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Flashcards in 6. finer points of cell cycle control Deck (67)
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1

phosphorylation can be inhibitory or stimulatory, TRUE or FALSE?

TRUE

2

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

3

where is cdk2 phosphorylated? and how can this be identified?

the T loop (threonine loop)
> mass spectrometry

4

what is needed in order for Cdks to be active?

the phosphorylation of the T loop

5

describe the T loop when it is in-phosphorylated

its disordered

6

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

7

what phosphorylates the T loop of cdk2? and what is this not regulated by?

cyclin H/cdk7
this is not regulated by the cell cycle, it is always present and constantly active

8

although the T loop phosphorylation doesn't result in a massive conformational change, what fold does it increase enzyme activity?

many thousand fold

9

what two functions does phosphorylation of the T loop have?

>promotes active site access - by removing T loop steric hindrance
>promotes correct substrate alignment

10

what is the cdk phosphorylation consensus sequence?

S/TPxR/K

11

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

12

why is cyclin H/cdk7 a good anti-cancer target?

inhibiting it would stop the phosphorylation of cdks and could stop cell proliferation

13

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

14

what was giving rise to Nurse's Wee phenotype in fission yeast?

a lack of inhibitory phosphorylation which allows yeast to proliferate too fast

15

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

16

what does wee1 do?

> phosphorylates cdks at two sites
> tyrosine 15 and threonine 14

17

what is odd about wee1 specificity?

it is unusual to have a kinase that can phosphorylate these two residues

18

what motif does wee1 phosphorylate? and where is this conserved motif found?

GxGxxG motif
>in the ceiling of the ATP binding site

19

where are the tyrosine and the threonine that are phosphorylated located? (cdks)

near the exit part of the ATP binding site

20

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

21

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

22

what enzyme removed the phosphate from inactive cdks? and what does this do?

cdc25
this activates the enzyme

23

when was cdc25 first defined?

in Nurse’s original screen in yeast - these enzymes are conserved in humans

24

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

25

how many different types of cdc25 are there and what are they called? and how are they implicated in cancer?

three
a, b and c
one of them is normally over expressed in cancer

26

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

27

what is a Quinone arrangement?

opposite double bound oxygen across a 6 membered ring

28

how do these small molecules inhibit cdc25? and what does this mean?

quinones react irreversible with the active site cysteine
>they lack specificity

29

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

30

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