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BMG2002 CC > L8 > Flashcards

L8 Flashcards

1
Q

Functions of cyclin-dependent kinase inhibitors (CKIs)

A

Cdc28 (S. cerevisiae) and Cdc2 (S. pombe) belong to the group of eukaryotic kinases called cyclin-dependent kinases (CDKs)

Cyclin-dependent kinase inhibitors (CKIs) bind and inactivate the cyclin bound forms of CDKs

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2
Q

CKIs in S. cerevisiae

A

Far1 – binds and inhibits Cdc28-Cln complexes

Sic1 – binds and inhibits Cdc28-Clb complexes

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3
Q

CKIs in S. pombe

A

Rum1 - binds and inhibits Cdc2-Cdc13 (CyclinB) complexes

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4
Q

What does Far1 do in S. cerevisiae?

A

Binds and inhibits Cdc28-Cln complexes

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5
Q

What does Sic1 do in S. cerevisiae?

A

Binds and inhibits Cdc28-Clb complexes

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6
Q

What does Rum1 do in S. pombe

A

Binds and inhibits Cdc2-Cdc13 (CyclinB) complexes

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7
Q

Why is it important that the CKI are specific to a cyclin?

A

It allows you to block specific points in the cell cycle

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8
Q

What does a cdc7-ts mutation do in S. cerevisiae?

A

cdc7-ts mutation blocks right before replication – doesn’t stop budding or spindle pole body duplication

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9
Q

What does a cdc4-ts, cdc34-ts or a cdc53-ts mutation do in S. cerevisiae?

A

Role of cdc4-ts, cdc34-ts, cdc53-ts is analogous

Not involved in replication but isn’t needed for budding or spindle pole body duplication

Still blocks DNA replication

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10
Q

Sic1 and the regulation of initiation of DNA replication

A

CDC34 encodes a ubiquitin conjugating enzyme suggesting protein degradation is important for the initiation of DNA replication

When you knock out the activity of the ability of a protein to add ubiquitin, then you’re not degrading something that you should be degrading before replication occurs

This suggests an inhibitor – needs to be got rid of by ubiquitination before replication occurs

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11
Q

Biochemical & genetic evidence that Sic1 is the inhibitor of Cdc28-Clb complexes

A

In cdc34-ts mutation Cdc28-Cln activity is high. However, mutations which increase stability of G1 cyclins do not arrest cells.

Strains deleted for all six CLB genes looks like cdc34-ts mutations

In the cdc34-ts mutant at the non-permissive temperature no Cdc28-Clb activity can be detected

Sic1 protein is present in early G1, vanishes shortly before S phase and doesn’t reappear until nuclear division.

Sic1 protein is at high levels in cdc34ts mutants at the non-permissive temperature

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12
Q

What is the essential function of Cdc28-Cln at START?

A

To target Sic1 for degradation

cln1Dcln2Dcln3D cells are dead but are rescued by deletion of the SIC1 gene

Will divide & undergo cell division

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13
Q

Regulation of Sic1

A

The transcription of the SIC1 gene is present throughout the cell cycle but peaks at the end of mitosis

Cdc28-Cln phosphorylates Sic1 on multiple sites and this targets it for degradation via Cdc4, Cdc34 and Cdc53
– Sic1 has 9 phosphorylation sites of Cdc28-Cln
– It only gets degraded when any 6 of the 9 are phosphorylated
– Sic1 then disappears & the cell can enter S phase

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14
Q

How many phosphorylation sites does Sic1 have on Cdc28-Cln?

A

9

It only gets degraded when any 6 of the 9 are degraded

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15
Q

Function of Sic1

A

sic1∆ mutants are not dead but enter S phase early and have a longer S phase

Hence Sic1 is involved in the timing of S phase along with the other elements of initiation of DNA replication induced at START

Don’t want to spend lots of time in S phase – lots of opportunity for DNA damage

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16
Q

What options does S. cerevisiae have in G1 phase?

A

Starved cells enter G0 arrest

Haploid cells mate

Diploid cells sporulate

17
Q

What does mating pheromone do?

A

Mating pheromone inhibits START blocking budding, DNA replication & spindle pole body duplication

18
Q

What happens if we add mating pheromones to yeast cells of 2 different mating types – ‘a’ and alpha?

