Transcript Receptor Tyrosine Kinases

Enzyme-linked Cell Surface
Receptors
30 April 2007
Non-Receptor Tyrosine Kinases
• NRTK’s associate with membrane receptors or multiprotein
complexes which regulate their activity.
• Activation involves both conformational changes and tyrosine
phosphorylation of activation loop residues by heterologous
kinases or autophosphorylation.
• NRTK’s contain domains that mediate binding to proteins,
lipids, or DNA.
– proteins: SH2, SH3, FERM
– lipids: Pleckstrin homology (PH)
– DNA: example, Abl
Nonreceptor tyrosine kinases
• This kind of receptors lack intrinsic enzymatic activity.
Instead they are non- covalently associated with
intracellular protein tyrosine kinases.
• N-terminal extracellular ligand binding domain
• single transmembrane  helix
• cytosolic C-terminal without tyrosine kinase
activity
Non-Receptor (Cytoplasmic) Protein Tyrosine Kinases
From Hunter (2001) Nature 411,355.
Signalling Pathways Involving src Kinases
• Broadly expressed: fyn, c-src, c-yes, and yrk
• hematopoietic lineages: blk, c-fgr, hck, lck, and lyn.
• C-src associates with receptor and non-receptor
tyrosine kinases via its SH2 domain:
– PDGFR, EGFR, IGF-1R, FAK, CSF-1 etc.
• C-src associates with transmembrane proteins that
are not kinases:
– interleukin receptors, T and B cell receptors
Receptor and Non-receptor tyrosine
(cytokine) kinases
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For both receptor types,
dimerization brings two
intrinsic kinases, which
then phosphorylate each
other on a Tyr residue in
the activation lip
Catalytic site is then
exposed to ATP or protein
substrate
P* of other Tyr residues,
which become docking
sites for signaling proteins
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SH2, PTB domains
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Some cytokine receptors
(IL-4R) and some RTKs
bind multidocking
proteins (i.e IRS1) via PTB
domain
P* of docking protein
recruits SH2-domain
containing signaling
proteins
These proteins too are P*
by activated receptor
Cytokines control many aspects of
cell growth and differentiation:
Prolactin: differentiation of ductular epithelial
cells into milk secreting acinar cells
 IL-2: T Cell proliferation
 IL-4: B Cell antibody production
 Interferon-: resistance to viral infection
 Differentiation of blood cells
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G-CSF, Thrombopoietin
Erythropoietin: proliferation and
differentiation of erythroid progenitor
cells
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Progenitors cells are
saved from death, each
generating more than
50 red blood cells
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All cytokines are
structurally similar:
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All cytokine receptors are
also similar
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Four  helices
Two subdomains, each
consisting of seven ß
strands
Both cytokines and their
receptors are thougt to be
derived from a common
ancestor
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All cytokine receptors activate similar signaling pathway;
BUT different cellular responses arise depending on the
TFs sets, chromatin structures
For instance, prolactin expression in erythroid progenitors
results in cell division and differentiation but not milk
secretion
JAK-STAT Signaling Pathway
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Cytoplasmic tyrosine kinases: Jaks (Janus activated
kinases)
Latent gene regulatory protein: STAT(Signal
Transducer and activator of transcription)
Ligand receptor interaction leads to phosphorylation of
Jaks.
Jaks, then phosphorylate and activate STATs, which are
normally inactive located on the plasma membrane.
Activated STATs then migrate to nucleus and activate
gene transcription.
SIGNALING
RECEPTOR-ASSOCIATED STATS ACTIVATED SOME RESPONSES
LIGAND
JAKS
g -interferon
Jak1 and Jak2
STAT1
a -interferon
Tyk2 and Jak2
STAT1 and STAT2 increases cell resistance
to viral infection
Erythropoietin
Prolactin
Jak2
Jak1 and Jak2
activates macrophages;
increases MHC protein
STAT5
stimulates production of
erythrocytes
STAT5
stimulates milk
production
Growth hormone Jak2
STAT1 and STAT5
stimulates growth by
inducing IGF-1 production
GM-CSF
Jak2
STAT5
stimulates production of
granulocytes and
macrophages
IL-3
Jak2
STAT5
stimulates early blood cell
production
JAK-STAT Signalling Pathway
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Activated JAKs P* Tyr
residues on the receptor,
which then become
docking sites for STATs
STATs contains:
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N-terminal SH2 domain,
which binds to Tyr-P* on
the receptor
Central DNA binding
domain
C-terminal Tyr residue,
which is P* by JAK
JAK-STAT activated by alfa interferon
Modulation of Signaling
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As in other signaling pathways, cells must
turn off signals generated by JAK-STAT
pathway
Two classes of proteins dampen signaling
One over the short term (minutes)
 The other over longer time
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SHP1 phosphatase: short term
modulation
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Removes P* from a
particular P*-Tyr
residue on JAK and
inactivates it, unless
another cytokine binds
to cell surface receptor
Long term regulation: SOCS (CIS)
proteins
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Their transcription is
induced by STATs
Act in two ways:
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1. SH2 domains in several
SOCSs bind to P*-Tyr on
receptor and prevent the
