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THE CELL CYCLE II
Overview of CDKs:
1
...
There are a lot of CDKs within the eukaryotes that have expanded throughout
evolution and whose functions remain but most likely link physiological processes to
the cell cycle state
...
START is the major cell cycle checkpoint in yeast and ensures irreversible cell cycle
entry
...
A cells commitment to this transition is regulated via external nutrients, presence of
mating factors and stress
...
One scientist found that mating factors induced cell cycle arrest similar to mutation
of cdc28/cdc2/Cdk1
...
Cells exposed to mating factors early in the cell cycle were arrested in G1, whereas
those exposed to the factors late in the cycle were arrested in the next G1
...
This suggests that there is an event early in the cell cycle where the cell decides
whether to enter the next cycle or not
...
Cyclins 1,2 and 3 function with Cdk1 (Cdc2/Cdc28) at the START checkpoint to all cell
cycle entry, and are thus referred to as G1 cyclins
...
These cyclins lack a destruction box, but are unstable and degraded by the
proteasome
...
Overall, the G1 cyclin:CDK complex:
- Triggers progression through START
- Initiate DNA replication
- Initiate spindle pole body duplication (important in mitosis initiation)
- Bud emergence (in budding yeast)
Commitment to G1/S transition in Mammals:
1
...
2
...
e
...
e
...
3
...
4
...
Cyclin D Family:
1
...
2
...
3
...
4
...
5
...
6
...
7
...
8
...
9
...
10
...
11
...
Control of Cyclin D Levels:
1
...
Subsequently, m-RNA levels of the above all fall upon GF withdrawal
...
Levels of the cyclins are also dependent upon translation, whereby translation is
enhanced by GF stimulation involving PI3-kinase dependent activation of elF-4E
(transcription initiation factors)
...
Additionally, the levels also depend upon proteolysis: the half-life of cyclin D is 30
minutes due to proteasome-mediated degradation
...
PI3-kinase activation or Wnt signalling can reduced this degradation
...
Phosphorylation of cyclin D1 by GSK3b creates a recognition site for SCF ubiquitin
ligase
...
SCF-dependent ubiquitination of cyclin D1 allows is to be degraded by the
proteasome
...
PI3-kinase signalling through PKB/Akt phosphorylates and inhibits GSK3b,
preventing ubiquitination and degradation of cyclin D1
...
Levels of cyclin D can rapidly be changed by altering the rate of degradation
...
Cyclin D binds to CDK4 and CDK6 and the cyclin D/CDK4,6 are substrates for the Rb
family, p107 and p130
...
As well as acquisition of GF-independent, R-point passage involves biochemical
modification of pRb
...
That is, the degree of phosphorylation of Rb modulates whether or not the cell can
progress through G1
...
5
...
7
...
pRb binds a number of protein necessary for cell cycle progression via a conserved
‘pocket’ region
...
This family drives expression of S-phase genes, in that, E2F bound by pRb inhibits the
expression of S-phase genes, whereas inactive Rb allows E2F be activated and thus Sphase genes can be expressed
...
Furthermore, E2Fs bind to DNA as heterdimeric complexes with dimerization
partners
...
Within the 8 member E2F family, there are 3 transcriptional activators (1-3a) and 3
transcriptional repressors (3b-5), which regulate cell progression through G1
...
Early/mid G1 unphosphorylated/hypophosphorylated pRb binds and sequesters E2F
proteins, preventing them from activating transcription
...
Upon hyperphosphorylation of pRb by cyclin D:CDK4/6, and Cyclin E:CDK2, E2F
proteins are released
...
Release of E2F causes the transcription of genes necessary for completion of G1 and
entry into S phase
...
Oncoproteins (i
...
viral gene proteins) mimic pRb hyperphosphorylation, thus
preventing them from sequestering E2F proteins, and causing constitutive cell cycle
progression
...
A triple knockout of pRb, p130 and p107 are needed for the loss of cell cycle control
...
Cyclin E levels rise toward the end of G1 and fall during S phase
...
Cyclin E complexes with CDK2, forming the S-phase cyclin, thus suggesting that it
might play a role in the transition from G1 to S phase
...
Cyclin E can phosphorylate pRb and contributes to the passage of the restriction
point in G1 but is not induced by GFs
...
Cyclin E is ubiquinated by SCF and degraded rapidly à its expression in the cell cycle
is transient unlike that of cyclin D1
...
Cyclin D/CDK4,6 initiates
phosphorylation of pRb and causes
partial derepression of E2F leads to
expression of cyclin E
...
Cyclin E/CDK2 also phosphorylates pRb,
producing more cyclin E (positive
feedback mechanism)
...
Cyclin E/CDK2 phosphorylation of pRb
causes more E2F family members to be
transcribed (positive feedback
mechanism)
...
These initiated positive feedback cycles
account for how passage through G1
becomes GF-dependent (cyclin
D/CDK4,6) to GF-independent (Cyclin
E/CDK2), i
...
passage of restriction point
within G1
...
Upon exit from G1, cyclin E/CDK2 phosphorylates cyclin E, causing its ubiquitination
and proteolysis
...
Cyclin E levels drop upon S-phase entry and E2F causes transcription of cyclin A, due
to fully phosphorylated Rb during the RP passage
...
Cyclin A levels rise and remain high until mitosis
...
Cyclin A binds to CDK2 (and to a limited extent CDK1) à forming the S-CDK
complexes
...
DNA replication must happen once per cell cycle
...
6
...
