Download Cell Cycle Mitosis and Meiosis

Regulation of Cell Cycle
• The entry into and the exit from the cell cycle are
controlled by multiple extra-cellular signals
(nutrition, mitogens, growth factors etc.)
• The progression of the cell cycle through various
phases is regulated by transition through specific
‘check points’, mediated by specific phophorylating
enzymes called cyclin-dependent kinases (CDKs)
- A checkpoint represents a surveillance system, allows
detection of
- an incomplete previous step
- damage to the genome / mitotic spindle
- When a damage is sensed, cells arrest at specific phases
- allow time to repair the damage
1
Three Major Regulatory Pathways

Start Checkpoint
• The key checkpoint which
determines whether or not the cell
will duplicate



Regulated by growth factors,
nutrients and integrity of DNA
Rb is the primary regulatory
protein of this checkpoint
G2/M Checkpoint
• G2 to M transition is blocked until
all the DNA is duplicated


Controlled by MPF (maturation
promoting factor) is blocked until
DNA replicated
Spindle Checkpoint
•
Metaphase to anaphase transition
 Makes sure that all the chromosomes are
properly attached to microtubules
 Anaphase-promoting complex (APC) is a
key regulatory factor of this checkpoint
2
Growth Factors

Stimulatory growth factors
• Activate the Ras pathway


Ras mutations commonly found in pancreatic, colon, lung,
and bladder cancers, and occurs in about 25-30% of all
cancers
Inhibitory growth factors act through Cdk
inhibitors
• TGF-beta causes an increase in Cdk inhibitor
p15 and p21
• The Cdk inhibitor p21 plays a key role in
preventing cells containing damaged DNA from
passing through the G1 checkpoint
3
Check-points, CDKs

Each CDK phosphorylates and thereby modulates the
activity of a subset of target proteins specific for
individual transition within the cell cycle
• e.g. S-phase CDK may phosphorylate proteins involved
in DNA replication

Mammalian cells have several CDKs, viz.
• cdc2 (= CDK1), CDK2, CDK3, CDK4, CDK6, and CDK7
 act at different transitions in the cell cycle


Studies of Cdk’s and cyclins in genetically modified
mice reveal a high level of plasticity, allowing different
cyclins and Cdk’s to compensate for the loss of one
another.
Cdk1 is capable of substituting for the all the
other Cdk’s.
4
CDKs & Cyclins

Activation of CDKs require their association with
another group of proteins called ‘Cyclins’
• Cyclin D, H, E, A, B
• contribute to CDK substrate specificity
• The levels of different cyclins vary during the
cycle
• e.g. Cyclin E accumulates in late G1, associates
with CDK2, and is destroyed as cells enter S
phase

The activity of the Cyclin-CDK complex is further
subject to positive or negative regulation
• Phosphorylation/dephosphorylation
• Inhibitors of CDKs (e.g. p21 etc.)
• Proteolysis of cyclins and inhibitors
(APC/C)
5
6
7
The regulation of Cdk activity by
inhibitory phosphorylation


The active cyclin-Cdk
complex is turned off
when the kinase Wee 1
phosphorylates two
closely spaced sites
above the active site.
Removal of these
phosphates by the
phosphatase Cdc25
activates the cyclin-Cdk
complex
8
CDK inhibitor proteins (CDKI)

The cyclin D/CDK4/CDK6, Cyclin
E/CDK2, and Cyclin B/CDk1 are
inhibited by a group of CDK
inhibitor proteins (CDKI) that
include p21 & p27.
• CDKIs also impair CDK activating kinase
activity (CAK)
9
Restriction point control : G1 to S progression



The transition from G1 to S phase is regulated by a
checkpoint called “Restriction point” or Start
- a very important regulatory step, to ensure repair
of genome damage before initiation of DNA
replication
The initiation of cycle, i.e. entry of cells into G1 phase,
is determined by extra-cellular signals (mitogens,
nutrients & growth factors)
Growth factors induce synthesis of D-type cyclins
(D1, D2, D3)
• D cyclins associate with CDK4 and CDK6 in G1
• Cyclin D/CDK4/CDK6 complexes are activated through
phosphorylation by an enzyme complex called CAK (CDK
activating kinase)
11
Induction of D-type cyclins




Proliferation of animal cells is regulated
largely by extracellular growth factors
that control progression through the
restriction point in late G1.
The activity of cyclin-CDK
complexes is tightly regulated by
CDK inhibitors. Some Growth
factors shut off production of these
inhibitors.
Growth factors stimulate cyclin D1
synthesis through the
Ras/Raf/MEK/ERK pathway, and are
synthesized as long as growth factors
are present.
Cyclin D1 is also rapidly degraded, so
the intracellular concentration rapidly
falls if growth factors are removed.
Cyclin D & Cell Cycle Progression



As long as growth factors are present
through G1, Cdk4,6/cyclin D1
complexes drive cells through the
restriction point.
Defects (overexpression) in cyclin D1
regulation could contribute to the loss
of growth regulation characteristic of
cancer cells.
Many human cancers arise as a result
of defects in cell cycle regulation.
Rb controls the G1 to S transition



