BMSC339 - Regulation of Cdks.

How does transcription of cyclin D lead to more E2F activity?

--> increased G1-Cdk activation --> increased Rb phosphorylation --> increased E2F activity.

How does transcription of SCF lead to more E2F activity?

--> increased p27 phosphorylation --> increased G1/S-Cdk activation --> increased Rb phosphorylation --> increased E2F activity.

Name 2 molecules which regulate proteolysis of cell cycle molecules?

1) APC/C (anaphase promoting complex / cyclosome). 2) SCF.

Which 2 things might p53 do when it is activated by this pathway?

1) Cause cell cycle arrest. 2) Cause apoptosis.

Name 2 families of Cdk inhibitor proteins (CKIs) and give examples.

1) Cip/Kip family (e.g. p27, p21). 2) Ink4 (e.g. p16).

The transcription of which other 3 genes is activated by Myc?

1) Cyclin D. 2) Cdc25 (activates Cdk1). 3) SCF (degrades CKIs).

What are the 6 kinds of regulation of Cdks?

1) Cyclin binding. 2) CAK. 3) Wee1 and cdc25. 4) CKIs. 5) Proteolysis. 6) Rb and p53.

Which 2 things may eventually happen to a cell which arrests in G1?

1) DNA damage may be repaired --> continue cell cycle. 2) Apoptosis.

Which 2 things activate cdc25?

1) M-Cdk (positive feedback). 2) Myc (oncogene). (So cdc25 is often overexpressed in tumours).

Which 2 things regulate cdc20?

1) M-Cdk. 2) SAC (spindle assembly complex).

Name 2 other factors which contribute to the rapid division of ESCs.

1) No expression of CKIs. 2) Rb is inactive, so E2F is active throughout the cell cycle.

Name 2 kinds of cells which express high levels of p27^Kip1.

1) Terminally differentiated cells in G0 (quiescent). 2) Contact inhibited cells.

Name 2 kinds of cells which express high levels of p21^Cip1.

1) Terminally differentiated cells in G0 (quiescent). 2) Senescent cells.

How many subunits does APC/C have?

11 - large complex.

How many subunits does SCF have?

3.

What kind of protein is p53?

A tumour suppressor. (Mutated in more than 50% of cancers.)

What activates Chk1/Chk2 kinases?

ATM/ATR kinases.

What happens if there is a lesion in the Wee1 gene?

Acceleration of mitosis (short G2), leading to very small cells.

Does M-Cdk activate or inhibit cdc20?

Activates it (i.e. M-Cdk signals its own destruction).

If DNA cannot be readily repaired, what does p53 do?

Activates the Bax gene and suppresses the Bcl2 gene --> apoptosis.

What does phosphorylated p53 do?

Acts as a transcription factor to turn on genes such as p21 (CKI). (Also genes which retard rate of DNA replication or block G2/M progression.)

What is the action of APC/C?

Adds ubiquitin to M-cyclin and securin during mitosis, targeting them for degradation.

How are cyclins expressed in ESCs?

All cyclins (except cyclin B) expressed in comparable amounts throughout the cell cycle, rather than oscillating. Expression of all cyclins is significantly higher than in somatic cells.

What does excessive Myc production activate?

Arf (alternative reading frame from p16).

When does cdc25 dephosphorylate Cdk1?

At the onset of mitosis.

What does active Arf do?

Binds Mdm2, inactivating it and releasing p53.

What is the action of CKIs?

Block active sites of Cdks to regulate entry from G1 to S.

What activates APC/C during mitotis?

Cdc20.

What is the other name for CAK?

Cdk-7.

If these regulatory mechanisms are absent in ESCs, what may be controlling their cell cycle?

Cell autonomous mechanisms.

What does Rb control?

Controls the G1/S checkpoint by binding and inactivating E2F.

Which molecules are involved in maintaining Rb phosphorylation?

Cyclins A and E (positive feedback).

What activates ATM/ATR kinases?

Double stranded breaks --> ATM. Single stranded breaks --> ATR.

How does Rb return to its dephosphorylated, active state after M phase?

During the M to G1 transition, Rb is dephosphorylated by PP1 (protein phosphatase 1).

Other than DNA damage, what else activates p53?

Excessive mitogens (e.g. excess Myc production).

What happens if there is an excess of the Wee1 gene?

Extended G2 --> elongated cells.

Apart from G1-Cdk causing Rb to release E2F, what else increases levels of E2F?

Extracellular signals (mitogens).

