Cell Cycle
Throughout the life of an individual, but particularly during development, every cell
constantly faces decisions.
Should it divide? Yes
No--> Should it differentiate? Yes
No-->Should it die? Yes-->Apoptosis
No!
Proper development and tissue homeostasis rely on the correct balance between
division and cell death. Too much cell death leads to tissue atrophy such as in
Alzheimer’s disease. Too much proliferation or too little cell death can lead to cancer.
cancer = unregulated cell proliferation
apoptosis = programmed cell death
necrosis = unprogrammed cell death
The cell cycle is at the center of the decisions a cell makes. Dividing cells go through a
cycle consisting of, G1 (growth or gap), S (DNA synthesis), G2 (growth) and M phase
(mitosis). Specific events must happen in a particular sequence for the cell to replicate.
During the G1 phase, the cell integrates mitogenic and growth inhibitory signals and
makes the decision to proceed, pause, or exit the cell cycle. S phase is defined as the
stage in which DNA synthesis occurs. G2 is the second gap phase during which the cell
prepares for the process of division. M stands for mitosis, the phase in which the
replicated chromosomes are segregated into separate nuclei and cytokinesis occurs to
form two daughter cells. In addition, the term G0 is used to describe cells that have
exited the cell cycle and become quiescent. When cells differentiate, they usually stop
dividing and therefore exit the cell cycle. Most cells that stop dividing to differentiate do
so in the G1 phase although some arrest in G2.
An important checkpoint in G1 is referred to as start in yeast and the
restriction point in mammalian cells. This is the point at which the cell becomes
committed to DNA replication and usually to completing a cell cycle. Before beginning
the cell cycle, the cell must assess whether sufficient growth has occurred (eg. are
there enough ribosomes?), whether there is cellular damage and whether there is the
proper complement of growth factor signaling. Another, checkpoint exists at the G2 to M
transition where cells become committed to divide. Again the cell must assess whether
the chromosomes are completely replicated and whether there is cellular damage and
proper growth factor signaling.
The events of the cell cycle are regulated by a protein complex consisting of a cyclin
dependent kinase (cdk) and a cyclin. Both the CDK and the cyclin are protein
kinases. Different CDK/cyclin complexes regulate different phases of the cell cycle. In
yeast, there is only one CDK that interacts with different phase-specific cyclins. In
mammals, there are different CDKs and different cyclins for different phases of the cell
cycle. Cyclins (and CDKs in mammals) are expressed cyclically; each cyclin is
expressed only in the appropriate phase of the cell cycle and then is rapidly degraded.
CyclinD is exceptional in that it does not oscillate with the cell cycle. When quiescent
cells re-enter the cell cycle and divide (i.e. go from G0 to G1), cyclinD is the first cyclin
to be activated. Growth factors regulate the expression of cyclinD. This is one of the
ways that growth factors feed information into the cell cycle. Thus cells cannot pass
start without growth factor signals. Many growth factors signal to the cell cycle through
ras and the MAP kinase pathway.
At each stage the CDK/cyclin complex performs 3 important functions:
1. They activate the cellular activities that are associated with that particular
phase in the cell cycle.
2. They activate the CDK/cyclin complex that controls the next stage in the cell
cycle.
3. They inactivate the CDK/cyclin complex from the previous stage in the cell
cycle. Often they trigger their own inactivation or degredation.
In this way, they control the forward progression of the cell cycle as well as the events
of that particular phase.
CDK/cyclin complexes are regulated in 4 major ways:
1. Association of the CDK and the cyclin (cyclin activates CDK)
2. Phosphorylation
3. Binding to cyclin dependent kinase inhibitors (CKIs)
4. Proteolytic degradation
Phosphorylation
Association of a CDK and cyclin is necessary but not sufficient for the formation of an
active complex. The proper phosphorylation status is also required for activity.
Phosphorylation of CDK/cyclin complexes can be either activating or inhibitory. As an
example we will consider the regulation of MPF activity. (MPF=maturation promoting
factor, or mitosis promoting factor). MPF consists of a CDK (cdc2) and cyclinB. After
association of these 2 proteins, the CDK is phosphorylated on a specific residue
(tyrosine 15) by a kinase called wee1. This is an inhibitory phosphorylation therefore
wee1 ---| MPF. Then a second, activating phosphate is added to another residue
(threonine 161) by a kinase called CAK (CDK activating kinase). However MPF is still
not active because of the phosphorylated tyr15. MPF finally becomes activated when
the inhibitory phosphate is removed by a phosphatase called cdc25 (or string). Then
MPF is active and can trigger mitosis.
All the while, different cellular inputs are regulating the expression and activity of wee1,
CAK and cdc25, ensuring that division is not triggered until the appropriate time.
