p53 part 2 Flashcards
What are the domains seen in the P53 molecule?
A transactivation domain (TAD amino acids 1-40)
A proline rich domain (PRD) with five PXXP motifs that recruits histone acetyltransferases that regulate p53 stability (amino acids 60-90)
A DNA binding domain (DBD) that binds DNA consensus recognition elements (amino acids 100-300)
A tetramerisation domain (amino acids 324-355)
A C-terminal regulatory domain (CTD) that binds DNA non-specifically and may regulate the activity of the DBD domain (amino acids 360-393)
What P53 domain does MDM2 and MDMX interact with?
In unstressed cells these bind to TAD and will target the protein for degradation by ubiquitinating C-terminal lysines while MDMX prevents interaction with other transcription factors
What do cell culture studies suggest about p53 activation?
That it follows post-translational modifications performed by kinases, prolyl isomerases and acetylases
This can occur with the DNA damage response activating damage responsive kinases that phosphorylate S15 (ATM, ATR, DNAPK), T18 and S20 (Chk2) in the TAD these phosphorylations alter the atrucutre of the amphipathic alpha-heix with which MDM2 interacts, ATM also phosphorylates MDM2 to displace it
What occurs due to PRD phosphorylation?
This occurs at T81-P82 by the JNK stress kinase and allows binding of prolyl isoemrse PIN1 this generates cis-trans prolyl isomerisation disfavouring MDM2 binding
What does displacement of MDM2 from the p53 molecule allow?
Tetramisation which inturn hides a nuclear export signal on the molecule allowing nuclear entry
Association of the PRD with HATS and PXXP motifs, C-terminal lysines are acetlyated increasing p53 stability and DNA binding at specific target genes
How many phosphorylation sites are there on p53?
23 with many of these being phosphorylated by multiple enzymes meaning that it needs to be viewed in the light of clinical cancer observations, observations of transgenic mice models and observations from yeast expression
What observations can be made about p53 phosphorylation from clinica cancers?
The P53 mutations are concentrated in the DNA binding domain
S,T and K residues in the TAD, PRD and CTD domains contribute to less than 0.1% of mutations arguing against any one of these residues being critical for p53 function
What observations can be about p53 mutations from transgenic mouse models?
In transgenic mice p53 mutants lacking amino acids that are phosphorylated or acetylated show minor changes in transcription, cell cycle arrest, apoptosis and tumour suppression
This shows that these residues are not essential for but fine tune p53 activity
What observations can be made about p53 from yeast expression studies?
In yeast expression assays activity of more than 2000 p53 mutants was investigated with mutants occurring in the DBD having markedly reduced transcriptional activity but mutations in the TAD, PRD and CTD having only minor effects
What characterises the abnormal proliferation generated by oncogenes by carcinogenesis?
Replicative stress where replication may be initiated multiple times at replication origins, replication forks may stall or prematurely terminate leaving partially replicated DNA with extended lengths of single stranded DNA and potentially generating double strand breaks
What are the different stages of early bladder cancers?
Ta where there are early superficial tumours
T1 where there are early invasive cancers
T2-4 which are the more advanced cancer stages
What mutations that deregulate the cell cycle can induce the DNA damage response?
Activating mutations of FGFR3
Loss of function mutations of pRb
Over expression of Cyclin-E
How can we visualise how premalignant tumours have a continuously active DDR ?
Through immunohistochemically using phosphorylation-specific antibodies this includes
pS1981 of ATM
phosphorylation of the ATM substrate gammaH2AX
pT68 of Chk2
pS15 of p53 (and the appearance of p53BP1 in nuclear foci)
What can we learn from the fact that premalignant cancers have an almost constant DNA damage Response?
The presence of DDR, p53 activation and senescence are strongly correlated with progression to a carcinoma leading to a break down of this association
The DDR is maximal before genetic instability appears but decreases as tumours become more advanced this decrease correlates with the inactivation of components including ATM loss of heterozygosity and loss of expression, Chk2 mutation and loss of heterozygosity, p53 mutation and LOH at the p53 locus and loss of p53BP1 expression
What characterizes Epithelial to mesenchymal transition?
Loss of epithelial E-cadherin and gain of mesenchymal (vimentin) markers
Motile, invasive and potentially metastatic behaviour
Progenitor cell properties
A pro-survival, therapy resistant phenotype
