Cell growth + division Catherine

Question 1

Transcriptional activation of cell cycle

Answer 1

• +ve feedback (move to next stage and repress previous)
• pRb binds E2F to repress expression in G1, Cyclin D-Cdk4/6 phosph pRb
• E2F induces cyclin E expression → cyclin E further phosph pRb → transcribes cyclin A
• Cyclin A = -ve feedback, phosph E2F

Question 2

Degradation of mitotic proteins

Answer 2

1. SCF
   - Controls G1/S and G2/M boundaries
   - SCF substrates = cyclin D/E + CKIs like p27
2. APC
   - Co-activators = Cdc20 + cdh1
   - Activated cyclin B → activated Cdc20
   - If free kinetochores, APC/C is inhibited by MCC
   - MCC turned over by TRIP13 → free APC
   - When kinetochores are attached to microtubules, ↓ formation of MCC but turnover of TRP13 carries on, ↓ inhibition on APC/C
   - Cyclin B then degraded

Question 3

Regulation of proteolysis machinery

Answer 3

• APC/C needs Cdc20/Cdh1 to bind
• In early M, CDK phosph APC/C → Cdc20 binds → cyclins destroyed → ↓ CDK → anaphase
• Cdh1 is phosphorylated during S, G2 + M, x bind APC
• When exits M, Cdh1 is dephosph to activate APC. This ubiquit Cdc20 so x simultaneous activation of both

Question 4

CKIs

Answer 4

Yeast
- Sic1p phosph Cdc28-cyclin, prevents premature S phase entry

Mammals

• INK4 + CIP/KIP
• p27 highest in Go + early G1, ↓ ↓ in G1/S
• Prevent activation of cyclinE-Cdk2 or cyclinD-Cdk4
• E2F → p27 transcription → cyclin E/ cdk2 inhibited → x pRb phosph (-ve feedback)

Question 5

Regulation by phosphorylation

Answer 5

• Cdc2 = inhibited at Thr14/Tyr-15 + activated at Thr161
• Wee1/Cdc25
• CAK = activating phosph
• Cdk1 activates own activator Cdc25 + inhibits inhibitor, w/o cyclin B Cdk1 ↓, wee1 ↑ cdc25 ↓

Question 6

DNA replication control

Answer 6

• CDT1 needs to be free + dephosph
• Geminin sequesters CDT1 but destroyed by APC in A
• Cdt1 removed during DNA replication so PIC x re-assemble

Question 7

GFR as oncogene more

Answer 7

• Ligand binds to ectodomain → dimerisation → activates IC tyr kinase domain
• Phosphotyr activates ds components
• Point mutation to one that causes dimerisation + activated w/o ligand
• Or chromosomal translocation, replaces EC domain w/ segment that dimerises

Question 8

Intracellular transducer as oncogenes more

Answer 8

• Ras mutations in Q61, G12 + G13
• Q61 interfers w/ H20 coordination needed for nucleophilic attack
• G12/G13 prevent van der waal btw Ras + GAP through steric hindrance which affects orientation of catalytic Gly at 61
• Raf becomes oncogenic by mutation at Val600→ Glu600 (mimics activation loop)

Question 9

TF as oncogenes more

Answer 9

• Ras affects myc expression in 2 ways
  1. P13K = ds of Ras, inhibits GSK3 → prevents Myc being proteolysed
  2. Ras activates Raf-1 → activates MEK pathway → phosph of c-Myc
• Myc = 40% of tumours
• ↑ effects e.g. cyclin D, CDKs (activates Cdc25), E2F (myc stimulates CDK → phosph pRb → frees E2F, directly w/ E2F promoter) + CKIs

Question 10

Oncogenes not involved in cell proliferation

Answer 10

• Bcl-2 inhibits apoptosis
• Tumour suppressors = also important e.g. p53
• Telomerase + VEGF also oncogenes
• Protein acts is important e.g. at branch vs in linear pathway
  + how easy to mutate

Question 11

p53 vs pRb

Activation

Answer 11

• p53 = phosph + stabilised by several kinases, Mdm2, ARF

- pRb = phosph

Question 12

p53 vs pRb

Effects - cell cycle

Answer 12

• pRb acts through E2F target genes, p53 acts directly via p53 target genes
• pRb binds E2F and blocks transcription (can repair damage)
• p53 acts through p21 (inhibit Cdk2, prevents inactivation by Prb)
• p21 null mice are deficient in cell cycle arrest

Question 13

p53 vs pRb

Effects - CIN

Answer 13

• Experiments knockout
• BubR1 can physically assoc. w/ + activate -53 during SAC checkpoint activation + p53 activates BUBR1
• pRb loss affects mitotic fidelity

Question 14

p53 vs pRb

Effects - Apoptosis

Answer 14

1. Extrinsic
   - Phosph pRb means genes like caspase 7 are expressed
   - p53 induces expression of Fas TNF receptor
2. Intrinsic
   - pRb induces MOMP by binding BAX
   - p53 induces expression of pro-apoptic genes

Question 15

p53 vs pRb

Effects - metabolism

Answer 15

• E2F null mice
• p53 suppresses glucose transport directly by preventing GLUT1/4 transcription
• Oxidative metabolism

Question 16

p53 vs pRb

Effects - angiogenesis

Answer 16

• E2F target genes = bFGF
• Loss of pRb → activation of Id2
• p53 inhibits HIF

Question 17

Apoptosis initiation

Answer 17

Extrinsic pathway

• TM receptor-ligand or NK or cytotoxic T cell-mediated injection of granzymes
• Death receptor - ligand → IC domain recruits adaptor
• FADD recruits initiator caspases → form DISC → form executive caspases

Intrinsic pathway

• Bax/Bad inhibited by Bcl2/Xl but BH3 sequesters Bcl2.XL → free Bax/Bad to homodimerise → outer membrane pore
• Frees cytc which binds APAF1 + forms apoptosome → CARD domain recruits caspase 9 → activates executioner caspases like 3 + 7
• Smac/Diablo → AIF + endonuc G

Question 18

Apoptosis execution

Answer 18

Nuclear effects

• MOMP → AIF + endonuc G
• Caspase dependent = RAIDD + RIPK1 compete for binding to PIDD + lead to survival or apoptosis
• Caspase 2 = us of caspase 3 which cleaves ICAD → DNA fragment
• Nuclear lamina degraded by caspase

Cytoskeletal effects

• Caspases cleave ↑ components e.g. actin + myosin
• Caspase-mediated proteolysis of ROCK1

Question 19

Apoptosis degradation

Answer 19

• Phospholipid asymmetry + externalised PS
• Bridge molecules = phagocyte recognition ligand
• Some receptors bind directly to apoptotic cell
• Internalise particle

Question 20

Apoptosis regulation

Answer 20

• Bcl2/Xl block apoptosis by sequestering Bak/Bax
• Pro-survival genes must be overwhelmed
• Mit fission
• Bcl2 = regulated by p53