binding of other signaling
proteins. SOCS-1 binds to
P*-Tyr on the activation lip
of JAK
2. All SOCSs contain a
SOCS box domain that can
recruit E3 ubiquitn ligases
Protein Tyrosine Phosphatases (PTPs)
Protein Tyrosine Kinase
Substrate + ATP
Substrate-P + ADP
Protein Tyrosine Phosphatase
(PTP)
Protein Tyrosine Phosphatases (PTPs)
Receptor-like or Transmembrane PTPs
 CD45
 PTP
 LAR
Non-receptor or Cytoplasmic PTPs
 PTP1B
 SHP1
 SHP2
Regulation of PTP Activity
Mechanism
Effect
Example
Regulated expression
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DEP-1, LAR, PTP1B etc
Tyrosine phosphorylation
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SHP1,SHP2, PTP1B
Association with substrates 
SHP1, SHP2
via SH2 domains
Dimerization
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PTP, CD45
Oxidation of essential Cys
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PTP1B
with H2O2 or ROS
Association with cell matrix 
PTP(DEP-1)
Ligand interactions
? LAR, PTP
Selected Functions of PTPs
• The obvious - dephosphorylation of phosphotyrosine
residues
• counter-regulates TK-dependent reactions
• suppresses growth factor, cytokine, integrin receptor
pathways
• essential for mitogenic effects of growth factor
receptors (eg.PDGFR, EGFR)
• tumor suppressors
TGF-ß Pathway
Overview
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Play widespread roles in the development
Bone morhogenetic protein (BMP7) induce
bone formation in cultured cells
Many others BMP contribute to the formation
of mesoderm and earliest blood-forming cells
TGFß1 stimulates the transformation of some
cells in culture
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Other isoforms of TGFß have antiproliferative
effects on mammalian cells
Loss of TGFß receptors, thereby induce tumor
formation by releasing inhibitory pressure of these
isoforms
BUT, TGFß proteins also triggers the secretion of
GFs from cells, counterbalancing their inhibitory
effect
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Drosophila homolog dpp controls dorso-ventral
patterning
Other members activin and inhibin regulate
early development of genital tract
Despite this diversity, the signaling pathway by
TGFß superfamily proteins is simple
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Direct P* and activation of transcription factors
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In humans, there are 3 TGFß isoforms
TGFß1, TGFß2, TGFß3
 Each encoded by unique gene and expressed in
tissue specific fashion
 Synthesized as a large precursor with a prodomain
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Although cleaved, this prodomain remains
associated non-covalently with TGFs after
secretion
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TGFß is stored in the ECM as an inactive
complex containing the cleaved prodomain and
Latent TGFß Binding Protein (LTBP)
Binding of LTBP by thrombospondin or
integrins affects its conformation and release
matuer dimeric TGFß
Another way is the digestion of LTBP by matrix
metalloproteases.
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Intrachain S-S bonds render monomeric TGFß
resistant to denaturation
Homo-, heterodimer formation occurs via S-S
bonds between N-terminal Cys residues on both
monomers
Sequence variation among TGFß isoforms is
observed in the N-terminal region
Ser-Thr Kinase Activity of TGFß
Receptors
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Receptors were identified by 125I-labelled TGFß
Three receptors:
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Rı, RII and RIII having MW 55, 85 and 280 kD,
respectively
Most abundant RIII is a proteoglycan also
named ß-glycan
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Binds and concentrates TGFß near the cell surface
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Type I and type II are dimeric receptors with
cytosolic Ser-Thr kinase activity
RII is constitutively active and can P* itself
Upon TGFß binding, a complex consisting two
copies each of RI and RII forms
RII then P* RI cytoplasmic subunit, activating
its kinase activity
Activated RI P* Smad TFs
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Three types of Smad proteins
Receptor-regulated Smads (R-Smads)
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Co-Smads
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Smad2, Smad3
Smad4
Inhibitory Smads (I-Smads)
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Smad7
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All mammalian cells secrete one TGFß isoform
and most express TGFß-R
Why are cellular responses different?
Different cells have different sets of TFs.
Response diversity is also generated by binding
of different TGFß isoforms to their related
receptors and thereby activating different Smad
proteins
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i.e. BMP bind to a different receptor, activating
Smad1
I-Smads and Negative Feedback
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SnoN and Ski regulate Smad signaling
They relieve growth-inhibitory effects of TGFß
signaling
They don’t affect DNA binding of Smad complexes,
but rather they block the transcription activation effect
of Smads
The expression of SnoN, Ski, as well as Smad-I are
induced by TGFß stimulation
SMAD 7 (I-Smad) blocks the P of R-Smads by RI
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Inactivation of TGFß receptors and Smad
proteins is a common event in human cancer
Smad4 mutation in pancreatic cancer
Abrogated TGFß pathway is unable to induce
transcription of growth-inhibitory genes such as
p15 and myc