Signalling mechanisms in growth and division

Question 1

How does the ERK cascade regulate gene expression and lead to progression through G1 of the cell cycle?

Answer 1

At end of cascade, last kinase (ERK) phosphorylates a no. of proteins (e.g. TFs) and changes their activity
Phosphorylated TFs go on to regulate gene expression
Important genes turned on = c-Myc –> cell proliferation (raised conc when cell moves from G0 to G1)

Question 2

How do ligands activate tyrosine kinase receptors?

Answer 2

E.g. peptide growth factors
Tyrosine kinase receptors normally sit on plasma membrane as monomers
Most GFs are dimeric
When dimeric GF binds to 2 receptor tyrosine kinase molecules, this brings them closer together
When Rs close together, tyrosine kinase domain cross-phosphorylates tyrosine residues in proteins on partner receptor using gamma phosphate of ATP
Phosphorylated domains on tyrosine kinase Rs act as docking sites for adaptor proteins

Question 3

What do adaptor proteins do once bound to activated tyrosine kinase receptors?

Answer 3

E.g. Grb2
No enzymatic activity
Have domains that are important in molecular recognition - bring other proteins together
Contribute to downstream signalling
Grb2 always has Sos (exchange factor) bound
Binding to RPTK allows Sos to activate Ras

Question 4

What is Ras?

Answer 4

GTP-binding (G) protein

Question 5

Why is Ras a powerful ‘molecular switch’?

Answer 5

On - GTP bound
Off - GDP bound
When on, Ras binds to a kinase and sets off a protein kinase cascade

Question 6

How is the cycle of Ras (and other GTP binding proteins) controlled?

Answer 6

Under the influence of appropriate signals, exchange factors (e.g. Sos) can replace GDP with GTP to make Ras active
GTPase activating proteins stimulate hydrolysis of GTP to inactivate Ras
Ras can also switch itself off

Question 7

How do cyclins and their kinases regulate the cell cycle?

Answer 7

Cell cycle control is based on cyclin-dependent kinases (Cdks)
Present in proliferating cell thr/o cell cycle
Not activated until bound to activating protein, cyclin
Different cyclin-Cdk complexes trigger different events in the cell cycle
Activated Cdks phosphorylate proteins on serine or threonine to drive cell cycle progression

Question 8

What are cyclin-dependent kinases?

Answer 8

Serine-threonine kinases

Question 9

When are cyclins present during the cell cycle?

Answer 9

Cyclins are transiently expressed at specific points in cell cycle
Once they have activated Cdks, they are degraded
Cyclins are regulated at the level of expression
Resynthesised during next mitosis

Question 10

How are Cdks regulated by phosphorylation?

Answer 10

Example: M-phase promoting factor
Cdk1 binds to cyclin B*
This Cdk-cyclin complex is usually inactive on its own
2 phosphorylation reactions also regulate Cdk1 activity
Cdk activating kinase (CAK) puts an activating phosphorylation onto Cdk1*
Wee1 puts an inhibitory phosphorylation onto Cdk1
Even though Cdk1 is bound to cyclin, the inhibitory phosphate must be removed before it can function
Cdc25 takes off inhibitory phosphate* put on by Wee1
Mitosis initiation can be regulated at 3 points on this complex(*)

Question 11

Explain the positive feedback mechanism of MPF and Cdc25

Answer 11

Dephosphorylation of inhibitory site of Cdk1 by Cdc25 activates it towards end of interphase
Active MPF is able to phosphorylate Cdc25 to increase its activity
As soon as you get some active Cdk1 then you activate more Cdc25, which, in turn, leads to more dephoshorylation of the inhibitory site
This positive feedback reinforces activation of MPF and drives mitosis

Question 12

How does MPF regulate mitosis?

Answer 12

MPF, when active at end of metaphase, phosphorylates a no of key substrates involved in mitotic process
This phosphorylation puts mitosis “on hold”
Can’t progress to next stage until a signal is sent saying that metaphase has been correctly achieved
Once kinetochores are correctly attached to microtubule spindles, a signal is released that causes cyclin B to be degraded
Cyclin B is degraded –> Cdk1 inactived –> key substrates dephosphorylated –> mitosis can progress

Question 13

Different cyclins and different Cdks are required at different stages of the cell cycle. Cdk2 is activated in G1 (by cyclin E) AND S (by cyclin A). How is the same Cdk used if they need to do different jobs?

