Flashcards in 6. finer points of cell cycle control Deck (67):
phosphorylation can be inhibitory or stimulatory, TRUE or FALSE?
when cdk2 from G0 and late G1 phase is run on a gel what is seen? what does this suggest and how is it proved?
predominantly one band for cdk2 in G0 cells and two bands in the late G1 sample
>cdk2 has been phosphorylated, phosphatase can be used to revert late G1 phase cdk2 to single band
where is cdk2 phosphorylated? and how can this be identified?
the T loop (threonine loop)
> mass spectrometry
what is needed in order for Cdks to be active?
the phosphorylation of the T loop
describe the T loop when it is in-phosphorylated
what happens to the T loop when cyclin E binds cdk2?
>the T loop becomes ordered
>it undergoes conformational changes which make it more susceptible to phosphorylation
>phosphorylation induces further conformational changes at opens up the active site
what phosphorylates the T loop of cdk2? and what is this not regulated by?
this is not regulated by the cell cycle, it is always present and constantly active
although the T loop phosphorylation doesn't result in a massive conformational change, what fold does it increase enzyme activity?
many thousand fold
what two functions does phosphorylation of the T loop have?
>promotes active site access - by removing T loop steric hindrance
>promotes correct substrate alignment
what is the cdk phosphorylation consensus sequence?
how does T loop phosphorylation promote correct substrate alignment?
>R and K carry a net positive charge at physiological pH
>the phosphorylation introduces a negative change which interacts with this
why is cyclin H/cdk7 a good anti-cancer target?
inhibiting it would stop the phosphorylation of cdks and could stop cell proliferation
what are the draw backs of using cyclinH/cdk7 inhibits as a therapeutic? and how can this be overcome?
that cyclin H/cdk7 is also required in the phosphorylation of the C terminal domain of RNA polymerase II, converting it from initiation phase to elongation phase of transcription - inhibiting it may abolish transcription
>there is a widow of opportunity to target T loop P over the transcriptional process, but even this still has off target effects
what was giving rise to Nurse's Wee phenotype in fission yeast?
a lack of inhibitory phosphorylation which allows yeast to proliferate too fast
how do you screen for mutants in fission yeast?
>apply a mutagen to the system at a dose which mutates one gene per yeast
>this is likely to be an inactivating mutation
what does wee1 do?
> phosphorylates cdks at two sites
> tyrosine 15 and threonine 14
what is odd about wee1 specificity?
it is unusual to have a kinase that can phosphorylate these two residues
what motif does wee1 phosphorylate? and where is this conserved motif found?
>in the ceiling of the ATP binding site
where are the tyrosine and the threonine that are phosphorylated located? (cdks)
near the exit part of the ATP binding site
when the tyrosine and threonine are phosphorylated what does this result in? (cdks)
altered access of ATP and substrate to the ATP binding site
>blocks substrate recruitment
> substrates need to come into close proximity to the gamma phosphate
what is wee1 kinase activity physiologically important for? (2)
>to ensure that cdks are only active at the right stage.
>for growth arrest in response DNA damage
what enzyme removed the phosphate from inactive cdks? and what does this do?
this activates the enzyme
when was cdc25 first defined?
in Nurse’s original screen in yeast - these enzymes are conserved in humans
why might cdc25 be a good potential therapeutic target?
it activates the cell cycle
>dual specificity phosphatase and so will be easier to drug as its unusual
how many different types of cdc25 are there and what are they called? and how are they implicated in cancer?
a, b and c
one of them is normally over expressed in cancer
what do cdc25 inhibitors target? and describe what many of the inhibitors have in common?
the active site cysteine
>many of these small inhibitors have a Quinone arrangement
what is a Quinone arrangement?
opposite double bound oxygen across a 6 membered ring
how do these small molecules inhibit cdc25? and what does this mean?
quinones react irreversible with the active site cysteine
>they lack specificity
why is it hard to inhibit cdc25? and what does this mean?
it is hard for medicinal chemists to make flat, water soluble drugs with binding specificity to the shallow unfeatured surface/active site
>this means its not necessarily a good drug target
why was cyclinE/cdk2 through to be essential for entry into S phase?
it phosphorylates serine 567 on Rb to fully activate E2F and drive cell cycle into S phase
what experimental evidence is there that cyclin E/cdk2 is essential?