A

It interacts with the cell of the opposite type

When it does that it inhibits START – don’t get budding, replication or spindle pole body duplication

It behaves like a cdc28 ts mutation
Behaves like an inhibited triplet Cln mutations

Mating pheromone interferes with the cdc28-G1 cyclins in some way

19
Q

Why do haploid cells arrest the cell division cycle in G1?

A

If you have a cell in G1 mating with a cell in G2 then you’re making something that has 3 copies of the chromosomes – not making a diploid

If you take a cell in G2 with another G2 cell, then you get 4 copies

By mating 2 cells in G1 you get a diploid

Only phase in the cell cycle when you can mate to get a diploid cell is in G1

Coordinate the cell division cycles with each other

20
Q

How do haploid cells arrest the cell division cycle in G1?

A

Mating pheromone from one cell in G1 is ejected into the media & arrests the other cell of the opposite mating type

Both act on each other to arrest the cell division cycle – coordinate with each other to allow mating in G1

21
Q

What controls the cell cycle arrest of haploid cells in G1?

A

The cell cycle arrest depends on a protein kinase cascade and the function of a CKI called Far1 which inhibits Cdc28-Cln1 and Cdc28-Cln2

22
Q

Regulation of Far1

A

The expression of Far1 is regulated by the Ste12 transcription factor

Far1 is phosphorylated by Fus3 & this possibly activates the Far1 protein

Far1 has several layers of regulation

23
Q

What transcription factor regulates Far1?

A

Ste1 transcription factor

24
Q

What is Far1 phosphorylated by?

A

Fus3

This possibly activates the Far1 protein

25
Q

Function of Far1?

A

Far1 blocks the activity of Cdc28-Cln1 and Cdc28-Cln2

The mechanism of action of Far1 is unclear

The phosphorylated form of Far1 appears to bind the Cdc28-Cln1 and Cdc28-Cln2 complexes and induce the degradation of Cln1 and Cln2

Fus3 may phosphorylate Cln3 and target it for degradation

This would block Cln1 and Cln2 expression

26
Q

How does the mating pheromone work?

A

1) 2 mating pheromones – ‘a’ cells look for an alpha cell
The alpha factor binds to a receptor on the cell surface of the ‘a’ cell called the Ste2
2) ‘a’ factor binds to a receptor on surface of the alpha cell called Ste3
3) Ensures that an ‘a’ cell & ‘a’ cell don’t mate & an alpha & an alpha don’t mate
4) Can only detect the opposite mating pheromone
5) Then all feeds into a common pathway
6) Once bound to other pheromone it activates the trimeric binding protein releasing the G-alpha bound to GTP
7) Beta-gamma complex binds to a protein kinase called Ste20 which activates a MAP kinase cascade which is called Ste11, Ste7 & Fus3
8) Ste5 is a scaffold pathway for the cascade – holds the proteins together – gives a strong amplification signal

27
Q

What are the 2 outcomes of mating pheromones?

A
  1. Phosphorylates Ste12 – activates the transcription factor and induces expression of Far1 (the inhibitor)
  2. Also directly phosphorylates Far1 – this inhibits cdc28-Cln kinase

Ensures both cells are in G1

28
Q

Role of Rum1 (a CKI) in S. pombe

A

Appears to be to prevent mitosis in the absence of DNA replication

Rum1 also appears to have a role in size control at START

Makes sure mitosis only occurs after replication has occurred

29
Q

Biochemical and genetic evidence of Rum1 function

A

Overexpression of rum1+ stimulates DNA replication in the absence of mitosis

A rum1∆ strain undergoes mitosis if blocked at START

Cdc2-Cdc13 is required for the G2/M transition

Rum1 can inhibit Cdc2-Cdc13 form of the kinase in vitro

A rum1∆ strain has a shorter G1 phase

30
Q

Regulation of Rum1

A

The Rum1 protein is present only in G1 phase and is absent in S, G2 and M phases

Expression levels are relatively constant hence cell cycle regulation is presumably translation and/or proteolytic

Protein acts analogously to Sic1

Regulated similar to Sic1

31
Q

CDKs are regulated by a variety of mechanisms, what are they?

A
  • Cyclins
  • Protein kinases
  • Protein phosphatases
  • CKIs
  • Gene expression
  • Protein degradation

BMG2002 CC (10 decks)

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