CS - Receptor tyrosine kinases + MAPK pathway

Question 1

Examples of Receptor Tyrosine Kinase (RTK)

Answer 1

~ insulin receptor
~ vascular endothelial GF receptor
~ platelet derived GF receptor
~ epidermal GF receptor

Question 2

RTK facts

Answer 2

~ Receptor Tyrosine Kinase (RTK)
~ large family
~ posses intrinsic tyrosine protein kinase activity
~ no endogenous enzyme activity

Question 3

Types of RTK

Answer 3

~ receptors linked to tyrosine kinase

~ receptor associated kinases

Question 4

Epidermal growth factor

Answer 4

~ EGF = 53 a.a. polypeptide

~ regulation of cell growth, proliferation and differentiation

Question 5

Epidermal growth factor receptor (5)

Answer 5

~ ligand = epidermal growth factor (EGF)
~ single polypeptide
~ 1 transmembrane helix
~ 1 extracellular EFG binding domain
~ 1 kinase domain (intrinsic kinase activity)

Question 6

EFG receptor w/o bound

Answer 6

~ ligand bingin site = empty

~ tyrosine kinase = minimal activity

Question 7

Dimerisation of EGF receptor

Answer 7

~ induced by ligand binding
~ creates a conformation change
~ dimerisation induces trans autophosphorylation on tyrosine residues on activation lip of tyrosine kinase

Question 8

Phosphorylation of additional tyrosine residues

Answer 8

~ tyrosin kinase phosphorylates additional tyrosine residues in the RTK cytoplasmic domain
~ creates binding sites for additional signalling proteins

Question 9

Kinase domain dimer + EGF binding

Answer 9

1. kinase active site is normally blocked by activation loop
2. EGF binding + receptor dimerisation = asymmetric kinase dimer
3. asymmetric kinase dimer removes activation loop from kinase active site
4. active kinase phosphorylates tyrosine residues

Question 10

MAPK pathway (2+8)

Answer 10

Mitogen-activated protein kinase
~ enzyme cascade
1. EGF + EGF receptor
2. phosphorylated receptor
3. EGF receptor-SOS complex
4. activated Ras
5. activated Raf
6. activated MEK
7. activated ERK
8. phosphorylated transcription factors = changes in gene expression

Question 11

Creation of the EGF receptor-SOS complex

Answer 11

1. phosphotyrosine residues on RTK creates a binding site for the SH2 domain of GRB2 to bind to
2. Sos binds tot he SH3 domain on GRB2
3. Sos binds to Ras, activating it

Question 12

GRB2

Answer 12

~ a SH2 domain-containing protein
~ binds to phosphotyrosine residues
~ contains SH3 domain that binds to Sos

Question 13

Sos

Answer 13

Son of sevenless

~ GEF –> ‘pries’ Ras open, allowing GDP to diffuse out and be replaced by GTP –> activates Ras

Question 14

Ras

Answer 14

~ acts as a molecular switch
~ active/ON form = GTP bound = allows interaction betweens switches I + II
~ inactive/OFF form = GDP bound
~ conversion results from GEFs or GAPs

Question 15

GEFs

Answer 15

~ Guanine nucleotide-Exchange Factor

~ exchange GDP for GTP

Question 16

GAPs

Answer 16

~ GTPase-Activating Protein

~ accelerates GTP hydrolysis

Question 17

Activation of Raf

Answer 17

~ activated Ras dissociates form Sos
~ active Ras binds to N-terminal domain of Raf
~ hydrolysis of Ras-GTP –> Ras-GDP activates Raf via de-phosphorylation of a serine binding Raf to 14-3-3 (protein)

Question 18

Activation of MEK

Answer 18

a.k.a. Mitogen activated protein kinase kinase (MAPKK)

~ active Raf activates MEK by ATP –>ADP

Question 19

Activation of ERK

Answer 19

a.k.a. mitogen activated protein kinase (MAPK)

~ MEK activates MAPK by phosphorylating it

Question 20

EFG signalling and stimulation of cell proliferaiton

Answer 20

1. active MAPK dimer phosphorylates kinase p90
2. MAPK and p90 migrate to nucleus where they phosphorylate TCF and SRF
3. phosphorylated TCF and SRF bind to SRE in early response genes e.g. c-fos

Question 21

TCF

Answer 21

Ternary Complex Factor

Question 22

SRF

Answer 22

Serum Response Factor

Question 23

SRE

Answer 23

Serum Response Element