L11: RTKs and the MAPK pathway

Question 1

Responses RTK is involved in, RTK features

Answer 1

1. Insulin receptor
   -> glycogen synth
2. Vascular endothelial GF rec.
   -> new blood vessel growth
3. Platelet driven GF rec.
   -> embryonic dev, cell proliferation and migration
4. Epidermal GF rec.
   -> growth and proliferation and differentiation

• Possess intrinsic tyrosine protein kinase activity
• Huge family of proteins

Question 2

Epidermal GF receptor (about)

Answer 2

• A typical receptor tyrosine kinase. Single pp, crosses PM once. has an EGF-binding domain, Kinase domain provides intrinsic kinase activity. C-terminal tail is tyrosine rich (in cytosol)
• Ligand: epidermal GF (53 aas in humans)
• Regulation of cell growth, proliferation, differentiation

Question 3

Epidermal GF receptor (mechanism) + asymmetric kinase domain dimer formation

Answer 3

• Ligand binding induces receptor dimerization (HER1)
• Dimerization induces autophosph. of kinase activation lip
  -> partial activation of kinase activity
• Phosph. of tyrosine residues fully act. tyrosine kinase -> phosph. additional T residues in RTK cyt. domain
• Creates BS for additional signalling proteins

1. Kinase activity site blocked by activation loop
2. Asymmetric kinase dimer removes activation loop from acceptor kinase AS
3. Active kinase phosp. tyrosine residues

Question 4

Ras (basic structure, switch activity, single point mut.)

Answer 4

• Monomeric G protein, guanyl binding site conserved across G proteins
• Has 2 elements called switch I and II which are involve in switch activity. They don’t interact w/ GDP in inactive form; Sos binds and forces protein open, GDP diffuses out to be replaced w/ GTP (active)
• Sos displaced
• A single point mutation in Ras interferes w/ GTPase activity (implications for colorectal cancer - 50% arise from this)

Question 5

GRB2

Answer 5

• Cytosolic adaptor protein
• Contains SH2 domain, binds phototyrosine residues (RTK autophosph. creates phototyrosine resiudes)
• Also has a SH3 domain which binds Sos

Question 6

EGF signalling

Answer 6

• EGF binds receptor (Ras inactive)
• Phototyrosine resides on RTK permit SH2 domain of GRB2 to bind receptor (via autophosph. to create BS)
• GRB2 recruits Sos through SH3 domain
• Sos binds to Ras, activating it (exchange of GDP for GTP at Guanyl BS)

Question 7

Enzymes affecting Ras activation

Answer 7

• GEFs (Guanine nt-exchange factor) promote GTP exchange
• GAPs (GTPase-activating protein) accelerate GTP hydrolysis

Question 8

Activated Ras in EGF pathway

Answer 8

• Binds the N-terminal regulatory domain of Raf, activating it
  (Raf = serine threonine kinase, linked to 14-3-3 protein in inactive form; deposph. of serine causes Raf activation)
• Hydrolysis of Ras-GTP to Ras-GDP releases active Raf

Question 9

Protein kinase cascade in EGF pathway

Answer 9

• Active Raf kinase activates MEK
  (MEK is a MAPKK)
• This in turn phosph. ERK on tyrosine-185, threonine-183
  (ERK is a MAPK)
• Active ERK dimer phosph. protein kinase p90
• Active ERK dimer and p90 migrate to nucleus and phosp. TCF (Ternary complex factor) and SRF (Serum response factor) in early response genes
  e.g. c-fos