Cell Cycle 6

Question 1

What is the site of phosphorylation required to activate all cdks?

Answer 1

Threonine ~160 in the t loop

Question 2

What is the affect of cyclin binding cdk on t loop?

Answer 2

Causes T loop to undergo conformational change, which is then phosphorylated by CAK (cyclinH/cdk7)

Question 3

What phosphorylates cdk t loop?

Answer 3

cdk7/cyclin H (CAK)

this further opens the binding site

Question 4

Other than opening the binding site, what does the ~160Y phosphorylation?

Answer 4

The negative charge is important in aligning the consensus sequence - (S/T)Px(R/K) -, which has a positive residue, in the binding site

Question 5

What is the consensus sequence for cdks?

Answer 5

(S/T)Px(R/K)

Question 6

What else does CAK (cyclin H/cdk7) target?

Answer 6

RNA polymerase

Question 7

What is the function of Wee1?

Answer 7

Inhibits cdk2 by phosphorylation. This prevents early division.

Question 8

What are the human equivalents of wee1?

Answer 8

Myt1, Mik1

Question 9

What type of kinase is Wee1?

Answer 9

tyrosine/theorine

Question 10

What is the consensus sequence of wee1?

Answer 10

GxGxxG - the ceiling of the ATP binding site. Phosphorylation blocks access to ATP
on cdks t14/15

Question 11

Name an agonist of Wee1?

Answer 11

cdc25

Question 12

What activates Wee1?

Answer 12

DNA damage - as a result cyclinD/cdk4 gets phosphorylated so Rb can’t be phohsphorylated

Question 13

Is wee1 a tumour supressor?

Answer 13

yes

Question 14

What is cdc25?

Answer 14

A phosphatase which reactivates cdks after inhibitory phosphorylation - target T/Y

Question 15

What 3 pieces of evidence is there that Cyclin-E/cdk2 phosphorylation results in release of E2F?

Answer 15

1) antibody initiation results in cells accumulating in G1
2) Inhibition with Roscovitine (ATP analgoue) causes G1 arrest
3) Expression of dominant negative cdk2 (145D –> N) which can not longer coordinate Mg2+ ion causes G1 arrest

Question 16

What happens when a KO mouse for cyclin E1/2 is created?

Answer 16

Mostly normal development - slightly slower division

Question 17

Why do cyclin E1/2 KO mice have mostly normal development?

Answer 17

Other cyclins take there place. Only cyclin required in cyclin is cyclin A and cdk1

Question 18

What is the affect os roscovitine in Rb null cells?

Answer 18

No affect because no downstream pathway

Question 19

What is roscovitine?

Answer 19

cdk2 inhibitor (ATP analogue) - hence can inhibit other cdks (also can inhibit pyridoxal kinase also inhibited)

Question 20

What is bortezomib?

Answer 20

Proteasome inhibitor (inhibits chymotrypsin part of proteosome which is involved in p27) - not specific to p27

Question 21

What 4 molecules can stabilise p27?

Answer 21

C1, C2, C16, C20

Question 22

How do C1, C2, C16, C20 stabilise p27?

Answer 22

Interrupts p27 interaction with skp2