The Eukaryotic cell cycle 2: LECTURE 3

Irreversibiility, proteolysis and checkpoints

p34^cdc2/cdc28 also known as

Cdk1

What does it mean that cdc28/cdc2 are required for START and M phase in fission yeast?

• Maybe cdc28 can act in a stage specific manner??

How to test this and work this out?

1. Genetic screens in budding yeast

   • identified a number of new genes

   • encoding proteins related to cyclins

2. Mutant phenotypes and temporal expression of these genes was looked at

   • observation: They were required to activate Cdc28

   • In a stage specific manner

     • for G1/S and for G2/M transitions

What was overall found out

G1, S and M-phase cyclins: stage specific cyclins

In budding yeast

CDK:

1. Cdc28 → constant

2. Cyclins→ oscillating for activation

   • stage specific cyclins

Fission yeast

CDK:

1. Cdc2 → constant

2. Cyclins→ oscillating for activation

   • stage specific cyclins

Analogous START control found in animal cells

Restriction Point

What is the Restriction point

• regulatory mechanism in late G1

• shifts cells between proliferative and quiescent states

  • depending on:

    • nutrients and growth factors

Can they come out of quiescence?

• may resume proliferation if have

  • nutrients and growth factors

  • restored

Cancer cell restriction point?

Have lost their restriction point control

What the link between START and restriction point suggests?

• molecular mechanisms must be conserved too

How we found out more about cell cycle in human cells

• genetic and biochemical approaches

  • screens for functional complementation of yeast cell cycle mutants

  • with human cDNA

  • (as done for cloning human cdc2 previously)

Overall: want to find similar functions found in yeast of the proteins we understand to the one found in humans:

• so we can know what genes are invovled!

Results from this research

1. Cdk2, 4 and 6 found

   • what this means: must be stage-specific Cdk too!

2. Human cyclins D and E

   • What this means: G1 controls in yeast and humans are conserved!

Overall info we know about human cell cycle

• stage specific cyclin

and

• stage specific cdk subunits!

Together: make stage specific CDK complexes

• play critical role in ordering evening cell cycle

• controlled by oscillating cyclins

  • how work??

Stage specific accumulation of cyclins is controlled

1. Transcriptionally

and

2. Post-transcriptionally

How are these cell cycle transitions made irreversible?

Ubiquitin-dependent proteolysis

• targeted destruction of cell cycle regulators

• ensures that the regulators (cyclin) oscillators to get from one phase to the next?

contributes to sharp and irreversible cell cycle transitions

How works?

Steps

1. Ubiquitin activation

2. conjugation

3. ligation

What is involved?

• E1, E2 and E3 ensymes

What happens?

1. E1 and E2 prepare a ubiquitin moiety for..

2. ligation by the E3 to a lysine side-chain in the target protein

   • adds a ubiquitin to the protein

3. Repeated

   • forms a polyubiquitin chain on the target

4. Poly-ubiquitinated target then recognised by proteasome

   • ‘cellular chamber of doom’

   • protein subject to proteolysis

The specical role of E3?

• Governs substrate specificity

2 Examples of E3

1. SCF

2. APC/C

Play critical roles in targeting cell cycle regulators

SCF

Multisubunit

• with core components

  • Skp1 and Cdc53

  • and variable F-box protein

    • for substrate recognition

APC/C

• anaphase promoting complex/cyclosome

• associated to mutually exclusive activators

  • Cdc20 or Cdh1

E3s’ mechanism for substrate recognition?

These two examples differ in how they do it but…

Both based on distinctive sequence signatures found in the target:

• degrons

Role of SCFs in yeast

1. Destruction of bound CDK inhibitors (CKIs)

2. Destruction of G1 and G1/S-cyclins

Overall for: G1→ S phase

1. Destruction of bound CKI

1. S-phase CDK is built during G1 BUT

   • held inactive by bound CKI

2. G1-CDK activity meets threshold

3. CKI phosphorylated

   • generating a phospho-degron

4. This is recognised by SCF

   • targeted for proteolysis

5. Now S-CDK is released

6. This can phosphorylate the CKI too

   • accelerating the reaction:

   • positive feedback loop

7. OVERALL→ progression into S phase

This is in yeast btw

CKIs found in human cells

• found several families of them

• some also destroyed by ubiquitin-dependent proteolysis

  • via SCF

Why are CKI families useful?

