HC15 cellular signalling and cell division

Question 1

MAPK pathway

Answer 1

mitogen binds to receptor –> activation of Ras –> phosphorylation/activation of MAPKKK (Raf) –> phosphorylation/activation of MAPKK (Mek) –> phosphorylation/activation of MAPK (Erk) –> activation of transcription of a regulatory protein –> this protein/TF stimulates expression of Myc –> Myc upregulates cyclin D –> active G1-Cdk –> G1-Cdk phosphorylates Rb –> Rb dissociates from E2F –> E2F drives transcription of cell cycle progression genes –> from G1 phase to S phase.

Question 2

GPCR general pathway

Answer 2

G-protein coupled receptors –> activated by mitogen binding –> GPCR interacts with G-protein anchored into membrane –> affinity for GDP decreases –> G-protein binds GTP –> activated G-protein –> the alpha subunit dissociates from the beta+gamma subunit –> signal transduction .

alpha subunit has intrinsic GTPase activity –> GTP will be hydrolysed into GDP –> G-protein inactive

Question 3

phosphorylation on which amino acids in eukaryotes and prokaryotes?

Answer 3

eukaryotes: serine, threonine, tyrosine
prokaryotes: histidine

Question 4

GPCR: PIP2

Answer 4

G-protein is activated –> phospholipase C is activated –> PIP2 is cleaved into: diacylglycerol (DAG) and inositol triphosphate (IP3) –> IP3 mediates opening of calcium channels in ER –> cytosolic calcium levels increase –> DAG and calcium coactivate protein kinase C (PKC)

Question 5

Desentisation of GPCR

Answer 5

activated GPCR –> stimulates GPCR kinase (GRK) –> phosphorylation of GPCR on multiple sites –> arrestin can bind to phosphorylated GPCR –> desentisation

Question 6

RTKs

Answer 6

Receptor tyrosin kinases
signal protein binds –> dimerisation –> trans-autophosphorylation on tyrosines –> signal transduction

Question 7

activation of Ras via RTKs

Answer 7

adaptor protein Grb2 binds to phosphorylated tyrosines on RTK –> Ras-GEF can dock on Grb2 as well –> stimulates release of GDP of Ras –> Ras binds GTP –> active –> can bind to MAPKKK

Question 8

Cell-phases

Answer 8

G1: growing of cells just after cell division. need to accumulate resources and energy for DNA replication
S: DNA replication
G2: growing of cells to accumulate enough resources to be able to provide 2 daughter cells + determine whether DNA replication occurred
M: cell division/mitosis.

Question 9

cyclin-CDK: activation

Answer 9

cyclin dependent kinases form complexes with specific cyclins –> partial activation –> phosphorylation by CDK-activating kinase (CAK) –> fully active cyclin-Cdk complex.

These complexes need to be inactive but ready to go. therefore, phosphorylation of different sites:
- wee1 kinase –> inhibitory phosphate is placed on the Cdk
- Cdc25 phosphatase –> removes the inhibitory phosphate –> active state of cyclin-cdk complex

Question 10

p27 + regulation

Answer 10

CDK inhibitor: binds active cyclin-Cdk complexes –> inactivates them

These inhibitors also need to be degraded: active SCF complex can phosphorylate CDK inhibitors –> signal for ubiquination –> degradation by proteasome

Question 11

DNA replication only once initiated in the cell cycle: how?

Answer 11

cdc6 binds to origin of replication. cdc6 levels are under control of S-CDKs –> phosphorylate cdc6 –> degradation.

When DNA replication is not proceeding normally –> cdc6 is crucial –> halts cell in S-phase.

Question 12

DNA integrity not satisfcatory:

Answer 12

phosphorylation of p53 –> p53 is stabilised/activation (no longer degradation by mdm2) –> p53 binds to p21 gene –> p21 is a CDK inhibitor –> inactivates complexes of G1/S- and S-CDKs –> blocks DNA synthesis progression.

Question 13

Myc can bind to regulatory regions of many genes:

Answer 13

Leading to expression of these genes:
- cyclin D –> more G1-CDK-cyclin complexes –> Rb phosphorylation –> increased E2F activity

• SCF –> increased degradation of p27/other CDK inhibitors –> activation of G1/S cyclin-CDK complex –> Rb phosphorylation –> E2F activity
• E2F –> more E2F activity

This all leads to entry into the S-phase

Question 14

Apoptosis

Answer 14

degradation of nuclear lamins and DNA.

extrinsic pathway: interaction between death receptors and ligand (Fas receptor + Fas ligand) –> activation of caspase 8 –> activation of caspase 3

intrinsic pathway: mito’s are damaged –> cytochrome c is released into cytosol –> binds to Apaf-1 –> aggregation of Apaf-1 into apoptosome –> activation of caspase 9 –> caspase 3 activation.