Transcript The Eukaryotic Cell Cycle

Molecular
Biology II
The Cell Cycle
The Life Cycle of Cells
• The Cell Cycle Follows a Regular Timing Mechanism.
• Newly born cells grow and perform their functions.
• After reaching a specific point in their life cells divide via mitosis to
give rise to daughter cells.
• Cell Differentiation Creates New Types of Cells.
• Cells Die by Suicide
The size difference between a neuron
(from the retina) and a lymphocyte in a
mammal
Both cells contain the same amount of DNA.
A neuron grows progressively larger after it
has permanently withdrawn from the cell
cycle. During this time, the ratio of cytoplasm
to DNA increases enormously (by a factor of
more than 105 for some neurons). (Neuron
from B.B. Boycott, in Essays on the Nervous
System [R. Bellairs and E.G. Gray, eds].
Oxford, UK: Clarendon Press, 1974.)
Proteins that participate
Cell Growth Control
1. Growth Factors
2. Growth Factor Receptors
3. Intracellular Transducers
4. Transcription Factors
5. DNA Repair Proteins
6. Cell Cycle Control Proteins
7. Anti Death Proteins
Cell growth control through Nutritional stress
These graphs show the relationship between growth rate, cell size, and
cell cycle time. (A) If cell division continued at an unchanged rate when
cells were starved and stopped growing, the daughter cells produced at
each division would become progressively smaller. (B) Yeast cells
respond to some forms of nutritional deprivation by slowing the rate of
progress through the cell cycle so that the cells have more time to grow.
As a result, cell size remains unchanged or is reduced slightly. (A unit of
time is the cycle time observed when nutrients are in excess.)
Fresh Growth Medium Stimulates
Proliferation in a confluent cell monolayer
Cells in a confluent monolayer do not divide (gray). The cells resume
dividing (green) when exposed directly to fresh culture medium.
Apparently, in the undisturbed confluent monolayer, proliferation has
halted because the medium close to the cells is depleted of mitogens, for
which the cells compete
Serum induces new gene expression
and Protein synthesis
The stages of mitosis and cytokinesis in
an animal cell
The Eukaryotic Cell Cycle
In most growing cells, the four phases
proceed successively, taking from 10-20
hours depending on cell type and
developmental state.
Interphase comprises the G1, S, and G2
phases.
DNA is synthesized in S, and other
cellular macromolecules are synthesized
throughout interphase, so the cell roughly
doubles its mass.
During G2 the cell is prepared for the
mitotic (M) phase, when the genetic
material is evenly partitioned and the cell
divides.
Nondividing cells exit the normal cycle,
entering the quiescent G0 state.
The Cell Cycle
The cell cycle, is highly regulated in
multicellular organisms:
G0: Stationary or resting phase
M : Mitotic phase
G1: Growth phase I (cell growth and
preparation for the synthetic phase)
S:
Synthetic phase (replication of DNA)
G2: Growth phase II (Cell growth and
preparation for cell division)
The fate of a single parental
chromosome throughout the eukaryotic
cell cycle
The control of the cell cycle
The essential processes of the cell
cycle such as DNA replication,
mitosis, and cytokinesis are
triggered by a cell-cycle control
system. By analogy with a
washing machine, the cell-cycle
control system is shown here as a
central arm the controller that
rotates clockwise, triggering
essential processes when it reaches
specific points on the outer dial.
Checkpoints in the cell-cycle control
system
Information about the completion
of cell-cycle events, as well as
signals from the environment, can
cause the control system to arrest
the cycle at specific checkpoints.
The most prominent checkpoints
occur at locations marked with
yellow boxes.
1. G1/S checkpoint
2. G2/M Checkpoint
3. G0/G1 Checkpoint
Cell Cycle Checkpoints are regulated by
Cyclins and Cyclin Dependent Kinases
There are four classes of cyclins, each defined by the stage of the cell
cycle at which they bind Cdks and function. Three of these classes
are required in all eucaryotic cells:
1. G1/S-cyclins bind Cdks at the end of G1 and commit the cell to DNA
replication.
2. S-cyclins bind Cdks during S phase and are required for the initiation
of DNA replication.
3. M-cyclins promote the events of mitosis.
4. G1-cyclins, helps promote passage through Start or the restriction
point in late G1.