increases combinatorial control over the restriction point

2. Destruction of G1 and G1/S-cyclins

Also dependent upon phospho-degrons

1. G1/S-cyclin goes through restriction point

2. it is phosphorylated

   • forms a phospho-degron

3. Recognised by SCF

4. destroyed

5. so easily go onto the next stage

Role of APC/C

• controls ubiquitin-dependent proteolysis

• to trigger metaphase → anaphase

Way APC/C recognises its targets

2 sequence signals

1. destruction box (D-box)

2. KEN-box

many targets may contain both sequences

How does it work? (compared with SCF)

SCF ubiquination

• controlled by targeted phosphylation

BUT
with APC/C

1. M-CDK phosphykated the APC/C complex

2. promoting binding to its activator cdc20

   • form APC/C^Cdc20

3. This complex triggers metaphase to anaphase transition through 2 major steps…

How APC/C^Cdc20 triggers metaphase to anaphase?

1. ubiquitin-dependent proteolysis of “secruin”

2. destruction of S and M-cyclins

Step 1

1. securin is destroed by the complex

2. releases ‘separase’

3. separase destroyed cohesion between sister chromatids initiating anaphase

Step 2

S and M-cyclins are destroyed

1. Initiated by APC/C^Cdc20

2. Completed by APC/C^Cdh1

Allows the progession into G1 phase in the subsequent cell cycle

When should the progression of the cell cycle be stopped?

1. genome replication is ongoing/ impeded

2. DNA undergoes damage

3. spindle assembly is perturbed

How is in appropiate progression prevented?

Surveillance mechanisms: checkpoints

What does DNA damage block promoted by?

Rad9

(radiation sensitive)

How found this out?

1. collection of radiation sensitive (rad) mutants

2. Most arrest but are defective in repairing the damage

   • BUT

3. One mutant instead could not arrest upon damage

   • rad 9

   • proliferates normally despite DNA damage

   • THEREFORE: Rad9 is needed to stop proliferation after DNA damage!

     • rad9 is the prototypic ‘checkpoint’ mutant

     Then other mutations were identified to do the same thing

   • found there were two sepraate DNA surveliience mechanisms

Two checkpoints related to DNA found after finding more mutations

1. DNA replication checkpoint

2. DNA damage checkpoint

Found conserved in yeast and humans!

• makes senses coz if DNA damaged, it was be really crucial: so needs to be conserved

Checkpoints for DNA integrity are conserved in yeast and humans

Their significant is shown

• by the predisposition to cancer

• arising from inactivation of ATM or BRCA1

Check point (3)- Metaphase-anaphase transition

Spindle assembly checkpoint (SAC)

Why check?

• are all duplicated sisters

  • attached to the spindle?

  • bi-oriented?

• crucial for chromosomal segreation accuracy

  • to couple progression and spindle integrity

How was info about SAC found out?

1. MT poisons

   • disrupt mitotic spindle or

   • Observation: cell cycle arrest or delay

2. Single chromosome not achieving bi-orientation

   • cell cycle arrest/ delay

   SO there must be a checkpoint here but also need

3. Molecular insight

   • found with genetic screens with budding yeast

   • find the key conserved components and pathway

     • conserved in humans!

Molecular insight: looking at yeast

1. Wild type + Microtubule poison→ cells arrest @ metaphase

2. mad and bub mutant + Microtubule poison→cannot arrested

   • rebud and die with missegregated chromosomes

   • Mutants used:

     • mad= ‘mitotic arrest defective’

     • bub= ‘budding unihibited by benzimidazole’

So the overall SAC mechanism we now know! (for unattached kinetochores)

1.Unattached kinetochores

1. unattached kinetochores relay signals through the SAC

2. inhibit Cdc20 activation of APC/C

3. Prevents the destruction of securin

4. STOPS moving into anaphase

Extra SAC mechanism for attached kinetochores but not got tension

2.Error-correction mechanism

e.g( sister kinetochores are attached to MTs from the sample spindle pole)

1. attached but not under tension

2. phosphorylation by mitotic kinase Aurora B

   • destabilises such attachments

3. now unattached kinetochore engases the SAC

   • until correctly re-attached and tension sensed

4. Once all re-attached andbi-oriented under tension

   • SAC is satisifed and inhibition ends

5. can now move into → anaphase

   • Cdc20 activates APC/C

   • tagets securin for proeolysis

   • separase active

   • cleaves irreversible loss of sister chromatid cohesion

   • anaphase

Overall check points map

1. DNA damage check G1/S phase

2. DNA replication check (S-phase?) and G2

3. Spindle check (M-phase)

Overall what is enforcing order and irreversibility along the cell cycle?

1. oscillatory activity of CDKs

2. surveillance by checkpoints