4. p27 and quiescence Flashcards
give an example of a protein that hold a cell in quiescence? and name an additional method
Rb
Cdk inhibitors
how were Cdk associated proteins identified? explain how this works
yeast 2 hybrid
- DNA binding domain attacked to cdk in yeast cells
- binding partners attacked to activation domain
- when binding partner binds cdks there is expression of reporter gene
what does the yeast 2 hybrid system exploit?
the modular nature of gene activator proteins
DBD alone is not enough to drive gene expression, it requires the activation domain to recruit basal machinery
give an example of a reporter gene?
a gene that allows allow growth in absence of specific nutrients/amino acid
name 3 cdks inhibitors
and what family do they belong to?
p21
p27
p57
they belong to the Cip/Kip family of cdk inhibitors
compare Cip/Kip family of cdk inhibitors
they are structurally similar although they vary in molecular weights and so have slightly different length
what do all Cip/Kip family of cdk inhibitors have?
a structurally conserved cyclin/cdk binding domain at their N terminus
what does p27 bind?
it binds the catalytic region of cdk2 and substrate recruitment site on cyclin A
describe the catalytic domain of cdks
they largely consist of an alpha helix rich C terminal part and a beta sheet rich N terminal part, the junction between the two parts is a packet which is the catalytic cleft of the enzyme
what two things does p27 do?
it inhibits catalysis by cdk2 by blocking the catalytic site and block substrate recruitment by cyclin A
what motif does p27 use to bind cyclin A?
RxL motif on p27 binds substrate recruitment surface on cyclin A
what other things have been shown to bind cyclin A through this motif?
cyclins and other cdks have been shown to interact by the RxL motif
what do crystal structures show us about where p27 binds cdk2?
- p27 has a key tyrosine residue that occupies the same space as the purine ring of ATP and so forms the same hydrogen bonds
- this means that ATP cannot bind cdk2 when p27 is bound
describe the radioactive kinase assay used to measure kinase activity of cdk2 as concentration of p27 increases
- gamma phosphate of ATP is labelled 32P
the ability of cdk2 to transfer this labelled gamma phosphate to a substrate is measured - high levels of p27 can almost completely inhibit kinase activity
what cyclin and cdk combination do cdk inhibitors preferentially bind?
D cyclin cdk4
E cyclin cdk2
A cyclin cdk2
cdk inhibitors bind cdks in what manner? and what affect will this have an one version being knocked out?
in a stoichiometric manner
this means that if one version of the gene is knocked out, the individual will only have half as much cdk inhibitions
what happens when cdk inhibitors are over expressed in vivo?
G1 phase arrest
what happens when cdks are expressed while a cell is going though S phase?
this can still trigger cell cycle arrest
what is the function of cdk inhibitors?
to prevent cells from passing of the restriction point when they should not be proliferating
what can induce p21 expression?
DNA damage
what happens to mice when p21 is knocked out? and what gender is this phenotype more pronounced in?
- they are tumour prone
- this leads to early death but not a very strong phenotype
- this cancer phenotype is more pronounced in male mice
p21 KO are cancer prone, what does this suggest about p21?
it may be a tumour suppresser
how often are p21 and p27 mutated in human cancer?
not very often
what is the cosmic database?
human catalogue of mutations in cancer
how is p27 a prognostic marker for cancer?
high levels indicate slow proliferating cancers that are not highly malignant
if a person is haploid for a gene and this makes more likely to develop a disease than someone who is homozygous WT but less likely to develop it that someone who is homozygous null, what is this called?
give an example of a gene that behaves like this
haploinsufficentcy
p27
in addition to tumourigenesis, what is also observed in mice that are KO p27? and what does this show us about p27?
they are very large compared to WT as their cells have proliferated more
this shows us that p27 is important for controlling normal proliferation
name the disease which with p57 and what occurs in this disease?
Beckworth-Weidemann syndrome is a rare disease where there is over growth of certain parts of the body due to additional cell division at localised points. it is associated with deformed skeletal structure and although it is not a cancer phenotype, suffers don’t have a good life expectancy
cyclin dependent kinases have the potential to be good tumour suppressers, why can we not say that they are?
they do not fit the proper description and there is no good evidence that they are.
what is the main role of p27?
to maintain quiescence and prevent aberrant proliferation by cdks
for the switch between G1 and G0, what two factors are expressed in a reciprocal manner? when there is no mitogenic stimulation what are the relative levels of these two things?
p27 and cyclin D
in G0 p27 is high and cyclin D is low
when do p27 levels drop?
levels decline as cell starts to proliferate
what causes levels of p27 to decline?
when cell cycle is stimulated cyclin D cdk4 expression overwhelms p27. when cyclin E is expressed cdk2 is activated and this phosphorylates p27 at threonine 187. this phosphorylation recruits ubiquitin machinery and tags p27 with poly-ubiquitin for ubiquitin-mediated proteolysis
what is the proteasome?
a mutli-subunit proteases
what does E1 enzyme do?
E1 activates ubiquitin and passes it on to E2
give an example of three proteins that E2 associates with
Rbx1, Cul1 and Skp1
what is the F-box protein?
an adaptor protein between E2 protein complex and phospho-substrates
what is E3 made up of?
Cul1, Skp1 and F-box
how does the F-box protein function?
it brings phospho-protein substrates into close proximity with the E2 complex so that it can transfer Ub onto the substrate and build up poly-Ub chains
what are two known oncogenes in this p27 degradation process?
Skp1 and F-fox protein are over expressed in some cancers
what two process occur through ubiquitin mediated proteolysis in the cell cycle? in the second example, what does this ensure?
elimination of p27 from quiescent cells after mitogenic stimulation
cyclin turn over
this is also due to phosphorylation recognition, each new cyclin causes the destruction of the previous one
this ensures directionality through the cell cycle