The structural basis of Cdk activation
These drawings are based on three-dimensional structures of human Cdk2, as
determined by x-ray crystallography. The location of the bound ATP is indicated. The
enzyme is shown in three states. (A) In the inactive state, without cyclin bound, the
active site is blocked by a region of the protein called the T-loop (red). (B) The binding
of cyclin causes the T-loop to move out of the active site, resulting in partial activation
of the Cdk2. (C) Phosphorylation of Cdk2 (by CAK) at a threonine residue in the Tloop further activates the enzyme by changing the shape of the T-loop, improving the
ability of the enzyme to bind its protein substrates.
The regulation of Cdk activity by
inhibitory phosphorylation
The active cyclin-Cdk complex is turned off when the kinase Wee1
phosphorylates two closely spaced sites above the active site. Removal
of these phosphates by the phosphatase Cdc25 results in activation of the
cyclin-Cdk complex. For simplicity, only one inhibitory phosphate is
shown. The activating phosphate is added by CAK,
The inhibition of a cyclin-Cdk
complex by a CK Inhibitor
This drawing is based on the three-dimensional structure of the human
cyclin A-Cdk2 complex bound to the CKI p27, as determined by x-ray
crystallography. The p27 binds to both the cyclin and Cdk in the
complex, distorting the active site of the Cdk. It also inserts into the ATPbinding site, further inhibiting the enzyme activity.
A simplified view of the core of the cellcycle control system
Cdk associates successively with
different cyclins to trigger the different
events of the cycle. Cdk activity is
usually terminated by cyclin
degradation. For simplicity, only the
cyclins that act in S phase (S-cyclin) and
M phase (M-cyclin) are shown, and they
interact with a single Cdk; as indicated,
the resulting cyclin-Cdk complexes are
referred to as S-Cdk and M-Cdk,
respectively.
Protein Degradation is Required for
progression through Cell Cycle
Passage through three critical cell-cycle transitions, G1-S phase, G2-M phase
(metaphase → anaphase, and anaphase → telophase and cytokinesis), is irreversible
because these transitions are triggered by the regulated degradation of proteins, an
irreversible process. As a consequence, cells are forced to traverse the cell cycle in one
direction only.
In higher organisms, control of the cell cycle is achieved primarily by regulating the
synthesis and activity of G1 Cdk complexes
Extracellular growth factors, called mitogens, induce the synthesis of G1 Cdk
complexes. The activity of these and other Cdk complexes is regulated by
phosphorylation at specific inhibitory and activating sites in the catalytic subunit.
The point in late G1 where passage through the cell cycle becomes independent of
mitogens is called the restriction point
Cell Phase Specific Cyclins
Activity of mammalian Cdk-cyclin
complexes through the course of
the cell cycle in G0 cells induced to
divide by treatment with growth
factors.
The width of the colored bands is
approximately proportional to the
protein kinase activity of the
indicated complexes. Cyclin D
refers to all three D-type cyclins.
Regulation of mitotic cyclin B levels in
M-Phase
The anaphase-promoting complex (APC)
is activated only when MPF activity is
high. Binding of the active APC and E2
covalently linked to a ubiquitin (not
shown) to the cyclin B destruction box
leads to the addition of multiple ubiquitin
(Ubi) molecules. As the polyubiquitinated
cyclin B is degraded, MPF activity
declines, triggering the onset of telophase.
Following cytokinesis, synthesis of cyclin
B occurs in the interphase daughter cells.
APC activity remains high until late in the
G1 of the next cell cycle when it is
inactivated by a G1 Cdk complex. When
the MPF activity rises enough, another
mitoses ensues.
Current model for regulation of the
eukaryotic cell cycle
Growth Promoter and Growth Suppressor
Genes control the Cell Cycle
Growth Promoters: EGF, PDGF, Myc
Growth Suppressors:Rb, P53, P16, etc.
Growth Factors Promote Cell Growth
In this simplified scheme,
activation of cell-surface receptors
leads to the activation of PI 3kinase, which promotes protein
synthesis, at least partly through
the activation of eIF4E and S6
kinase. Growth factors also inhibit
protein breakdown (not shown) by
poorly understood pathways
Growth Supressor Retinoblastoma (Rb)
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pRB is a DNA binding protein expressed in every cell type and
is an important regulator of the cell cycle.
Activated (Unphosphorylated) pRB acts as a ‘brake’ in the cell
cycle at G0/G1 boundary.
