Establishing irreversibility of G1/S transition

Question 1

When are the different cell cycle transitional checks?

Answer 1

~ Start (G1/S) transition
~ G2/M transition
~ Metaphase -anaphase transition

Question 2

Types of positive regulation of CDKs (activation)

Answer 2

~ mitogen activated signalling
~ transcriptional regulation of cyclin expression
~ binding of cyclin subunits
~ phosphorylation of CDK
~ removal of phosphorylation sites

Question 3

Types of negative regulation (inhibition)

Answer 3

~ CDK inhibitor proteins
~ inhibitory phosphorylation (Wee1)
~ Degradation of cyclin subunits

Question 4

Mitogen dependent signalling

Answer 4

1. mitogen binds to receptor
2. Ras activated
3. Activates MAP kinase
4. leads to transcriptional activation of Myc

Question 5

Myc

Answer 5

~ gene regulatory protein

~ promotes cyclin D and E2F expression

Question 6

E2F

Answer 6

~ major transcriptional regulator of S-phase genes and cyclin E/A
~ inactivated by binding to Rb protein

Question 7

Cyclin binding + CAK

Answer 7

1. CDK is inactive
2. cyclin is added + interacts with T-loop = partly active
3. CAK adds phosphate group to T-loop =conformational change = fully active

Question 8

CAK

Answer 8

CDK activating kinase

Question 9

CDKI (4 + name)

Answer 9

CDK inhibitor proteins
~ binds to SPF to prevent S-phas entry
~ binds to cyclin + CDK subunits
~ it prevents substrate binding + phosphorylation
~ needs to be degraded into order for S-phase to start

Question 10

Ubiquitin mediated protein degradation

Answer 10

~ involves 3 couples enzyme complexes (E1-3)

1. Ub is bound to E1 (requires ATP)
2. E1 transfers Ub to E2
3. E2 binds to E3 awa target protein
4. E2/E3 transfer of Ub to substrate

Question 11

E1

Answer 11

ubiquitin activating enzyme

Question 12

E2

Answer 12

conjugating enzyme

Question 13

E3

Answer 13

ubiquitin ligase

Question 14

Ternary complex + ubiquitin

Answer 14

Ternary complex = E2, E3 and substrate

1. successive rounds of ubiquitination label substrate for degradation
2. substrate released after full modification
3. proteosome-mediated degradation

Question 15

Proteosomal-mediated degradation…

Answer 15

~ regulates cell cycle transitions

~ each cell cycle phase is regulated by a specific ubiquitin ligase

Question 16

G1 ubiquitin ligase

Answer 16

APC-Cdh1

Question 17

S-phase ubiquitin ligase

Answer 17

SCF

Question 18

G2-anaphase ubiquitin ligase

Answer 18

APC-Cdc20

Question 19

Proteasomal activity, regulation + G1/S transition

Answer 19

1. S phase Cdk is inhibited by binding of Sic1
2. Sic1 is phosphorylated on multiple sites by G1/S Cdk
3. Phosphorylation of Sic1 targets for degradation by SCF.
4. S phase Cdk is now activated
5. S phase Cdk promotes DNA replication initiation