Lecture 11

Question 1

Describe the receptor tyrosine kinase family

Answer 1

• Very large
• Involved in many responses

Question 2

What responses are receptor tyrosine kinases involved in?

Answer 2

• Insulin receptor (synthesizes glycogen)
• Vascular endothelial growth factor receptor (new blood vessel growth)
• Platelet derived growth factor receptor (embryo development, proliferation and migration)
• Epidermal growth factor receptor (growth, proliferation and differentiation)

Question 3

What do RTKs possess?

Answer 3

Intrinsic tyrosine protein kinase activity

Question 4

Describe the epidermal growth factor receptor

Answer 4

Ligand is epidermal growth factor (EGF)

EGF = 53 amino acid polypeptide

• Regulates cell growth, proliferation and differentiation
• Single polypeptide crosses plasma membrane once
• Intrinsic tyrosine kinase activity

EGF-binding domain - Transmembrane helix - Kinase domain - C-terminal tail

Question 5

Stage 1: What occurs when the EGF receptor is without bound?

Answer 5

• Ligand binding sites empty
• Tyrosine kinase exhibits minimal activity

Question 6

Stage 2: Dimerization of EGF receptor

Answer 6

• Ligand binding sites induces receptor dimerization
• Dimerization induces trans autophosphorylation on tyrosine residues on activation lip of tyrosine kinase

Question 7

What occurs to the side chain of tyrosine?

Answer 7

Phosphorylated

Serine –> Tyrosine kinase and ATP hydrolysis –> Phosphoserine

Question 8

Stage 3: Phosphorylation of addition tyrosine residues

Answer 8

• Tyrosine residue phosphorylation = Full activation of tyrosine kinase
• Additional RTK cytoplasmic domain tyrosine residues are phosphorylated by tyrosine kinase
• Creates binding sites for additional proteins

Question 9

What occurs when EGF activates EGF receptor

Answer 9

• Asymmetric kinase domain dimer forms

1. Kinase active site blocked by activation loop
2. Asymmetric kinase dimer removes activation loop from acceptor kinase active site
3. Active kinase phosphorylates tyrosine residues

Question 10

Mitogen associated Protein kinase pathway (MAPK) pathway - Stage 1

Answer 10

Recruitment of downstream signalling proteins

• G-protein Ras needs to be activated
  -EGF binds to receptor
• Ras inactive
• Phosphotyrosine residues on RTK permit SH2 domain of GRB2 to bind receptor
• Autophosphorylation of RTK creates binding site for SH2 domain containing cytosolic adapter protein GRB2
• Ras and Sos (Son of sevenless) remain inactive
• GRB2 binds to RTK via it’s SH2 domain
• Sos binds SH3 domain on GRB2
• Sos binds Ras

Question 11

Explain what GRB2 contains

Answer 11

SH2 (Src homology 2) domain-containing proteins

Binds phosphotyrosine residues on proteins

Also contain SH3 which binds Sos

Question 12

Explain Ras

Answer 12

Monomeric G-protein
Act as molecular switches and timers

Ras ON -> GAPs and GTP hydrolysis -> Ras OFF

Ras OFF -> GTP hydrolysis and GEFs -> Ras ON

GEF: Guanine nucleotide-exchange factor: Promote GDP-GTP exchange

GAP: GTPase activating protein: Accelerates GTP hydrolysis

Question 13

3 different forms of Ras

Answer 13

Ras-GDP - Switch I and II do not interact directly with GDP

Ras-Sos - Sos ‘pries’ Ras open, GDP to diffuse out

Ras-GTP: Sos displaced, switch I and II interact with GTP

Question 14

Stage 2 of MAPK pathway

Answer 14

Activation of Ras

Sos promotes GTP binding to Ras, activating it

Active Ras dissociates from Sos

Ras activates free downstream signalling components

Question 15

Ras associated with colorectal cancer

Answer 15

50,000 cases of colorectal cancer/year

50% of tumours - single activating point mutation in K-ras oncogene

Question 16

Stage 3

Answer 16

Raf activation

Ras-GTP binds N-terminus of Raf

Partial activation of Raf by dephosphorylation of 1 out of 2 serines either binding or releasing Raf from 14-3-3

Ras-GTP -> Ras-GDP, releasing Raf from Ras

Dimerization and phosphorylation fully activate Raf

Question 17

What occurs when Raf is active - Stage 4

Answer 17

Activates MEK - MAPKK (mitogen activated protein kinase kinase) - tyrosine and serine/threonine kinase

Activates MAPK - Mitogen activated protein kinase (ERK - serine/threonine kinase)

Question 18

Stage 5

Answer 18

Mitogen activated protein (MAP) kinase (ERK)

MEK phosphorylates tyrosine-185 and threonine-183

Question 19

Stimulation of cell proliferation

Answer 19

Protein kinase p90 phosphorylated by active MAPK

Active MAPK and p90 migrate to nucleus and phosphorylates TCF (ternary complex factor) and SRF (serum response factor)

Phosphorylated TCF and SRF bind SRE in early response genes