Week 8(Cell Cycle Activities of CDKs and the Ubiquitin Ligases)

Question 1

When do G1/S cycling accumulate?

Answer 1

During G1

Question 2

What is Retinoblastoma (Rb)?

Answer 2

A pocket protein that sequesters (holds out of action) members of the E2F family of transcription factors.

Question 3

What do G1/S CDK do?

Answer 3

phosphorylates Retinoblastoma (Rb)

Question 4

What does E2F stimulate?

Answer 4

stimulates the transcription of genes that promote entry into S-phase such as G1-cyclins, S-cyclins and DHFR (dihydrofolate reductase).

Question 5

What is Phosphorylating Rb part of?

Answer 5

a Feedback Loop which Promotes Entry into S phase

Question 6

Why might we want to prevent entry into S-phase?

Answer 6

The arrest of the cell cycle in G1 or G2
following DNA damage reduces the risk that
a neoplastic change will be passed onto the
daughter cells.

Question 7

What can cause DNA damage?

Answer 7

DNA damage includes damage done by
ultraviolet and x-irradiation, and
chemotherapeutic drugs.

Question 8

How does the DNA damage checkpoint work?

Answer 8

Severe DNA damage in G1 increases the activity
of p53.

p53 is a transcription factor.

In undamaged cells, p53 is unstable and is present
at low concentrations since it is bound by Mdm2,
a ubiquitin ligase that targets p53 for degradation.

Question 9

What does DNA damage activate?

Answer 9

DNA damage activates kinases (ATM/ATR and
Chk1/Chk2) that phosphorylate p53,
releasing it from Mdm2.

Question 10

p53 transcribes CKIs

Answer 10

Once stable and active, p53 turns on the expression of genes that encode CKIs (inhibitors of Cdk-cyclin complexes) such as p21 (p21 is similar to p27, a CKI normally present in early G1).

When p21 or p27 is bound to G1/S-Cdk or S-Cdk the complex is inactive.

Question 11

What is Mdm2?

Answer 11

A ubiquitin ligase

Question 12

describe the pathway from DNA Damage to Cell Cycle Arrest

Answer 12

DNA Damage

Stable p53

Transcription of p21 mRNA

p21 protein inhibits CDK-cyclin

Rb is unphosphorylated

No Free E2F

No entry into S phas

Question 13

How is M-CDK activated?

Answer 13

-M-cyclins accumulate during G2.

-By the end of G2, the concentration of M-Cdk is high,
but the complex is inactive due to inhibitory phosphates
added by Wee1.

-Cdc25 removes the inhibitory phosphates.

-Cdc25 is activated by Polo kinase and by M-Cdk itself,
generating a positive feedback loop (M-Cdk also inhibits
Wee1).

Question 14

Is M-CDK Is Activated by Positive Feedback?

Answer 14

Yes

Question 15

What does M-Cdk do?

Answer 15

-Assembly of the mitotic spindle—
phosphorylates proteins that regulate
microtubule behaviour
-Chromosome condensation—phosphorylates the
condensin complex
-Nuclear envelope breakdown—phosphorylates
lamins
-Rearrangement of actin cytoskeleton and Golgi
-(Other kinases are also important).

Question 16

DNA Replication Checkpoint Prior to Mitosis

Answer 16

Unreplicated DNA or unfinished replication forks send
a negative signal to block M-Cdk activation to
prevent mitosis.

The final target of the negative signal is Cdc25. As long
as cdc25 is inhibited, M-CDK will be inactive.

Question 17

What is APC?

Answer 17

A ubiquitin ligase

Question 18

When is APC active?

Answer 18

APC is active when bound to the activating subunit Cdc20.

Question 19

What does the phosphorylation of APC by M-Cdk promote?

Answer 19

Cdc20 binding

Question 20

Is there a delay between M-Cdk activation and Cdc20-APC activation?

Answer 20

No

Question 21

What does Cdc20-APC targets securin for?

Answer 21

Degradation

Adds ubiquitin to securin

Question 22

What does Securin bind and inhibit?

Answer 22

separase (a protease)

Question 23

What happens after separase is free from securase?

