5. p16 and senescence Flashcards
what has p16 been shown to interact with? and how was this shown?
cdk4 and cdk6
>this was shown in a Yeast-2-hyrbid screen using a histidine reporter
>the then proved this interaction in mammal cells using immunoprecipitation
how often is p16 mutated in cancer cells lines? what sort of mutations are seen and what does this imply?
it is mutated very frequently in cancer cells lines
> these mutations are mostly deletions
> this implies that a tumour suppresser has been knocked out
describe the structure of p16
4 ~ 33-residue Ankyrin repeats
these are helix turn helix motifs that interact and stabilised each other
what part of p16 provides binding specificity?
the loops provide binding specificity of protein-protein interactions
what is seen when p16 is added to the cdk cyclin complex that it binds?
p16 kicks cyclin D off cdks4
> there is reciprocal binding of p16 and cyclin D to cdk4
what might an inactivating mutation of p16 found in tumours result in?
it no longer being able to interact with cdk4 and cdk6
a kinase assay was done titrating p16 with cdk4/6, what was observed? and what is observed with mutant p16?
high levels of p16 inactivate kinase
mutant p16 does not bind cdk4/6 and so kinase activity remains the same/is uninhibited
describe how p16 binds cdks
> p16 binds the opposite side of cdk4/6 to cyclins
how does the binding of p16 affect cdks
p16 affects cyclin binding site in an allosteric way and does not directly interact with the binding surface
describe what happens to the structure of cdks when p16 binds
> the N lobe of the kinase rotates by 15 degrees, this misaligns ATP binding site
15 degrees twist in alpha helix which is important for cyclin binding is displaced so that cyclin can no longer bind
what two things does the binding of p16 to cdks inhibit? and how does p27 differ from this?
cdk4/6 binding to ATP and cyclin D
> p27 sterically blocks the ATP binding site and the substrate recruitment by the cyclin
what can be deduced from the amount of DNA in a cell?
the stage in the cell cycle
what phases of the cell cycle will cells have double as much DNA?
G2 and M phase
what can affect the proportion of cells in different cell cycle phases when p16 is added?
whether Rb is knocked out
when p16 is added to Rb WT cells, what is seen in flow cytometry experiments? and how is this different to mutant p16?
there is a large peak in the G1 phase with no cells in G1 and M phase
> populations of cells in different stages of the cell cycle are similar to that of the control with no p16
what is seen when p16 is added to Rb null cells? and what does this indicate?
the addition of WT and mutant p16 has no affect on what stage of the cell cycle Rb null cells are in
what is the only essential substrate of cdk4 to allow cell cycle progression? and why is this?
Rb - along with additional phosphorylation this allows the release of E2F
why does p16 have no affect on Rb null cells?
E2F is already free and active therefore inhibiting cdk4 has no affect
how can p16 indirectly have an affect on cdk2?
> there is always a basal expression levels of p27 which binds all cdks/cyclins
when p16 is added it displaces p27 from cdk4
this frees up p27 to bind other cdk/cyclins and so increases inhibition of cdk2 which reduces number of cells in S phase
what is normally expressed in high levels in Rb null cells, and what affect would adding more of this have?
p16
adding more would have little effect as there are no cyclinD/cdk complexes remaining
what happens when p16 is over expressed in Rb positive cells?
it causes G1 cell cycle arrest
how common are p16 mutations in primary tumour material? and comment on why this might be different from cell lines and how this might then compare to cell lines?
it is mutated less common than in cell lines
>p16 can also be inactivated by promoter methylation - this inactivates transcription
>this would still not equate to the number of mutations seen in cells lines
when DNA is methylated, what is made and what enzyme catalyses this reaction? and what do this do?
5-methyl-cycteine
DNA methyl transferases
> generally this represses transcription
what can be coupled with methylation to further silence a gene?
histone methylation by HDACs to compact chromatin
what can be seen when doing a restriction digest with enzymes sensitive to DNA methylation at p16 locus of carcinoma cells?
some cells have strong bands indicating methylation which is not seen in control cells
> this indicated the promoter is supressed in some cancer cells
what are the four other family members of the p16 family? comment on their structure and function
p15, p16, p18 and p19
> they have Ankyrin repeat loops that bind to cdk4 and cdk6
which two family members of p16 are involved in wide spread cell cycle regulation and what are the other two used for?
p15 and p16
p18 and p19 are involved in specific cell type proliferation and inhibition during development, and so are not associated as much with cancer
what do double KO p16 mice have a predisposition to?
tumours
why is there a difference in the number of time p16 is mutated in cells line and primary tumours? and why is this?
loss of p16 is required to establish a cells line
> this is because p16 is known to impose senescence
> when culturing cells you are selecting for cells are proliferating i.e. have lost p16
