Lecture 11 Flashcards
Describe the receptor tyrosine kinase family
- Very large
- Involved in many responses
What responses are receptor tyrosine kinases involved in?
- 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)
What do RTKs possess?
Intrinsic tyrosine protein kinase activity
Describe the epidermal growth factor receptor
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
Stage 1: What occurs when the EGF receptor is without bound?
- Ligand binding sites empty
- Tyrosine kinase exhibits minimal activity
Stage 2: Dimerization of EGF receptor
- Ligand binding sites induces receptor dimerization
- Dimerization induces trans autophosphorylation on tyrosine residues on activation lip of tyrosine kinase
What occurs to the side chain of tyrosine?
Phosphorylated
Serine –> Tyrosine kinase and ATP hydrolysis –> Phosphoserine
Stage 3: Phosphorylation of addition tyrosine residues
- 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
What occurs when EGF activates EGF receptor
- Asymmetric kinase domain dimer forms
- Kinase active site blocked by activation loop
- Asymmetric kinase dimer removes activation loop from acceptor kinase active site
- Active kinase phosphorylates tyrosine residues
Mitogen associated Protein kinase pathway (MAPK) pathway - Stage 1
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
Explain what GRB2 contains
SH2 (Src homology 2) domain-containing proteins
Binds phosphotyrosine residues on proteins
Also contain SH3 which binds Sos
Explain Ras
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
3 different forms of Ras
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
Stage 2 of MAPK pathway
Activation of Ras
Sos promotes GTP binding to Ras, activating it
Active Ras dissociates from Sos
Ras activates free downstream signalling components
Ras associated with colorectal cancer
50,000 cases of colorectal cancer/year
50% of tumours - single activating point mutation in K-ras oncogene
Stage 3
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
What occurs when Raf is active - Stage 4
Activates MEK - MAPKK (mitogen activated protein kinase kinase) - tyrosine and serine/threonine kinase
Activates MAPK - Mitogen activated protein kinase (ERK - serine/threonine kinase)
Stage 5
Mitogen activated protein (MAP) kinase (ERK)
MEK phosphorylates tyrosine-185 and threonine-183
Stimulation of cell proliferation
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