Answer 23

separase cleaves the cohesin complex that holds the sister chromatids together

Question 24

Entrance to Anaphase=

Answer 24

Activation of Separase

Question 25

Describe the Spindle-Attachment Checkpoint that inhibits Anaphase

Answer 25

Free kinetechores block progression to anaphase (a kinetechore is a specialised region of the chromosome that attaches to the microtubules of the spindle). Mad2 binds free kinetechores. The presence of Mad2 inhibits Cdc-20-APC So anaphase doesn’t take place (Note this checkpoint works on a ubiquitin ligase instead of a CDK).

Question 26

What is the function of Mad2?

Answer 26

Mad2 binds to unattached kinetochores and sends a negative signal to Cdc20-APC to inhibit it

Question 27

In response to DNA damage, what does S-Cdk promote?

Answer 27

DNA replication

Question 28

What does p53 indirectly inhibit?

Answer 28

G1/S CDK

Question 29

How does M-Cdk promote mitosis?

Answer 29

In response to unreplicated DNA, cdc25 is inhibited and so fails to activate M-CDK. In response to small size, wee1 inhibits M-CDK.o

Question 30

What does APC-cdc20 promote?

Answer 30

anaphase | -In response to free kinetechores, Mad2 inhibits APC-cdc20.

Question 31

What are mitogens?

Answer 31

Typically growth factors that stimulate cells to divide

Question 32

Explain how phosphorylation retinoblastoma is part of a feedback loop that promotes entry into S phase?

Answer 32

1. A mitogen (growth factors that stimulates cells to divide) binds to tyrosine kinase receptors and cause a MAP kinase cascade 2. MAP kinase phosphorylates transcription factors (activates them) so they can start transcribe immediate early genes 3. One of those immediate early genes is a transcription factor called MYC 4. MYC promotes the transcription of cyclins 5. Retinoblastoma bound to E2F protein then an active CDK cyclin phosphorylates retinoblastoma causing it to release E2F 6. E2F can start transcribing genes and in a positive feedback loop, more E2F is produced 7. Eventually S-CDK is activated and DNA synthesis occurs

Question 33

What happens to G1/S-Cdk or S-Cdk when p21 or p27 is bound?

Answer 33

The complex is inactive

Question 34

What happens in the p53 pathway to prevent entry into S phase?

Answer 34

1. DNA is damaged (e.g. by x-rays) 2. ATM/ATR kinases are activated which activate CH1/CH2 3. The kinases phosphorylate P3 thus releasing it from MDm2 4. P53 levels rise 5. P53 causes the transcription of P21/P27 6. The mRNA is translated to produce the P21/P27protein which is a cyclin dependent kinase inhibitor protein 7. This protein inactivates the G1/S-CDK 8. Rb is unphosphorylated so remains bound to E2F 9. No entry into S phase

Question 35

What is the function of Cdc25?

Answer 35

It removes inhibitory phosphates

Question 36

What activates Cdc25?

Answer 36

Cdc25 is activated by Polo kinase and by M-Cdk itself, generating a positive feedback loop (M-Cdk also inhibits Wee1).

Question 37

Explain what happens in the M-CDK activation pathway?

Answer 37

1. CDK binds to a cyclin but is inactive 2. CDK activating kinases adds phosphate to T loop 3. Wee1 adds the inhibitory phosphate 4. Cdc25 removes the extra phosphate 5. Now the M-CDK is active 6. Positive feedback encourages the phosphorylation of CDC 25 making more of it active which leads to a rise in M-CDK activity and an inhibitory effect on wee1

Question 38

What does target mean in this context?

Answer 38

Adds ubiquitin ligase to a substrate

Question 39

Explain the pathway that promotes entrance into anaphase?

Answer 39

1. An inactive APC is phosphorylated by M-CDK and that allows Cdc-20 to bind and activate APC 2. The APC-Cdc20 complex (ubiquitin ligase) adds ubiquitin to sequin which is then degraded in the proteasome 3. Separase, once inactive (bound to securin) is now active 4. Separase ‘chops up’ the cohesion’s between sister chromatids thus making it possible for the chromatids to be pulled apart in anaphase

Question 40

What is a kinetochore?

Answer 40

a kinetechore is a specialised region of the chromosome that attaches to the microtubules of the spindle