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Receptor tyrosine kinases

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Receptor tyrosine kinases Introduction: Protein phosphorylation Recruitment of kinases in signalling pathways Consequences of protein phosphorylation – PowerPoint PPT presentation

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Title: Receptor tyrosine kinases


1
Receptor 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

2
Receptor tyrosine kinases
  • RTK-mediated pathways
  • Ras-Raf-MAP kinase pathway, use of dominant
    negative mutants to map pathway
  • R7 photoreceptor development pathway in
    drosophila

3
Introduction
  • 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

4
Introduction
  • 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.)

5
RTK 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

6
RTK 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

7
RTK structure/function
Regulatory domains
8
RTK ligands
  • Typically small soluble proteins
  • Work in autocrine and paracrine manner
  • Dimerize (may aid in receptor dimerization)
  • Some RTK ligands membrane-bound

9
Receptor 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.)

10
RTK autotransphosphorylation
11
Src homology (SH) 2 SH3 domains
  • SH2 domains bind P-Tyr-containing sequences
  • SH3 domains bind to pro-rich (PxxP) sequences

12
SH2 SH3 domains
13
RTK-mediated pathways one pathway with two very
different functions
  • Ras-Raf-MAP kinase pathway
  • R7 photoreceptor development in drosophila
    (fruitfly)

14
The 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
15
Use 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.

16
Dominant 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
17
Combine 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
18
R7 photoreceptor development
  • Fruitfly (Drosophila melanogaster)
  • Compound eye (800 ommatidia)
  • Each ommatidium has 8 photoreceptor cells each
    detects a different wavelength of light

19
R7 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)

20
The R7 photoreceptor developmental pathway is a
RTK-MAP kinase cascade
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