The Retinoblastoma protein, Rb, is phosphorylated by Cyclin
D/CDK4/CDK6, which is necessary to drive the cell past the
restriction point
• Once the cell crosses the restriction point, mitogenic stimulation
is no longer needed, and the entry into the S phase is ensured
In its hypophosphorylated state, the Rb is complexed with
E2F family of transcription factors (E2F1 – E2F5)
Phosphorylation of Rb dissociates E2F
14
Cell cycle regulation of Rb and E2F



Rb plays a key role in coupling
cell cycle machinery to the
expression of genes required
for cell cycle progression.
In G0 or early G1, Rb binds
to E2F transcription factors,
which suppresses
expression of genes
involved in cell cycle
progression.
Rb is phosphorylated by
Cdk4,6/cyclin D complexes
as cells pass through the
restriction point, and
dissociates from E2F,
allowing transcription to
proceed.
Restriction point control and S phase



Cyclin E/CDK2 kinase
is needed to maintain
Rb in its
hyperphosphorylated
state
As Cyclin E/CDK2
activity decreases,
cyclin A synthesis is
induced.
Accumulation of the
Cyclin A/CDK2
complexes signals
entry into S phase.
16
G2 to M Checkpoint

The mitotic Cdk1-cyclin B complex
(MPF) controls the G2 checkpoint
by phosphorylating proteins involved
in the early stages of mitosis
• MPF = Maturation-promoting factor
• Activated by multi-step process
• MPF phosphorylates lamin
proteins of the nuclear lamina
(causing breakup of nuclear
membrane)
• MPF phosphorylates condensin
complex which may trigger
chromosome condensation
17
MPF regulation





MPF is regulated by
phosphorylation and
dephosphorylation of Cdk1.
Cyclin B is synthesized and
forms complexes with Cdk1
during G2.
Cdk1 is phosphorylated and
inhibited, leading to
accumulation of inactive
Cdk1/cyclin B complexes
throughout G2.
Dephosphorylation activates
Cdk1, which phosphorylates
several proteins that initiate
the events of M phase.
Cyclin B is degraded by
ubiquitin-mediated proteolysis.
Cyclic MPF activity
19
DNA damage checkpoints


DNA damage
checkpoints ensure
that damaged DNA
is not replicated and
passed on to
daughter cells.
The cell cycle is
arrested in response
to damaged or
unreplicated DNA.
Arrest at the DNA damage checkpoints





DNA damage checkpoints are mediated
by related protein kinases, ATM and
ATR, that are activated in response to
DNA damage.
They activate a signaling pathway that
leads to cell cycle arrest, DNA repair,
and sometimes, programmed cell
death.
ATM is activated by double-strand
breaks, ATR is activated by singlestranded or unreplicated DNA.
They phosphorylate and activate the
checkpoint kinases Chk2 and Chk1.
Chk1 and Chk2 phosphorylate and
inhibit Cdc25 phosphatases, which are
required to activate Cdk1 and Cdk2.
• Inhibition of Cdk2 results in cell cycle arrest in
G1 and S.
• Inhibition of Cdk1 results in arrest in G2.
Mdm2 controls the levels of p53 in the nucleus


Mdm 2 controls the level of p53
in the nucleus by binding p53
and shuttling it out of the
nucleus where it is destroyed
by the ubiquitin-dependent
pathway
If p53 is phosphorylated by
the ATM/ATR pathway and
is acetylated, it will no
longer interact with Mdm2
• Thus, the levels of p53
increase in the nucleus
22
***p53 ***



p53 plays a pivotal role in cell cycle arrest in
response to DNA damage
When DNA is damage ATM & ATR (two
DNA-dependent protein kinases) leads to
the phosphorylation of p53, preventing its
degradation
• Thus, DNA strand breaks by UV or ionizing
radiation increase levels of p53 protein
• P53 is a transcription factor
p53 then stimulates the expression of p21
(a CDK inhibitor) that causes cell cycle
arrest!!!!!
23
Role of p53 in G1 arrest





In mammalian cells, arrest at the
G1 checkpoint is also mediated by
protein p53, which is
phosphorylated by both ATM and
Chk2.
p53 is a transcription factor, and
its increased expression leads to
induction of Cdk inhibitor p21.
p21 inhibits Cdk2/cyclin E
complexes, leading to cell
cycle arrest in G1.
The gene encoding p53 is
frequently mutated in human
cancers.
Loss of p53 prevents G1 arrest in
response to DNA damage, so the
damaged DNA is replicated and
passed on to daughter cells.
Li-Fraumeni Syndrome (LFS)






Rare “Cancer Families” with a history of many
different forms of cancer (bone and soft tissue
sarcoma, breast cancer, brain tumor,
leukemia, and adrenocortical carcinoma)
Usually afflict patient at an unusually early age
Inherited in an autosomal dominant pattern
> 70% of families have a mutant form of the
TP53 gene (codes for p53)
LFS can occur both as a sporadic and familial
form
Mutations in both alleles are necessary
to inactivate the TP53 gene
25