Once the Rb protein becomes highly phosphorylated at about the start checkpoint, for how long does it remain in this state?

For the remainder of the cell cycle, until after M phase.

How is the control of G1/S by Rb passed to allow entry into S phase?

G1-Cdk (cyclin D + Cdk4) phosphorylates Rb, causing it to release E2F.

What role does this return to its active state allow Rb to fulfill?

Gatekeeper of the cell cycle.

Where is Rb found?

In the nucleus.

What is the action of p16?

Inhibits G1-Cdk by displacing the cyclin.

What does p21 do?

Inhibits G1/S phase Cdks, reducing phosphorylation of Rb and hence causing arrest in G1.

Which Cdks does p27^Kip1 inhibit and when?

Inhibits G1/S-Cdk and S-Cdk during G1.

Which Cdks does p21^Cip1 inhibit and when?

Inhibits G1/S-Cdk and S-Cdk following DNA damage.

What is the role of Nim1?

Inhibits Wee1 by phosphorylating it in response to nutritional status. Hence accelerates mitosis.

Does SAC activate or inhibit cdc20?

Inhibits it if there are any centromeres which are unattached to microtubules - preventing progression through anaphase.

What controls the activity of SCF?

It is expressed constantly throughout the cell cycle, but its activity is regulated by the phosphorylation state of its target proteins. (Also stimulated by Myc.)

How common is loss or mutation of Rb in cancer?

Lost or mutated in at least 1/3 of all human tumours. Lost or mutated in all cases of retinoblastomas.

Via which pathway do mitogens activate transcription of E2F?

MAP kinase cascade activates the oncogene Myc. Myc acts as a transcription factor for genes that promote cell growth, including E2F.

Describe an experiment which confirms the in vivo genetic interaction of Mdm2 and p53.

Mdm2 knockout is lethal for mouse embryonic development. Simultaneous knockout of Mdm2 and p53 is not lethal.

How does p53 expression differ between normal cells and cells with DNA damage?

Normal cells express low levels of p53 (made and then degraded to keep at low concentration). p53 expression rapidly rises if DNA is damaged.

What are usual target proteins of SCF?

Phosphorylated CKIs (p27, p21) - releases S-Cdk in G1 and leads to S phase. Also cyclin E.

What inhibits cyclin-Cdks?

Phosphorylation at Thr-14 and Thr-15 by Wee1 kinase.

What activates p53?

Phosphorylation by Chk1/Chk2 kinases.

Binding of cyclins only partially activates Cdks. What else must occur for full activation?

Phosphorylation on Thr-161 by Cdk-activating kinase (CAK).

How does phosphorylation activate p53?

Phosphorylation prevents p53 from binding to Mdm2 (which is an E3 ubiquitin ligase and so tags it for degradation).

What is the result of the degradation of securin?

Separase is released and cleaves the cohesin protein that holds sister chromatids together, allowing anaphase to occur.

How does the cell cycle of embryonic stem cells differ to the cell cycle of somatic cells?

Shorter G1 and G2 and so faster division. Divide rapidly without growth.

Why is Mdm2 overexpressed in some tumours?

Tags p53 for degradation (E3 ubiquitin ligase), meaning that even if there is DNA damage, cancer cells will keep replicating.

What determines the target proteins of SCF?

The F-box subunit, which recognises target proteins.

Which structure keeps Cdk inactive when it is not bound to a cyclin?

The T loop blocks the active site (the ATP binding site).

What happens to the T loop when CAK phosphorylates the Cdk?

The T loop improves the binding of the substrate.

What happens to the T loop when cyclin binds?

The T loop moves out of the active site.

What happens in a cdc20^- deletion mutant?

The cells arrest in M phase, with unseparated sister chromatids.

In which context was the Rb protein first discovered?

The first tumour suppressor gene identified through studies of retinoblastoma (inherited eye cancer in children).

When the cyclin-Cdk is required, what removes the inhibition by Wee1 kinase?

The phosphatase cdc25.

Which gene codes for p16?

The tumour suppressor mts1 / Ink4a. (Frequently mutated in human tumours.)

Why does Wee1 kinase inhibit Cdk-1 before mitosis?

To allow the cell time to grow before it divides. Hence plays a role in cell size control.

What is the action of the released E2F?

Turns on transcription of >30 genes required for transition to S phase.

What kind of molecule are both APC/C and SCF?

Ubiquitin ligases (E3).

What activates transcription of Mdm2?

p53 (i.e. p53 signals its own destruction).

What is activated if there is DNA damage in G1?

p53.