1. antibody microinjection against cdks2 leads to accumulation of cells in G1 phase
2. inhibitors such as roscovitine (molecule based on adenine)that binds to cdks ATP binding site and causes G1 arrest
3. 3. expression of dominant negative cdk2- these are proteins with all feature of WT protein but lack activity
in order to make a dominant negative, what do you need to consider?
the catalytic mechanism of the enzyme
describe the catalytic mechanism of cdk2
>ATP sits in the binding site of cdk2
>negative phosphates neutralised by magnesium
>magnesium coordinated by Asp145 and Asn132
>magnesium abstracts charge from oxygen on phosphate
>this causes are rearrangement that frees up the gamma phosphate to react with charge hydroxyl on substrate
which residue provides a large negative charge to keep this magnesium ion in position in cdk2?
how is the dominant negative cdk2 made?
mutate aspartic acid to asparagine
>the side chains are basically the same you just exchange the O- with an NH2
how is this negative dominant no longer catalytically competent?
the negative charge is lost and so the magnesium is no longer held in position, this means that the enzyme is no longer catalytically competent
what happens in cells that have this dominant negative cdk2?
cells pile up in G1 phase
what is required for proliferation in Rb null cells? describe an experiment that shows this. what does this suggest? and what makes this a good therapeutic target?
>adding roscovitine to Rb null cells and they pile up in G1 phase
>this suggest that cyclin E/cdk2 has activity required for G1/S transition
>targeting this should be effective in Rb null cells
how many cyclin E genes are there in humans and mice? name the genes in mice
two genes in each
E1 and E2
what is the phenotype when cyclin E1 is KO in mice?
what is the phenotype when cyclin E2 is KO in mice? and why is this the case?
>reduced male fertility
>cyclin E2 is largely restricted to expression in gonads so this is not suppressing
what is the phenotype when cyclin E1/E2 is KO in mice?
>embryonic lethality due to placental defects
>not lethality due to lack of cells only that the placenta did not develop properly
what was used in order to have normal embryonic development in cyclin E1/E2 KO mice? what one thing was not normal with these mice?
>placenta rescue through other genetic mutations
>they have reduced cardiac tissue
what do these mice double KO suggest about cyclin E?
cyclin E is not the major cell cycle regulator as cells have obviously gone through lots of cell divisions
>E1 is expressed in all tissues, double KO confirms that when E1 is KO, E2 does not replace it
what is seen when cyclin E1/E1 KO cells are grown in culture? and what does this show?
they grow slower than wild type cells but they still proliferate.
>this shows that cyclin E is not required for normal cell division.
is cyclin E required for cells to come out of quiescence?
what can make cells quiescent?
starving them of growth factors
how can S phase be measured?
looking at 3H-thymidine incorporation into cells
what are cdk2 double KO mice deficient in?
meiosis - they are incapable of making gametes
is cdk2 required for cells to come out of quiescence?
no, double KO are capable of getting back into the cell cycle (even if this is a little less efficient)
comment on the experiments proving and disproving cycli E/cdk2 importance?
they show the limitations in the experimental approaches used
e.g. KO show the opposite to antibody microinjection experiments, roscovitine and dominant negative cdks
what is expressed early in order to compensate for lack of cyclin E and cdk2?
what combinations of cyclins and cdks are capable to drive G1/S transition?
cyclin E/A and cdk2/1
KO mice have confirmed that only one cyclin and one cdk are required for cell cycle progression, what are they? and what does this suggest in terms of therapeutics?
cyclin A and cdk1
>small molecule inhibitors of cdk1 or things that disrupt the cyclin A/cdk1 interaction are good cancer targets as these are universal
>if you inhibit other cyclins and cdks then cells can by-pass therapeutics
what makes the cell cycle most efficient?
when cells have a full compliment of cyclins and cdks
name four proteins that promote quiescence and are anti-proliferative
name five proteins that promote and drive proliferation and thus are potential good drug targets
when p27 is KO in mice, what is their appearance? when cyclin D is KO in mice what is their appearance? and what is seen when they are both KO?
>mice are larger than WT
>mice are smaller then WT
>mice are same size as WT
where does roscovitine bind cdk2? how many kinases are there in our body and what implication does this have?
the ATP binding site
>there are 500 different kinases in our genome that have the same catalytic fold and so there is a high chance roscovitine targets more than one type of kinase
what else does roscovatine bind? and why is this strange? and what might this affect?
this kinase does not share the same catalytic fold as cdk2
>inhibiting this may affect metabolism
name a second inhibitor that is being used in clinics, describe its appearance
>it has a peptide like arrangement
what does Bortezomid inhibit?
>it inhibits the chymotrypsin activity in the proteasome
what is Bortezomid good at treating? and why is this the case?
>it is not known why multiple myelomas are susceptible to inhibition of degradation
>it may be because there is increased degradation in these cells which if stopped will reduce proliferation
what is a draw back of Brotezomid? and what could be done to overcome this?
it is a general proteasome inhibitor. blocking degradation has its draw backs, there are lots of things that we want to get rid of in cells
>it would be better to specifically prevent depredation of p27 as it can be targeted for the proteasome, additionally methods to stabilise p27 could be used e.g. cell might be degrading apoptotic factors
when compounds for specific inhibition of p27 degradation are being tested, what is looked at?
inhibition of degradation of p27 and not inhibition of degradation of other things
>trying to target the specific interaction
what binds to p27 and causes its ubiquitin mediated degradation? and how can this be used to stabilise p27?
small molecules can disrupt interactions made between p27 and skp2