cell cycle regulation

Question 1

what are the main regulators of the cell cycle?

Answer 1

cyclins and CDKs (Cyclin-dependent kinases)

Question 2

what kind of kinases are CDK?

Answer 2

Serine/threonine kinases

Question 3

How is CDK activity regulated?

Answer 3

• CDKs lack activity until bound by their specific cyclin subunits
• the activity of CDKs is modulated by several activators (family of cyclins) and Cdk inhibitors (CKI such as Ink4 and p21/Kip1)

-Cdk activity can also be regulated by inhibitory tyrosine phosphorylation which blocks phosphate transfer to substrates;
Phosphorylation–> CAK1, Wee1
Dephosphorylation–> Cdc25

Question 4

How is it ensured that in each stage of the cell cycle only the corresponding Cyclin/Cdk complexes exist?

Answer 4

• Cyclins exhibit specificity for particular Cdks
• different cyclins are expressed at different phases of the cell cycle; so cyclins are regulated tightly at the levels of synthesis (transcription) and (ubiquitination) degradation

Question 5

What are the different types of CDKs?

Answer 5

Over 20 types of CDKs have been identified!

family of CDKs that directly promote cell cycle progression=> CDK1, CDK2, CDK4, CDK6

an additional family that regulates transcription=> CDK7, CDK8, CDK9

CDK5 (and some others) have functions in specialized tissues

Question 6

What defines cyclins? what allows for their differential regulation and functional diversity?

Answer 6

• cyclins are remarkably diverse group of proteins classified solely on the existence of a cyclin box that mediates binding to CDKs
• the sequence variations outside the cyclin box allows for differential regulation and functional diversity

Question 7

What are the different cyclins?

Answer 7

CycD, CycE, CycA, CycB

Question 8

which cyclin is expressed at the start (G1 phase)? Which CDK(s) does it bind to and activate?

Answer 8

CyclinD;

[G1/S-CDK]= Cdk4, Cdk6

Question 9

Which cyclins are expressed in S phase? Which CDK(s) do they bind to?

Answer 9

CyclinE, CyclinA;
both CycE and A bind CDK2
[S-CDK]

Question 10

Which cyclins are present in M phase? which CDK(s)?

Answer 10

CyclinB (synthesized) and Cyclin A (persists from S phase)

they both bind to CDK1 [M-CDK]

Question 11

what are the types of CKIs? on what basis are they classified?

Answer 11

there are two families of CKIs–>

1. Ink4 family [p16, p15, p18, p19]
2. Cip/Kip family [p21, p27, p57]

they are classified based on their structure and the specificity of binding to CDKs

Question 12

how is the activity of the two families of CKI different?

Answer 12

-the Ink4 family CKIs primarily target Cyclin D-CDK complexes [Cdk4 and Cdk6];
these bind to an active Cyclin-CDK complex to inactivate the complex

-the Cip/Kip family members are more promiscuous and interfere broadly with the activities of all the cell progression CDKs (4,6,2,1); these bind to the Cdks to prevent them from binding to corresponding cyclins

Question 13

what is RB? what is its function?

Answer 13

Rb is retinoblastoma tumour suppressor protein;

It is a regulatory protein–>

when RB is unphosphorylated, it binds to and inhibits E2F (a transcription factor which allows progression to the late stage of G1)

CDK4 and 6 (upon activation by binding to CycD) phosphorylate RB which leads to its dissociation from E2F, which allows progression through R point

Question 14

What is the role of E2F?

Answer 14

E2F plays a major role in G1/S transition of the cell cycle

it activates the transcription of cyclins, CDKs, checkpoint regulators, DNA repair and replication proteins etc.

cyclins: A and E
CDK: 2
replication proteins: Mcm, Cdc6, Cdt1

Question 15

how many cell cycle checkpoints are there? what are they?

Answer 15

there are 4 cell cycle checkpoints:

1. the G1/restriction checkpoint
2. the DNA damage checkpoint
3. the G2 checkpoint
4. the spindle assembly checkpoint

Question 16

what is the Restriction checkpoint?

Answer 16

• the G1 phase can further be divided into an early and late phase, which is separated by the R point
• this checkpoint determines whether the cell will enter the cell cycle (if the cell is large enough and the environment is favourable then it will)

Question 17

what is the role of mitogenic signals?

Answer 17

• mitogens are extrinsic growth factors that induce a cell to enter the cell cycle (and eventually mitosis)
• the early G1 phase (mitogen-dependant) and the G1 checkpoint is controlled by mitogens but after crossing this checkpoint mitogens are no longer needed
• in the presence of mitogens sufficient cycD can be produced so that CycD-CDK4/6 complexes can form which promote entry into the late G1 (mitogen-independent) phase
• the mechanism of mitogenic signals is that it triggers signal transduction pathways involving MAPK (mitogen-activated protein kinase)

Question 18

what are the steps in DNA replication initiation?

Answer 18

1. recognition- ORC (origin recognition complex) binds to the origin and marks it, providing a “landing pad” for other proteins
2. Initiative assembly or ‘licensing’- takes place in G1 phase–Cdc6 and Cdt1 load the Mdm helicase onto the ORC to form the pre-RC
3. Unwinding- requires activation of the DNA helicase
4. Elongative assembly–takes place in S phase–loading of the replisome (including DNA polymerase, holoenzymes and SSB)

Question 19

what is the role of kinases in cell cycle regulation of DNA replication?

Answer 19

CDK and DDK are the two important kinases [the S-phase CDK triggers S phase]

in the late G1/early S phase–> phosphorylation of Mcm by DDK activates it

phosphorylation of ORC and Cdc6 triggers their degradation and nuclear export (prevents re-replication)

the essential CDK phosphorylation events are on the two protein- Sld2 and Sld3- which, when phosphorylated, help load the replisome so that origin firing can occur

Question 20

how is it ensured that re-replication doesn’t occur?

Answer 20

In S-phase–>

• phosphorylation of ORC and Cdc6 triggers their degradation and nuclear export
• Cdt1 is inhibited by geminin

Question 21

When does geminin start accumulating in the cell? when is it degraded? why is it degraded then?

Answer 21

• geminin starts accumulating in the S phase
• it is degraded by APC/C in the M phase
• it is degraded so that Cdt1 can again function in the next G1 phase

Question 22

when can pre-RC formation occur? why?

Answer 22

it can only occur in the G1 phase because ORC, Cdc6 are degraded and Cdt1 is inhibited in the other phases

Question 23

when can helicase activation occur? why?

Answer 23

helicase activation can only occur in late G1/early S phase

this is because helicase activation depends upon the activity of S-CDK and DDK which are only active then

Question 24

CAK1

Answer 24

A CDK activating kinase that phosphorylates CDKs on a conserved Thr residue
Important so that cyclins can bind to CDKs efficiently

Question 25

Wee1 kinase

Answer 25

is a negative regulator of the G2/M transition — Prevents entry into mitosis

Specifically phosphorylates and inactivates CDK1