Lecture 10: p53 Flashcards
p53 is the safety-brake of the cell cycle
*p53 Defective in numerous Tumours
*Familial cancer syndrome Li Fraumeni syndromesusceptibility to a wide variety of cancers
p53 is a nuclear protein that normally exists in the cell as a
homotetramer (ie an assembly of 4 identical polypeptide subunits)
Mutant p53 acts in a
dominant negative manner to ‘block’ the activities
of wild type (WT) p53
When have mutant p53 allele and WT p53 allele-
-function of p53 in cell
is almost gone-mixture of mutant and wild type p53-if tetramer has a
single mutant p53 subunit-interfere with function of the tetramer as a
whole
Direct DNA binding
Most mutations
affecting p53 cause the
p53 protein to lose its transcription
activating powers.
DNA binding is by
p53 tetramers.
DNA binding is central to p53 function
How is p53 expression and activity controlled?
1 Gene transcription
Stimuli such as DNA damage or irradiation increase p53
transcription
2 Protein stability
p53 is unstable, & rapidly degraded in normal cell (little p53
protein seen)
In response to DNA damage p53 levels increase in the cell
3 Post translational modification
For full activity, phosphorylation at key serine residues is
required.
4 Location
For full activity, p53 needs to be in the nucleus.
5 Structural interactions/oligomerization
For full activity, p53 needs to form a tetramer-(ie for p53
an assembly of 4 identical polypeptide subunits)
- How is p53 expression and activity
controlled?
Mdm2 protein can bind to and inactivate p53.
Following DNA damage (eg X-rays, UV, chemotherapeutic drugs) levels of
p53 protein increase dramatically in the absence of any marked changes
in mRNA levels of p53-indicates post-translational modification of p53
stabilises p53 protein
- p53 protein levels increase (see 8h post-radiation in Western blot
below) following phosphorylation events mediated by several kinases
(stabilize p53)(MDM2 not able to bind p-p53).
These kinases can become activated following DNA damage.
p53 is a transcriptional activator
p53 regulates transcription positively
p53 functions as a TF that halts cell cycle advance in
response to DNA damage and attempts to aid in
the repair process
Transcriptional
Activation of=
p21 (CDKI)-important target p53
GADD45 (role=involved in repair DNA
damage)
BAX, Bid, APAF1(role=Apoptosis)
Thrombospondin-1 (anti-angiogenesis)
MDM2 (role=p53’s negative
regulator!)
The great majority of the mutations
affecting the p53 gene cause the p53
protein to
lose its transcription-activating
powers
* amino acid substitutions in the DNA-binding
domain of p53.
Progression through G1
CDK2-E cyclin
inactive
—CDK2-E cyclin
active—RB—Rb-PPP (Hyperphosphorylated pRb)-
Allows for S-phase gene expression
By E2F TFs.
If DNA damage is detected in G1 then p53 will halt cell cycle
progression through transcriptional upregulation of
CDKI p21 (ie increase
in p21).DNA repair machinery then activated
DNA replication
p21 competes with DNA
polymerase d for PCNA
directly inhibits DNA
replication.
Proliferating Cell Nuclear
Antigen (PCNA) regulates
DNA replication
- p53 Targets CyclinB-CDK1
p53-another target is 14-3-3s protein sequesters
cyclinB-CDK1 complex in the cytoplasm, prevents
it from moving into nucleus-Prevents Mitosis
until DNA repair has been successful
* GADD45 (p53 target) binds to and dissociates
CDK1-cyclinB kinase
* P21 CDKI (v. important p53 target)
P53 and apoptosis
- Extrinsic
– p53 increases transcription of Fas
– p53 enhances Fas transport to cell surface - Intrinsic
– p53 induces BAX, PUMA and NOXA (pro-apoptotic proteins)
– p53 induces APAF-1 (see apoptosome)
– Inhibits BCL2 and BCL2-XL (direct binding)
p53 is mutated in a high
percentage of cancersLeads to deregulation of apoptosis
pathways and increases tumorigenesis
