Transcript Receptor Tyrosine Kinases

Enzyme-linked Cell Surface
Receptors
16 April 2007
Mechanism of PDGF signal transduction
PDGF
dimerize &
phosphorylate each
other
P
ras
MAPK
CELL
GROWTH
MAPK
P
phosphorlyates multiple
target proteins
Enzyme-linked Cell Surface Receptors
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Receptor Tyrosine kinases: phosphorylate specific tyrosines
Tyrosine kinase associated receptors: associate with
intracellular proteins that have tyrosine kinase activity.
Receptorlike tyrosine phosphatases: remove phosphate
group
Receptor Serine/ Threonine kinases: phosphorylate specific
Serine/ Threonine
Receptor guanylyl cyclases: directly catalyzes the production
of cGMP
Histidine kinase associated receptors: kinase phoshorylates
itself on histidine and then transfers the phosphate to a second
intracellular signaling protein.
Receptor Tyrosine Kinases (RTKs)
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Intrinsic tyrosine kinase activity
Soluble or membrane-bound ligands:
Nerve growth factor, NGF
 Platelet-derived growth factor, PDGF
 Fibroblast growth factor, EGF
 Epidermal growt factor, EGF
 Insulin
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Downstream pathway activation:
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Ras-MAP kinase pathway
TYROSINE KINASE RECEPTORS
• these receptors traverse the membrane only once
• respond exclusively to protein stimuli
– cytokines
– mitogenic growth factors:
• platelet derived growth factor
• epidermal growth factor
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Functions include:
Cell proliferation, differentiation
 Cell survival
 Cellular metabolism
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Some RTKs have been discovered in cancer research
Her2, constitutively active form in breast cancer
 EGF-R overexpression in breast cancer
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Other RTKs have been uncovered in studies of
developmental mutations that block differentiation
Outline
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Activated RTKs transmit signal to Ras protein
Ras transduces signal to downstream serinethreonine kinases
Ultimate activation of MAP kinase
Activation of transcription factors
Ligand binding to RTKs
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Most RTKs are monomeric
ligand binding to EC domain induces dimerization
FGF binds to heparan sulfate enhancing its binding
to receptor: dimeric receptor-ligand complex
Some ligands are dimeric: direct dimerization of
receptors
Insulin receptors occur naturally as a dimer
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Activation is due to the conformational change of the
receptor upon ligand binding
Protein Tyrosine Kinase
Substrate + ATP
Substrate-P + ADP
Protein Tyrosine Phosphatase
(PTP)
Tyrosine Protein Phosphorylation
• Eukaryotic cells coordinate functions through environmental signals soluble factors, extracellular matrix, neighboring cells.
• Membrane receptors receive these cues and transduce signals into the
cell for appropriate response.
• Tyrosine kinase signalling is the major mechanism for receptor signal
transduction.
• Tyrosine protein phosphorylation is rare (1%) relative to
serine/threonine phosphorylation.
• TK pathways mediate cell growth, differentiation, host defense, and
metabolic regulation.
• Protein tyrosine phosphorylation is the net effect of protein tyrosine
kinases (TKs) and protein tyrosine phosphatases (PTPs).
Subfamilies of Receptor Tyrosine Kinases
Protein Tyrosine Kinases (TKs)
Receptor tyrosine kinases (RTK)
– insulin receptor
– EGF receptor
– PDGF receptor
– TrkA
Non-receptor tyrosine kinases (NRTK)
– c-Src
– Janus kinases (Jak)
– Csk (C-terminal src kinase)
– Focal adhesion kinase (FAK)
TABLE 15–4 Some Signaling Proteins That Act Via Receptor Tyrosine Kinases
SIGNALING LIGAND
RECEPTORS
Epidermal growth factor (EGF) EGF receptor
Insulin
insulin receptor
SOME RESPONSES
stimulates proliferation of various cell
types
stimulates carbohydrate utilization and
protein synthesis
Insulin-like growth factors
IGF receptor-1
stimulate cell growth and survival
Trk A
stimulates survival and growth of some
(IGF-1 and IGF-2)
Nerve growth factor (NGF)
neurons
Platelet-derived growth factors PDGF receptors
stimulate survival, growth, and
proliferation of various cell types
Macrophage-colony-stimulating M-CSF receptor
stimulates monocyte/macrophage
factor (M-CSF)
proliferation and differentiation
Fibroblast growth factors
(FGF1 to FGF-24)
FGF receptors
(FGF-R1–FGFR4)
stimulate proliferation of various cell (FGFtypes; inhibit differentiation of some
precursor cells; inductive
development
VEGF receptor
stimulates angiogenesis
signals in
Vascular endothelial growth
factor (VEGF)
Ephrins (A and B types)
Eph receptors (A and B) stimulate angiogenesis; guide cell and
axon migration
Signaling from tyrosine kinase receptors
• Ligand induced dimerization
• Autophosphorylation
• Phosphorylation in the catalytic domain increase
the kinase activity
• Phosphorylation outside the catalytic domain
creates specific binding for other proteins.
