Signaling by Tyrosine Phosphorylation
in the
Nervous System
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
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.)
Receptor tyrosine kinases
• Introduction:
– Protein phosphorylation
– Recruitment of kinases in signalling pathways
– Consequences of protein phosphorylation
• RTK family:
– Classification and structure/function
– RTK ligands
– Receptor dimerization and
autotransphosphorylation
RTK family classification and
structure/function
• Four common structural features shared
among RTKs:
– Extracellular ligand-binding domain
– Single transmembrane domain
– Cytoplasmic tyrosine kinase domain(s)
– Regulatory domains
Seven subfamilies of receptor tyrosine kinases
RTK family classification and
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
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
RTK structure/function
Regulatory
domains
RTK Ligands
•
•
•
•
Typically small soluble proteins
Work in autocrine and paracrine manner
Dimerize (may aid in receptor dimerization)
Some RTK ligands membrane-bound
RTK Autotransphosphorylation
Receptor Dimerization and
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.)
Src homology (SH)2 and SH3 domains
Supposed Purpose:
Joins, combines, targets kinases and
phosphatases (see below) with activated
(ligand-bound) growth factor receptors.
SH2 and SH3 refer to domains that bind specific
peptides containing a phosphorylated tyr or
pro-rich sequence, respectively.
Src homology (SH)2 and SH3 domains
• Tyr phosphorylation allows recruitment of proteins
that possess domains that bind to specific peptide
sequences that encompass a phosphorylated tyr
and which  activating a variety of signalling
pathways.
• Such domains include SH2 and SH3 and P-tyr
binding (PTB) domains.
• These domains bind to the GF receptor only when
it is phosphorylated on tyr residues.
• SH2 domains: bind P-Tyr-containing sequences.
• SH3 domains: bind to pro-rich (PxxP) sequences.
• The proteins that contain SH2 domains belong to
several categories: PLCγ, PI-3K, Grb2, SHP-2, Src
(next slide):
SH2 and SH3 domains
RTK-mediated pathways: one pathway
with two very different functions
• Ras-Raf-MAP kinase pathway.
• R7 photoreceptor development in
Drosophila.
• RTK Signaling: Ras Pathway
The regulation of Ras activity, a famous
downstream molecule of RTK responsible for
cancer development
Three ways in which signaling proteins can cross-link
receptor chains
1. dimer, 2. monomer but brought together by
proteoglycan, 3. cluster on membrane
The importance of receptor
oligomerization
The docking of signaling molecules at RTK
The activation of Ras by RTK signaling
The MAP-kinase regulated by Ras
The Ras-Raf-MAP kinase pathway
SH2 domain
SH3 domains
Proline-rich regions (-PXXP-)
GDP
Tyr-P
GTP
Raf
Grb2
SOS
(inactive)
Nucleus
P
jun
Ras
Pi
(active)
P
MEK
P
fos
Ras
P P
P P
MAP kinase
Increase gene expression
DNA
MAP kinase
Use of oncogenic and dominant
negative mutants to map pathways
• Oncogenic Ras (V12Ras): defective GTPase
function. Always turned “on” (always GTPbound)
• 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.
Dominant negative Ras (N17Ras) sequesters SOS
and blocks pathway from Ras on down
SH2 domain
SH3 domains
Proline-rich regions (-PXXP-)
GDP
Tyr-P
GDP
Raf
Grb2
Sos
Ras
(inactive)
Nucleus
fos
N17Ras
jun
gene expression blocked
MEK
DNA
MAP kinase
Combine oncogenic and DN mutants to map
position of pathway components
SH2 domain
SH3 domains
Proline-rich regions (-PXXP-)
GDP
Tyr-P
Grb2
Sos
GDP
Ras
N17Ras
Raf
(inactive)
Nucleus
P
P
Oncogenic
Raf
P
fos
jun
MEK
P P
P P
MAP kinase
Increased gene expression
DNA
MAP kinase
R7 photoreceptor development
• Fruitfly (Drosophila melanogaster)
• Compound eye (800 ommatidia)
• Each ommatidium has 8 photoreceptor cells;
each detects a different wavelength of light
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 and ending with R7
(responds to ultraviolet light)
The R7 Photoreceptor Developmental Pathway
is a RTK-MAP Kinase Cascade
• RTK Signaling:
• PI 3-Kinase Pathway
The inositol phospholipids generated by PI3K
The recruitment of
signaling molecules with
PH domains to the plasma
membrane during B cell
activation
One PI3K pathway
PH domain: pleckstrin
homology domain
Another PI3K pathway to regulate cell survival
Intracellular Signaling Pathways activated by RTKs and GPCRs
RTKs – Some Additional Important Points
• See next slide:
• Low-abundance proteins, their activation exerts major
effects due to simultaneous activation of several
signaling pathways that are often synergistic and 
enhanced survival and growth.
• This is an important function of growth factors when
they are located on dendritic spines or shafts or on
nerve terminals.
• Ser/Thr kinases can alter gene expression.
