4. p27 and quiescence

Question 1

give an example of a protein that hold a cell in quiescence? and name an additional method

Answer 1

Rb

Cdk inhibitors

Question 2

how were Cdk associated proteins identified? explain how this works

Answer 2

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

Question 3

what does the yeast 2 hybrid system exploit?

Answer 3

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

Question 4

give an example of a reporter gene?

Answer 4

a gene that allows allow growth in absence of specific nutrients/amino acid

Question 5

name 3 cdks inhibitors

and what family do they belong to?

Answer 5

p21
p27
p57
they belong to the Cip/Kip family of cdk inhibitors

Question 6

compare Cip/Kip family of cdk inhibitors

Answer 6

they are structurally similar although they vary in molecular weights and so have slightly different length

Question 7

what do all Cip/Kip family of cdk inhibitors have?

Answer 7

a structurally conserved cyclin/cdk binding domain at their N terminus

Question 8

what does p27 bind?

Answer 8

it binds the catalytic region of cdk2 and substrate recruitment site on cyclin A

Question 9

describe the catalytic domain of cdks

Answer 9

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

Question 10

what two things does p27 do?

Answer 10

it inhibits catalysis by cdk2 by blocking the catalytic site and block substrate recruitment by cyclin A

Question 11

what motif does p27 use to bind cyclin A?

Answer 11

RxL motif on p27 binds substrate recruitment surface on cyclin A

Question 12

what other things have been shown to bind cyclin A through this motif?

Answer 12

cyclins and other cdks have been shown to interact by the RxL motif

Question 13

what do crystal structures show us about where p27 binds cdk2?

Answer 13

• 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

Question 14

describe the radioactive kinase assay used to measure kinase activity of cdk2 as concentration of p27 increases

Answer 14

• 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

Question 15

what cyclin and cdk combination do cdk inhibitors preferentially bind?

Answer 15

D cyclin cdk4
E cyclin cdk2
A cyclin cdk2

Question 16

cdk inhibitors bind cdks in what manner? and what affect will this have an one version being knocked out?

Answer 16

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

Question 17

what happens when cdk inhibitors are over expressed in vivo?

Answer 17

G1 phase arrest

Question 18

what happens when cdks are expressed while a cell is going though S phase?

Answer 18

this can still trigger cell cycle arrest

Question 19

what is the function of cdk inhibitors?

Answer 19

to prevent cells from passing of the restriction point when they should not be proliferating

Question 20

what can induce p21 expression?

Answer 20

DNA damage

Question 21

what happens to mice when p21 is knocked out? and what gender is this phenotype more pronounced in?

Answer 21

• they are tumour prone
• this leads to early death but not a very strong phenotype
• this cancer phenotype is more pronounced in male mice

Question 22

p21 KO are cancer prone, what does this suggest about p21?

Answer 22

it may be a tumour suppresser

Question 23

how often are p21 and p27 mutated in human cancer?

Answer 23

not very often

Question 24

what is the cosmic database?

Answer 24

human catalogue of mutations in cancer

Question 25

how is p27 a prognostic marker for cancer?

Answer 25

high levels indicate slow proliferating cancers that are not highly malignant

Question 26

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

Answer 26

haploinsufficentcy

p27

Question 27

in addition to tumourigenesis, what is also observed in mice that are KO p27? and what does this show us about p27?

Answer 27

they are very large compared to WT as their cells have proliferated more
this shows us that p27 is important for controlling normal proliferation

Question 28

name the disease which with p57 and what occurs in this disease?

Answer 28

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

Question 29

cyclin dependent kinases have the potential to be good tumour suppressers, why can we not say that they are?

Answer 29

they do not fit the proper description and there is no good evidence that they are.

Question 30

what is the main role of p27?

Answer 30

to maintain quiescence and prevent aberrant proliferation by cdks

Question 31

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?

Answer 31

p27 and cyclin D

in G0 p27 is high and cyclin D is low

Question 32

when do p27 levels drop?

Answer 32

levels decline as cell starts to proliferate

Question 33

what causes levels of p27 to decline?

Answer 33

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

Question 34

what is the proteasome?

Answer 34

a mutli-subunit proteases

Question 35

what does E1 enzyme do?

Answer 35

E1 activates ubiquitin and passes it on to E2

Question 36

give an example of three proteins that E2 associates with

Answer 36

Rbx1, Cul1 and Skp1

Question 37

what is the F-box protein?

Answer 37

an adaptor protein between E2 protein complex and phospho-substrates

Question 38

what is E3 made up of?

Answer 38

Cul1, Skp1 and F-box

Question 39

how does the F-box protein function?

Answer 39

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

Question 40

what are two known oncogenes in this p27 degradation process?

Answer 40

Skp1 and F-fox protein are over expressed in some cancers

Question 41

what two process occur through ubiquitin mediated proteolysis in the cell cycle? in the second example, what does this ensure?

Answer 41

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