Title: Receptor tyrosine kinases
1Receptor tyrosine kinases
- Introduction
- Protein phosphorylation
- Recruitment of kinases in signalling pathways
- Consequences of protein phosphorylation
- RTK family
- Classification structure/function
- RTK ligands
- Receptor dimerization autotransphosphorylation
2Receptor tyrosine kinases
- RTK-mediated pathways
- Ras-Raf-MAP kinase pathway, use of dominant
negative mutants to map pathway - R7 photoreceptor development pathway in
drosophila
3Introduction
- Protein phosphorylation represents the most
common form of posttranslational modification in
nature - Protein function altered by addition of a
negatively charged phosphate group to a Ser, Thr,
or Tyr residue - Binding properties
- Enzymatic activity if a catalytic protein
4Introduction
- Cell surface receptors recruit activity of
protein kinases in two general ways - Non-receptor tyrosine kinases Receptors lacking
self-contained kinase function recruit activities
of intracellular protein kinases to the plasma
membrane - Receptor tyrosine kinases Possess an intrinsic
tyrosine kinase activity that is part of the
receptor protein. Examples include receptors for
growth factors (PDGF, EGF, insulin, etc.)
5RTK family classification structure/function
- Implicated in diverse cellular responses
- Cell division
- Differentiation
- Motility
- At least 50 RTKs identified
- Subdivided into 10 subclasses based on
differences within extracellular, ligand-binding
domain of receptor - Oncogenic RTK mutants exist
- erbB gene encodes an N-terminal truncated,
constitutively active form of EGF receptor
6RTK family classification structure/function
- Four common structural features shared among
RTKs - Extracellular ligand-binding domain
- Single transmembrane domain
- Cytoplasmic tyrosine kinase domain(s)
- Regulatory domains
7RTK structure/function
Regulatory domains
8RTK ligands
- Typically small soluble proteins
- Work in autocrine and paracrine manner
- Dimerize (may aid in receptor dimerization)
- Some RTK ligands membrane-bound
9Receptor dimerization autotransphosphorylation
- Ligand-induced RTK activation induces Receptor
dimerization, leading to activation of catalytic
domains - Receptor autotransphosphorylation
- Further stimulates kinase activity
- Leads to phosphorylation of additional proteins
involved in receptor signalling pathway - Provides docking sites for downstream
signalling proteins (Grb2, PI3-kinase,
phospholipase Cg, etc.)
10RTK autotransphosphorylation
11Src homology (SH) 2 SH3 domains
- SH2 domains bind P-Tyr-containing sequences
- SH3 domains bind to pro-rich (PxxP) sequences
12SH2 SH3 domains
13RTK-mediated pathways one pathway with two very
different functions
- Ras-Raf-MAP kinase pathway
- R7 photoreceptor development in drosophila
(fruitfly)
14The Ras-Raf-MAP kinase pathway
SH2 domain
SH3 domains
Proline-rich regions (-PXXP-)
Tyr-P
Raf
Sos
Grb2
Ras (inactive)
Ras (active)
Pi
P
Nucleus
MEK
P
P
fos
jun
P
P
P
P
MAP kinase
MAP kinase
Increase gene expression
15Use of oncogenic dominant negative mutants to
map pathways
- Oncogenic Ras (V12Ras) defective GTPase
function. Always turned on (always GTP-bound) - Dominant negative Ras (N17Ras) can interact with
its immediate upstream partner (Sos), but cannot
become activated to transduce a downstream signal
(i.e., to Raf). Effect is to sequester Sos to
prevent it from activating endogenous Ras.
16Dominant negative Ras (N17Ras) sequesters Sos
blocks pathway from Ras on down
SH2 domain
SH3 domains
Proline-rich regions (-PXXP-)
GDP
Tyr-P
Raf
Sos
Grb2
Ras (inactive)
N17Ras
Nucleus
jun
MEK
fos
MAP kinase
gene expression blocked
17Combine oncogenic and DN mutants to map position
of pathway components
SH2 domain
SH3 domains
Proline-rich regions (-PXXP-)
GDP
Tyr-P
Raf
Ras (inactive)
Sos
Grb2
N17Ras
Nucleus
P
Oncogenic Raf
MEK
P
P
fos
jun
P
P
P
P
MAP kinase
MAP kinase
Increased gene expression
18R7 photoreceptor development
- Fruitfly (Drosophila melanogaster)
- Compound eye (800 ommatidia)
- Each ommatidium has 8 photoreceptor cells each
detects a different wavelength of light
19R7 photoreceptor development
- Photoreceptor cells recruited as an
undifferentiated precursor from epithelial sheet
of cells - Each photoreceptor develops in a specific order
beginning with R8 ending with R7 (responds to
ultraviolet light)
20The R7 photoreceptor developmental pathway is a
RTK-MAP kinase cascade