Cyclins can also be regulated by phosphorylation.
CDK inhibitors (CKIs)
There are 2 families of CKIs , the Cip/Kip family and the INK4 family.
• Members of the Cip/Kip family interact with all CDK/cyclin complexes. An
example of this family, p21, will be discussed below in relation to checkpoint
control.
• INK4 members interact with specific CDKs. For example, p16 interacts with the
G1 CDKs (cdc4 and cdc6 in mammals) and either prevent their association with
cyclinD or inhibit the preassembled complex. p16 is also important in checkpoint
control and will be further discussed below.
Ubiquitin dependent proteolysis
Ubiquitin dependent proteolysis is very important for rapidly degrading specific proteins.
Ubiquitin is a 76 amino acid peptide that is ligated (attached) to proteins targeted for
degradation. Additional ubiquitin peptides are sequentially added to the previous
subunit in a process called polyubiquitination. This is catalyzed by a protein complex
known as the ubiquitin ligase complex. Polyubiquitinated proteins are then
recognized and degraded by a proteolytic complex known as the 26S proteosome.
Ubiquitin dependent proteolysis is critical at 2 major steps in the cell cycle. Different
ubiquitin ligase complexes act at each step and the mode of regulation is different. The
progression from G1 to S-phase requires the action of a complex called SCF. SCF
recognizes proteins that are phosphorylated on PEST sequences, sites that are
common in proteins that are regulated by rapid turnover. Entry into S phase requires
the proteolytic degradation of an S phase inhibitor called Sic. Sic is phosphorylated on
it’s PEST motif by the G1 cyclinD. Thus Sic is targeted for polyubiquitination by SCF
and subsequent degradation by the 26S proteosome, removing the inhibitor and
allowing entry into S-phase. Ubiquitin dependent proteolysis is also important for
maintaining the forward progression of the cell cycle. SCF ubiquitinates cyclinD.
CyclinD is phosphorylated by the same CDK that it activates and this phosphorylation
targets it for ubiquitination by SCF and subsequent degredation.
Another ubiquitin ligase complex, APC (Anaphase Promoting Complex) is important for
the transition from metaphase to anaphase during mitosis. APC ubiquitinates M-phase
cyclins. The regulation of APC is very complex but at it is partly regulated by
phosphorylation of APC subunits. Phosphorylation of APC by the mitotic cyclin/CDK
activates it to ubiquitinate mitotic cyclins. It’s substrate specificity is controlled by
different subunits becoming part of the complex. For example, degradation of mitotic
cyclins is required for the onset of chromosome separation. Later, in telophase, APC
begins degrading proteins involved in anaphase. One subunit that provided specificity
for the mitotic cyclins is replaced by another subunit that confers substrate specificity
for the anaphase proteins. Thus, SCF activity is regulated by modification of the
substrates while APC activity is regulated by modification of the APC complex.
Checkpoint control
The most important checkpoint is called start in yeast or the restriction point in
mammalian cells. This is the point at which the cell commits to enter S phase. Since
most cells exit the cell cycle in G1 to differentiate, passing start generally commits a cell
to undergoing an entire cell cycle. Cells assess whether adequate growth has occurred,
whether proper growth factor signaling is present and whether any cellular damage is
present before reaching the decision to pass start. Because defects in cell cycle
checkpoint control can lead to unregulated cell division, many of the factors involved in
checkpoint control were first identified as oncogenes.
One central protein in regulating start is the retinoblastoma protein (Rb). Rb binds and
inhibits a transcriptional activator called E2F. E2F activates the transcription of many
cell cycle genes, including those involved in DNA synthesis as well as S phase cyclins.
Rb dissociates from E2F when it is phosphorylated by cyclinD, thereby allowing E2F to
activate transcription and initiate S phase. Rb is also a negative transcriptional regulator
of the CKI p16. In cells lacking Rb, p16 is overexpressed. The inactivation of Rb by
cyclinD allows expression of p16 which then inhibits the activity of the G1 cyclin. Thus
we see another example of how a cyclin promotes progression to the next phase while
inhibiting the current or previous phase.
CDK/cyclinD ——| Rb ——| E2F → S phase
Rb ——| p16 ——| G1 CDK
The myc transcription factor is also required for the G1 to S transition. myc activates
transcription of cdc25 which then activates the CDK complex, promoting cell cycle
progression to S phase.
Cells will not progress through the cell cycle if cellular damage is sensed. Cellular
damage induces the expression of a transcription factor called p53. p53 inhibits both
the G1 to S transition and the G2 to M transition. One of the genes whose transcription
is activated by p53 is the CKI p21. Thus:
Cellular damage → p53 → p21 ——| CDK/cyclin