Answer 13

Cyclin binding alters substrate specificity so that Cdk can phosphorylate diff substances
Substrate accessibility changes through cell cycle
This allows same Cdk to be used in diff stages of cell cycle

Question 14

What stimulates the cell to transition from G0 to G1?

Answer 14

Growth factor stimulation of signalling pathways
GFs bind to receptor protein tyrosine kinase
Through Ras, this triggers a kinase cascade
Leads to TF phosphorylation that turns on c-Myc expression
Myc = TF - stimulates cyclin D transcription
Cyclin D activates Cdk4 and Cdk6 to stimulate cyclin E synthesis
Cdk2/cyclin E is the G1/S checkpoint Cdk
Triggers cell cycle

Question 15

How do cyclins and Cdks give direction and timing to the cell cycle?

Answer 15

1st active Cdk4/6-cyclin D complex stimulates synthesis of next cyclin and they then become sequentially active
Each cyclin is involved in stimulating synthesis of next cyclin
Gives direction
Gives timing bc takes time for concentration of cyclin to build up so appropriate Cdk is activated

Question 16

How are Cdks regulated by specific Cdk inhibitor families?

Answer 16

INK4 family = G1 phase CKIs - inhibit Cdk4/6 by displacing cycD
CIP/KIP family = S phase CKIs - inhibit all Cdks by binding to Cdk/cyc complex
For cell cycle to progress, CKI must be degraded

Question 17

What activate Cdks?

Answer 17

Cyclins

Question 18

What else are Cdks controlled by other than cyclins?

Answer 18

Phosphorylation

CKIs

Question 19

What is c-Myc?

Answer 19

A transcription factor

Stimulates expression of cell cycle genes

Question 20

When does [c-Myc] in a cell rise?

Answer 20

If you trigger cell division (e.g. add a GF), [c-Myc] rapidly rises and then plateaus at intermediate level
Correlates with cell moving out of G0 into G1

Question 21

Where is Ras found?

Answer 21

Bound to plasma membrane (very important for it to work)

Question 22

How to receptor protein tyrosine kinases signal to Ras?

Answer 22

Grb2 binds to tyrosine domains on activated RPTK via its SH2 domain
Grb2 is always bound to Sos (exchange factor) via its Sh3 domain
Sos is close enough to membrane to activate Ras by allowing exchange of GDP for GTP
Changes conformation of Ras –> active state –> can signal downstream and allow propagation of signal

Question 23

How does Ras activate a protein kinase cascade?

Answer 23

GTP bound Ras binds to a kinase and then that kinase activates several other kinases
ERK cascade involved in growth stimulatory signalling:
Active Ras binds to Raf
Raf phosphorylates MEK
MEK phosphorylates ERK

Question 24

How does retinoblastoma regulate the cell cycle?

Answer 24

1. G0 - retinoblastoma is unphosphorylated - binds to and inhibits E2F TFs
2. E2F transcription factors are held in cytoplasm - everything is turned off
3. Myc induction –> Cdk4/6-cyclin D kinase targets retinoblastoma and phosphorylates it
4. Phosphorylated retinoblastoma loses its affinity for E2F and releases them
5. E2F TFs bind to promoters in nucleus of cell cycle progression genes (e.g. cyclin E - next cyclin need for cell cycle progression, proto-oncogenes (Myc))

Acts like a BRAKE in cell cycle = TSG

Question 25

Explain the positive feedback loop of retinoblastoma and cyclin E

Answer 25

Initial release of E2F after Rb phosphorylation allows transcription of cyclin E
Cyclin E forms complex with Cdk2
Rb = target for phosphorylation by active Cdk2-cyclin E complex
Rb becomes further phosphorylated and releases more E2F so cytoplasmic [E2F] increases

Question 26

How does the positive feedback loop of Rb and cyclin E lead to the cyclin A gene promoter being activated

Answer 26

To ensure that the cell cycle occurs in order, the cyclin A gene promoter is only activated once [E2F] is high enough