cell division

Question 1

describe how progress through the cell cycle is controled

Answer 1

• G1, Synthesis, G2, Mitosis, cytokinesis
• a series of checkpoints exist in which the cell does not move on to the next stage unless the conditions are appropriate and previous stage has been completed
  1) start (restriction point); should division processes begin?
  2) should mitosis begin (is DNA fully replicated and is the cell big enough)
  3) should anaphase of mitosis occur (are all chromosomes attached to spindle)
• Cyclin dependant kinases (Cdk’s) control progress by phosphorylation

Question 2

discuss Cdk structure and regulation

Answer 2

• cyclic binds to Cdks and moves the activation loop which exposes the active site
• Cdk can then phosphorylate target proteins at their serine/threonine

regulation

• cyclin can be synthesised/degraded for
• Cdk phosphorylation/dephosphorylation
• Cdk inhibitor proteins like p27 and p21 which block the active site

Question 3

G1-S transition

Answer 3

1) signal molecules such as mitogens (stimulate cell division) and growth factors (stimulate mass increase) bind to cell surface receptor tyrosine kinases and cause receptors to dimerase and phosphorylate each other (beocmes phosphotyrosines)
- epidermal growth factor (EGF) and platelet derived growth factor (PDGF)
- linear relationship between EGF concentration and final MAPK cascade activation

2) the Grb2 adaptor protein binds to the phosphotyrosine proteins which activates SOS guanine nucleotide exchange factor (GEF)
3) SOS binds Ras (G protein) which then releases GTPP and binds to binds to GTP which activates it
4) ras binds to and activates MAP3K (raf) by dephosphorylating its inhibiting sites
4) MAP3K phosphorylates and activates MAP2K (MEK)
5) MAP2K binds and activates MAPK (ERK)

6) ERK phosphorylates and activates transcription factors which then activate G1-cyclin (cyclin D) transcription.
- phosphorylation of the Cdk inhibitor (p27) causes to to promote assembly rather than inhibiting assembly
- also phosphorylates other kinases which alter apoptosis and cytoskeleton activities

7) G1-cdk phosphorylates and inactivates RBI, which releases E2F TF and activates G1/S- cyclin and S-cyclin
8) positive feedback loop; G1-cdk and S-cdk inactivate RB1 and phosphorylate p27 causing degradation

Question 4

discuss Ras

Answer 4

• causes activation of the MAPK cascade causing cells to move through cell cycle
• 30% of human tumours are due to hyperactive ras mutations caused by a single AA (doesnt hydrolyse GTP so is never inactivated)
• proto-oncogene (doesnt cause promote tumour formation but can when mutated)
• 40% of molecules form nanoclusters (approx 7 ras molecules), 60% are in monomer form. Ras can only bind to raf it ras is in cluster form
• the more ras present, the more nanoclusters present and the more activation of the MAPK cascade
• other activities such as apoptosis inhibition cytoskeleton organisation, cell migration
• different forms exist with different functions; H-ras, N-ras, K-ras (Kras4a and Kras4b); K ras in the only essential gene
• colon cancer associated with K-ras (50% of all cases) mutation where as skin cancer has an associaton with N-ras

Question 5

discuss Raf

Answer 5

• thought to be a proto-oncogene because mutated form (v-raf) is found in mouse tumours and when transferred to wild type cells it caused constitutively active MAPK cascade
• activation and deactivation is via removal and addition phosphates to inhibitory and activating sites

Question 6

what are limitations to experiments involviong this system

Answer 6

• in vitro studies are oversimplified
• overexpression to work out normal function is a flawed idea
• the signal transduction system works differently in different cell types and at different stages
• different isoforms exist which act in different wats
• inhibiting the pathway to work out function doesnt often work because the pathway is so complex

Question 7

what do the different cyclin-cdk compelxes do

Answer 7

G1-cdk; transition from G1 to S
G1/S-Cdk: initiates procedures in S phase such as formation of replication complexes
S-Cdk: initiates synthesis

Question 8

which cdk enzymes are needed for G1-S transition

Answer 8

Cd4 Cd 6 Cyclin D

Question 9

how does ERK cause activation of G1-Cdk

Answer 9

• activation of transcription factors of Cyclin D

- AP-1, c-Fos