• Autophosphorylated receptors bind to
signaling proteins that have SH2
(phosphotyrosine residues) domains
Receptor Dimerization and Kinase Activation
From Hunter (2001) Nature 411,355.
Consequences of receptor
dimerization
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Kinase in one subunit P* one or more tyrosine
residues on the other
Binding of ATP (insulin-R) or protein substrates (FGF-R)
 Enhanced kinase activity: P* of other sites on the
receptor
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P*-tyrosine residues become docking sites for
adapter proteins
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Small proteins with SH2, PTB and SH3 domains,
but without intrinsic enzymatic or signaling activities
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Coupling activated RTKs to components of signaling
pathways such as Ras
Ras upstream and downstream signaling.
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Through a variety of adaptor
proteins, these signals cause
guanine nucleotide exchange
factors to replace the GDP-bound
to inactive Ras with GTP. GAPs
trigger the hydrolysis of GTP back
to the inactive GDP-bound form.
GTP-bound Ras binds to a
plethora of downstream effector
molecules to stimulate intracellular
signaling of several pathways.
RTKs could activate Ras either by
aactivating GEF or inhibiting GAP.
Campbell and Der 2004
Ras
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Monomeric GTPase switch protein
Its activation is enhanced by GEF
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GDP-GTP exchange
Deactivation of Ras-GTP complex requires
GAP, which increases intrinsic GTPase activity
100 fold
Lifetime of Ras-GTP is higher than that of G
Ras is a small protein (170 aa. Vs 300 aa of G)
 G has a domain that functions like GAP
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Mutant ras proteins are associated with many
cancers
Mutant ras can bind GTP but can not hydrolyze
it, and thus remain constitutively in “on” state
Most oncogenic ras proteins contain a mutation
in codon 12 (Gly)
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This blocks the binding of GAP to ras, and prevents
GTP hydrolysis.
Linking ras to RTKs
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Experimental evidences
Fibroblasts were induced to proliferate with FGF
and EGF
 Anti-ras antibody microinjected: cell proliferation
arrest
 Injection of mutant ras proteins allows cell to
proliferate in the absence of growth factors.
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Ligand-bound RTKs activate ras! How?
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Two cytosolic proteins are involved: GRB2, Sos
SH2 domain in GRB2 binds to a P*-tyrosine
residue in the activated receptor
Two SH3 domains of GRB2 bind to and activate
Sos
Sos is GEF protein and convert inactive GDP-ras
into active GTP-ras
Developmental studies elucidated the role of
GRB2 and Sos in linking RTKs to ras activation
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Individual eyes of drosophila: ommatidia
Each ommatidium consists of 22 cells, 8 of
which are photosensitive neurons: retinula or R
cells (R1-R8)
The RTK sevenless is dedicated to the
regulation of R7 development
Flies with sevenless mutation lack R7 cells in
their eyes
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R8 cells express Boss (bride of sevenless) on
their surface which acts as a ligand for Sev RTK
on R7 cells
Studies with temperature sensitive sev mutants
allowed the discovery of downstream proteins
in Drosophila
SH2 containing GRB2
 Sos (GEF)
 Ras
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Introduction of mutant Ras proteins into sevmutant flies resulted in the development of R7
cells
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Protein domains of GRB2:
SH2, PTB: bind to phosphotyrosine residues
 SH3 (2) : bind to proline rich sequences
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Proline residues: extended conformation, fit to binding
pockets on SH3 domain
 Other residues determine binding-specificity
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Upon RTK activation, Sos is recruited to
membrane, near to its substrate, Ras.
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C-terminus of Sos inhibits its nucleotide exchange
activiyt; binding of GRB2 relieves this inhibition