of RNA polymerase II transcription and DNA repair.
Cdk8 C Regulation of RNA polymerase II transcription.
Cdk9 T Regulation of RNA polymerase II transcription.
Cyclin Inhibitors
Inhibitor Cdk Substrates Function
CKI: p21Cip1/Waf1 most Most Cdk-cyclin complexes Induced by p53 tumor suppresser. Cell-cycle arrest after DNA damage.
Cdk-cyclin complexes Binds PCNA (proliferating cell nuclear antigen; see Chapter 42) and
inhibits DNA synthesis. Promotes cell cycle arrest in senescence and
terminal differentiation. At low levels, may help assemble active
Cdk-cyclin complexes.
CKI: p27Kip1 Most Cdk-cyclin complexes Cell cycle arrest in response to growth suppressers like TGF- and in
contact inhibition and differentiation.
CKI: p57Kip2 Most Cdk-cyclin complexes Important in development of the palate.
INK4: p15Ink4b Cdk4, Cdk6 Cell-cycle arrest in response to transforming growth factor (TGF)-.
Altered in many cancers.
INK4: p16Ink4a Cdk4, Cdk6 Cooperates with the retinoblastoma susceptibility protein (pRb) in
growth regulation. Cell-cycle arrest in senescence. Altered in a high
percentage of human cancers. This gene overlaps the gene for p19ARF,
an important regulator of the p53 tumor-suppresser protein.
INK4: p18Ink4c Cdk4, Cdk6 Cell-cycle arrest in response to growth suppressers.
INK4: p19Ink4d Cdk4, Cdk6 Cell-cycle arrest in response to growth suppressers.
 CHAPTER 40 n Introduction to the Cell Cycle 711

APPENDIX 40.1

Inventory of the Enzymes of the Cell-Cycle Enginecontd

Other Components
Enzyme Substrates Functions
Wee1 kinase Cdk1 Y15 Nuclear kinase. Inhibits Cdk1-cyclin B in G2.
Myt1 kinase Cdk1 T14 + Y15 Cytoplasmic kinase. Inhibits Cdk1-cyclin B in G2.
Greatwall (Gwl) kinase Arpp19, ENSA (-endosulfine) Phosphorylated Arpp19 and ENSA inhibit PP2a, allowing active Cdk1-
(MASTL in humans) cyclin B to accumulate and trigger mitotic entry
Cdc25A phosphatase Cdk1 T14, Y15 Promotes G1 S transition and G2 M transition. Essential for life of
the cell.
Cdc25B phosphatase Cdk1 T14, Y15 Promotes G2 M transition. Essential in meiosis.
Cdc25C phosphatase Cdk1 T14, Y15 Promotes G2 M transition. Dephosphorylates Cdk1 complexed to
cyclins A, B at T14 and Y15. Not essential for life.
PP2A phosphatase Many proteins phosphorylated Regulated by ENSA/Greatwall. With its targeting subunits B55- and
by Cdk1-cyclin B B56- it regulates many activities during mitotic exit and cytokinesis.
PP1 phosphatase Many targets Associates with many targeting subunits, which can be, for example,
regulatory proteins, such as RepoMan or can be structural subunits
of the kinetochore (see Chapter 8). It is inactivated by Cdk
phosphorylation during mitosis, but has a key role in mitotic exit.
APC/CCDC20 Cyclin B, securin many others E3 ubiquitin ligase active during M. Requires high Cdk activity to
function. Destruction of cyclins and other substrates essential for exit
from mitosis. Contains 15 subunits plus the specificity factor Cdc20.
APC/CCdh1 Cyclins A, B, many others E3 ubiquitin ligase active during G1. Requires low Cdk activity to
function. Keeps Cdk activity low in G1 through cyclin proteolysis.
Contains 15 subunits plus the specificity factor Cdh1.
SCF Cyclin E, many others Class of E3 ubiquitin ligases containing Skp1 + cullin + Rbx1 + an F-box
protein. Humans have 78 F-box proteins (Caenorhabditis elegans
has more than 300), acting as specificity factors for substrates
phosphorylated at specific sites, including cyclin E and Cdk inhibitors.