• RTKs can/must be regulated (attenuated) – if not,
perhaps because of some mutation, such signaling
intermediates escape such control  cancer.
• Include mechanisms, such as desensitization,
degradation, and dephosphorylation of tyrs.
RTK Activation Lead to the Activation of Several Ser/Thr
Kinases with a wide Variety of Substrates:
CREB – end-point of several signaling pathways
Non-receptor Tyrosine Kinases
• Heterogeneous group of enzymes that share a
common conserved tyr cat domain and a lack
of extracellular ligand-bindiing domain.
• At least 32 known non-receptor tyr kinases in
humans distributed into 10 families.
• Diverse functions
• JAK/STAT-Cytokine receptors complex
resemble RTKs in their ability to transduce
signals in response to direct activation by
extracellular signals/ligands.
• Cytokine receptors are a receptor for CNTF
and for leptin.
Examples of Non-receptor Tyr Kinases
• Conserved catalytic domains – black
• Note that in JAKs, only the C-term tyr kinase
domain is catalytically active.
Activation Mechanism of a Cytokine Receptor coupled to JAK tyr Kinases and of STAT
Cytokine, hormone…
Receptor
Out
In
JAK tyr
kinase
P P
STAT
P P
STAT
Nucleus
P
P
STAT
STAT
STAT
P P
P
STAT
P
P
STAT
STAT
P
Transcription
STAT-regulated genes
Activation of c-Src
Myristic acid at N-term allows
Covalent attachment to
membrane
Myristoylation enriches Src
kinases in membrane rafts
Two modes of intrinsic inhibition
by interactions between:
(1) SH2 domain and
phosphorylated Y527;
(2) SH3 domain and
Polyproline region.
Regulation of Src Family of Kinases
• These kinases are attached to the membrane through an N-terminal
myristic acid.
• Maintained in an inactive state by intramolecular interactions that
can be alleviated as indicated.
• Note the unusual situation in which a tyr phosphatase can activate
a tyr phosphorylation pathway.
• Another way of activating Src is displacement of its SH2 domain
from the C-terminal phos’d tyr by a competing phosphopeptide.
Protein Tyrosine Phosphatases (PTPs)
• Overall, the various types of PTPs are rather
dissimilar (lacking sequence homology).
• 2 Types: Receptor-like (RPTP)s and Non-receptorlike PTPs.
• Among the RPTPs, there is a highly conserved cys
residue in the conserved catalytic domains (some
PTPs have 2 catalytic domains, although the Cterm one has little-to-no catalytic activity).
• Although PTPs tend to oppose tyr kinase
signalling, in some cases, they can activate
specific tyr kinases (above, Src).
Inactivation of MAP kinases (ERK)
by threonine or tyrosine dephosphorylation
Role of Protein Tyr Phosphorylation During
Development of the Nervous System
• Growth factors signaling.
• Synaptogenesis.
- NMJ – MusK (a tyr kinase, see preceding
slide) and agrin  AchR clustering.
• Eph receptors (see slide 6) and their ligands,
ephrins, participate in bidirectional signaling
between cells in the travelling growth cone.
Role of Tyr Phosphorylation in the
Regulation of Ion Channels and Receptors
NMDA
R
NMDA
R
P Tyr
P-Tyr
EphB2
Ca2+
PYK2/Cakβ
PKC
Src
TrkB ?
Role of Tyr Phosphorylation in Synaptic
Plasticity
• LTP.
• Synaptogenesis: Ephrins and their receptors: In
hipp cultures, Eph2 interacts with syndecan-2
(cell surface glycoprotein) to induce dendritic
spine formation assoc with NMDAR clustering
and synapse formation.
EphB activation increases NMDAR tyr
phosphorylation and glu-induced Ca2+ currents
through phosphorylation by Src kinases.
However, EphB2-/- appear/act normally
underscoring the redundancy of multiple phos
pathways in plasticity and development.
Role of Tyr Phosphorylation and
Phosphatases in Nervous System Diseases
• Refinement of PTP chromosomal positions
allows for genetic disease linkage studies:
• 19 PTP chromosomal regions are
frequently deleted in human cancers.
• 3 PTP chromosomal regions are
frequently duplicated in human cancers.
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PTEN
Tumor Suppressor
Mutated in various human cancers. Cowden
disease
Tyr kinase
Tau phosphorylation
Alzheimer’s
Fyn kinase Required for normal myelin formation.
DEP1
Tumor suppressor
Colon cancer susceptibility locus Scc1
(QTL in mice)
PTPk
Tumor Suppressor
Primary CNS lymphomas
SHP2
Noonan Syndrome
Developmental disorder affecting 1:2500
newborn
Stomach Ulcers
Target of Helicobacter pylori
•
Cdc25
Cell Cycle Control
primary breast cancer
•
PRL-3
Metastasis
•
FAP-1
Apoptosis
Upregulated in cancers, inhibits CD95mediated apoptosis
• Src
Stroke
Target of Myc and overexpressed in
Upregulated in metastases of colon cancer
Larger infarct size in mice for Src -/-