Cyclins

Question 1

What are cyclins?

Answer 1

Cylincs are cyclically activated proteins that are expressed at different levels during the cell cycle, when present, they bind to specific kinases to activate them

Question 2

What do cyclin dependent kinases do?

Answer 2

They phosphorylate proteins that are specific to certain stages of the cell cycle

Question 3

What gives the cell cycle directionality?

Answer 3

Its created via the sequential appearance and destruction of the cyclins

Question 4

What do cyclins do?

Answer 4

They direct kinases towards different substrates at different stages of the cell cycle
they allow the transition of a particular cell cycle phase

Question 5

What experiment a long time ago identified cyclins/cdka?

Answer 5

Experiment on a particular virus which fused cells together
they found that when you fused cells at different cell cycle phases- mitotic cells will try and induce mitosis in the other cells
identified via the observation of condensed chromosomes in the non mitotic cells
the result= death of the chromosomes

Question 6

In summary- what do mitotic cells do in terms of the cell cycle?

Answer 6

Mitotic cells cause interphase cells to join mitosis

Mitosis is dominant

Question 7

What was the critical experiment concerning frogs eggs and meiosis?

Answer 7

Injected the cytoplasm of a mature from egg (in meiotic metaphase) into an immature oocyte (arrested at G2)
this induced the maturation of the oocyte- could see the presence of a meiotic spindle
Something in the mature egg is inducing entry into M phase

Question 8

How can you tell meiosis has occurred in frogs eggs?

Answer 8

A little white spot on top of the egg is the meiotic spindle

Question 9

What did the maturation factor turn out to be- how did they find out?

Answer 9

They purified the factor from the other proteins via chromatography
The protein turned out to be a kinase- due to its ability to phosphorylate histone H1

Question 10

What critical experiment was carried out in sea urchin eggs?

Answer 10

Incubated the eggs with radioactive methionine (methionine would be incorporated into newly made proteins)
can see the radioactivity in the proteins via electrophoresis and autoradiography

Question 11

What did they find in the sea urchin eggs experiment?

Answer 11

Found a few proteins that were appearing and then disappearing
(unusual as most proteins were accumulating)
they plotted the data for the proteins and found that they were being created and destroyed in accordance to the cell cycle - synchrony to the cell cycle
CYCLINS*

Question 12

What experiment was carried out on linear yeast?

Answer 12

The creation of cell division cycle mutants
observed that CDC2 mutants were very long- they kept growing in G2
the same was observed in CDC25 mutants
observed that wee1 mutants, however, were very small
hypothesis was= cdc mutants are mutations which slow down the timing taken to induce mitosis
similiarly, wee1 mutants decrease this time

Question 13

What was concluded when they sequenced the genes in the linear yeast?

Answer 13

Cdc25 is a positive regulator of cdc2
Wee1 is a negative regulator of cdc2
cdc2 induces mitosis
Cdc2 was found to = Cdk1
MAKES SENSE- lose cdc2- wont go into mitosis- stay in G2= longer cells
lose wee1- go into mitosis quicker- shorter cells

Question 14

After sequencing- what was Cdc2 in yeast similar to?

Answer 14

The maturation promoting factor found in the mature frog eggs

Question 15

What did all 3 cell cycle driver experiments come together to conclude?

Answer 15

MPF is a protein kinase controlled by cyclins which are made and destroyed on a cyclical basis

Question 16

What was interesting about MPF and cyclin b activity?

Answer 16

The peaks of cyclin B activity didn’t exactly match up to MPF activity- which you would’ve thought it did considering cyclin B activates its Cdk
clearly something else is involved

Question 17

What does cyclin D interact with?

Answer 17

Cdk 4/6 = G1 Cdk

Question 18

What does cyclin E interact with?

Answer 18

Cdk2 = G1/S Cdk

Question 19

What does cyclin A interact with?

Answer 19

Cdk2/1 = S Cdk

Question 20

What does cyclin B interact with?

Answer 20

Cdk1 = M Cdk

Question 21

What are the two main functions of cyclins?

Answer 21

Targeting their catalytic subunit in the cell

activating the catalytic subunit

Question 22

How are Cdks activated?

Answer 22

Cyclins
CAKs- complex which phosphorylates a threonine residue on the cdk- activates proper catalytic activity
Cdc25- dephosphorylates the inhibitory phosphorylation what wee1 induces- activates the Cdk as a result

Question 23

How are Cdks inhibited?

Answer 23

CKIs- mainly operate in G1 and S

Wee1= phosphorylates adjacent threonine and tyrosine residues which inhibits the kinase

Question 24

Examples of CKIs and the cyclins they inhibit

Answer 24

P16 and 15 inhibits Cdk4/6

P21 + P27 inhibits Cdk2 and Cdk2/1

Question 25

How can antiproliferative signalling induce inhibitor expression?

Answer 25

TGFbeta binds to its receptor and induces the transcription of P15
P15 inhibits Cdk4/6- this prevents the transition from G1 to S
Neighbouring cells can also stop proliferation via contact inhibition

Question 26

How do mitogens regulate CKI function?

Answer 26

They inhibit the P21/27 system- so stops the inhibition of cell cycle progression
Akt (which is stimulated by the mitogen binding to its receptor) prevents P21 from reaching the nucleus

Question 27

What does cytoplasmic P27 do in low grade cancers?

Answer 27

Inhibits cell cycle progression

Question 28

How does nuclear and cytoplasmic P27 have different prognostic indications?

Answer 28

just nuclear P27= better prognostic outcome

nuclear and cytoplasmic p27= worse prognostic outcome

Question 29

What is the restriction point?

Answer 29

The specialised position within G1 where cells make the decision to exit the cell cycle into G0- or with the presence of proliferating signals- carry on through the cycle and divide

Question 30

What does Rb do and what are its implications in the absence of mitogens?

Answer 30

Rb is a DNA binding protein
inactivates E2F- transcription factor required for S phase entry
binds to a histone deacetylase which results in chromatin compaction

Question 31

What is the effect of mitogens on cyclin D, and thus Rb?

Answer 31

Mitogens induce the production of cyclin D

Cyclin D leads to the hyperphosphorylation of Rb- which prevents it from binding to the DNA and E2F

Question 32

In the presence of mitogens, what does Rb do?

Answer 32

Rb is hyperphosphorylated, can no longer bind to E2F or histone deacetylases
E2F is able to interact with RNA polymerase 2= transcription of genes required for S phase entry
= cell cycle progression