These origins comprise regions of recognised DNA that is bound to protein to form a
pre-RC (pre-replication complex) and the downstream site to DNA polymerase is
recruited to
...
In eukaryotes, 2 steps are necessary for the formation of a replication origin:
- Recognition of a sequence where replication occurs: the pre-RC site – Origin
Licensing
- Activation of DNA synthesis – Origin Firing
8
...
9
...
10
...
11
...
12
...
13
...
14
...
15
...
16
...
S-Phase Regulation:
1
...
e
...
2
...
- Geminin binds to CDT1 and prevents it acting in origin licensing
...
3
...
4
...
e
...
Low CDK activity means no inhibition of CDT1
...
After duplication of the genome in S phase, the cell enters into G2 (i
...
the second
growth phase of the cell cycle), where it undergoes growth and protein synthesis in
preparation for M
...
Progression from G2 to M is regulated by Cyclin B/CDK1 and as in M, CDK1 is
rendered inactive through Wee1 and Myt1-dependent phosphorylation, and is
activated through Cdc25-dephosphorylation
...
PLK1 (polo-like kinase), activated at G2/M transition, drives the activation of Cyclin
B/CDK1 complex:
- PLK1 phosphorylates Wee1, creating a phosphdegron that is recognised by the
SCF ubiquitin ligase complex
...
- PLK1 phosphorylates Cdc25, thus activating it
...
4
...
e
...
5
...
6
...
7
...
a
...
b
...
c
...
- Rapid means of arresting the
cell cycle without needing
protein synthesis and is this
p53-independent
...
A similar DNA-mediated cell cycle arrest can be achieved in G1 through ATM/ATR
mediated inactivation of Cdc25A and subsequent failure to activate Cyclin E/CDK2
...
The proto-oncogene, c-Myc, in partnership with Max, forms a transcription factor
that can promote either oncogenic transformation or apoptosis
...
This TF acts to enhance Cdc25A expression, and in turn this cdc25A/B can cooperate
with oncogenic Ras, or loss of Rb, to transform primary mouse fibroblasts
...
Cdc25 has been found to be overexpressed in a number of breast cancers, head and
neck cancers and non-hodgkin’s lymphomas
...
Overall: cdc25 is a proto-oncogene because it can drive cell cycle progression
...
Knockout studies have shown that only CKD1 is essential for the vertebrate cell cycle
and that cyclin A2 and cyclin B1 are the only essential cyclins
...
In the absence of CDK2, CDK4 and CDK6, CDK1 can bind to cyclin D to drive pRb
phosphorylation
...
However, CDK1/Cyclin D is not as efficient as CDK4,6 in most cell types
...
A CDK2 knockout would lead to adult mice deficient in meiosis, i
...
lacking sex cells,
whilst a CDK4 KO would cause deficiencies in pancreatic beta-cells and pituitary
mammatrophs
...
5
...
The Importance of CDK Inhibitors:
1
...
2
...
- Ink4 family – active in early and mid-G1 and inhibits cells that have not passed
the restriction point
3
...
4
...
The Cip/Kip Family:
1
...
2
...
3
...
4
...
5
...
6
...
7
...
8
...
- In early G1, there is a lot of p27 and p21 in the cell
...
This cyclin D creates more cyclin D/CDK4,6, which sequesters excess p21 and
p27, thus allowing full activation of cyclin E/CDK2 to drive R-point transition
...
Additionally, PKB/Akt-dependent phosphorylation of p21 and p27 inhibits them by
preventing their localisation to the nucleus, thus preventing induction of cyclin D and
G1 passage
...
Poorer survival has been found in breast cancer patients where p27 has accumulated
within the cytoplasm
...
This family consists of 4 closely related inhibitors of CDK4/6: p15, p16, p18, and p19,
that are expressed in a tissue-specific manner
...
They inhibit CDK4/6 by preventing the molecule from binding to cyclin D
...
P16 has been independently identified as the product of the MTS1 tumour
suppressor gene at 9p21, and a loss of this inhibitor is common in melanomas
...
In some tumours, p16 expression is eliminated by tumour methylation
...
Overall: p16 is a mediator of cell senescence and a tumour suppressor
...
The p16 locus encodes two unrelated proteins which are read from different
overlapping ORFs and with different 1st exons
...
The first protein is p16 and the second is called p14ARF (alternate reading frame)
...
ARF-/- mice are cancer prone and their cells fail to undergo senescence in culture
...
Furthermore, ARF induces a proliferation block via G1 arrest in a p53-dependent
manner
...
ARF is transactivated by E2F, binds to MDM2 and sequesters MDM2 into the
nucleus
...
Therefore, MDM2 can no longer ubiquinate p53 and p53 is not degraded
...
ARF has a central role in regulating the levels of p53 and is thus an important tumour
suppressor
...
In many tumours where p53 remains functional, p53’s function is eliminated by the
inactivation of ARF
...
Excessive ARF production can occur via oncogene activation thus leading to
deregulated proliferation
...
Mutations at the p16 locus have the ability to:
- Disrupt the regulation of CDK4/6, leading to deregulated proliferation through
the absence of CKI
- Counteracting the sustained proliferation through ARF- and p53-mediated
restraint on the cell cycle
...
Thus explaining why p16 is such an important tumour suppressor
...
TGF-b controls cell cycle machinery in part through its ability to modulate the levels
of CDK inhibitors
...
It is capable of inducing p15 expression to inhibit CDK4/6 but can also weakly
stimulate p21 expression to inhibit CDK1/2, thus inhibiting pre- and post-R point
cells