During G1, Phosphorylation of pRB (by Cdks) inactivates the
protein and allows cell cycle progression into S phase.
G0/G1
Growth Suppressor Functions of Rb
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The G0/G1 boundary is a particularly important
control point because this acts as a commitment to
cell division.
Cyclins (A, B, C, D) and Cyclin Dependent Kinases
(Cdks) are responsible for phosphorylating and
inactivating Rb.
These are in turn regulated by Cyclin dependent
Kinase Inhibitors like Cip/Kip and INK4 gene products
The G0/G1 Boundry
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The G0/G1 boundary is a particularly
important control point because this acts
as a commitment to cell division.
E2F as a growth Promoter
E2F is a transcription factor that activates several replication and growth related genes.
During G0/G1 transition, E2F is bound to activated or unphosphorylated RB, and as a
consequence is unable to activate transcription.
The RB-E2F complex can be disrupted by phosphorylation of RB or by DNA viral oncoproteins
that bind RB.
The release of free E2F leads to transcriptional activation of genes containing E2F binding sites,
such as the adenovirus E2 gene, cmyc, c-myb, dhfr, DNA polymerase a, of the transcriptional
machinery.
Mechanisms controlling S-phase initiation
in animal cells
G1-Cdk activity (cyclin D-Cdk4) initiates Rb phosphorylation. This
inactivates Rb, freeing E2F to activate the transcription of S-phase genes,
including the genes for a G1/S-cyclin (cyclin E) and S-cyclin (cyclin A).
The resulting appearance of G1/S-Cdk and S-Cdk activities further
enhances Rb phosphorylation, forming a positive feedback loop. E2F
acts back to stimulate the transcription of its own gene, forming another
positive feedback loop.
Restriction Point control
Growth supressor proteins such as
Rb and P16 control the cycle cycle
check points. These proteins
prevent excessive activation of
CycD and E2F at the G1/S phase
boundry.
Regulation of Rb and E2F
activities in late G1
Stimulation of G0 cells with mitogens induces expression of Cdk4, Cdk6, D-type
cyclins and E2F transcription factors (E2Fs), all encoded by delayed-response genes.
Interaction of E2Fs with hypophosphorylated Rb protein initially inhibits E2F activity.
When signaling from mitogens is sustained, the resulting Cdk4 cyclin D and
Cdk6 cyclin D complexes (Cdk4/6 cyclin D) initiate the phosphorylation of Rb,
converting some E2F to the active form. Active E2F then stimulates its own synthesis
and the synthesis of Cdk2 and cyclin E. Cdk2 cyclin E further stimulates Rb
phosphorylation releasing more E2F activity. These processes result in positive
feedback loops (blue arrows) leading to a rapid rise in both E2F and Cdk2 cyclin E
activity as the cell approaches the G1→S transition.
p53
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p53 has been called the ‘guardian of the genome’ as it directs cells with
damaged DNA that cannot be repaired to undergo apoptosis by inducing
the expression of apoptosis genes (P21, Bax, mdm-2)
G1/S Checkpoint
Cell-Cycle
Progression is
Blocked by DNA
Damage and p53 at
the G1/S checkpoint
p53-induced cell-cycle arrest in response
to DNA damage
The normally unstable p53 protein is stabilized by damaged DNA, so its
concentration increases. Acting as a transcription factor, p53 induces
expression of p21CIP, a cyclin-kinase inhibitor that inhibits all Cdk1-,
Cdk2-, Cdk4-, and Cdk6-cyclin complexes. Binding of p21CIP to these
Cdk-cyclin complexes leads to cell cycle arrest in G1 and G2.
Regulation of p53 Growth Suppressor
Functions
INK4 Locus (P14, P15, P16, P19)
Growth Suppressors
INK 4
• The INK4 locus at 9p21, gives rise to
multiple proteins after differential splicing.
• These proteins are involved cell cycle
control through Rb and P53 growth
suppressors.
E2F
P16
P14
Rb
P53
Cyc
D
Cyc
B
Bcl2
Bax
P21
An overview of the cell-cycle
control system
The core of the cell-cycle control system consists of a series of cyclinCdk complexes (yellow). The activity of each complex is also influenced
by various inhibitory checkpoint mechanisms, which provide
information about the extracellular environment, cell damage, and
incomplete cell-cycle events (top). These mechanisms are not present in
all cell types; many are missing in early embryonic cell cycles, for
example.