Transcript BPS 502

BPS 502
Chapter 15
• Cell Communication
• Signaling through enzymelinked cell surface receptors
Reading Assignment
MBC, Chapter 15
Levitzki A. 2003, EGF Receptor as a Therapeutic Target.
Lung Cancer, 41: S9-S14.
Objectives
• Be able to describe and give examples of each of
the six classes of enzyme-linked receptors
discussed.
• Understand the importance of these signaling
pathways in disease states, especially in cancer.
• Be able to discuss the various means of inhibition
of these signaling pathways in clinical use and in
various phases of clinical trials.
•Understand and describe the signaling pathways
that depend on regulated proteolysis.
Enzyme-Linked Cell-Surface Receptors
• They respond to
extracellular signaling
proteins called growth
factors that promote
growth, proliferation,
differentiation or cell
survival
Enzyme-Linked Cell-Surface Receptors
• They act as local mediators at low concentrations
(10- 9–10-11 M).
•The response can be slow (several hours) due to the
number of intracellular steps that are required leading
to changes in gene expression.
•Effects may be direct and rapid acting on the
cytoskeleton to control cell movement and shape
changes.
•Abnormalities in these fundamental signaling events
can lead to cancer.
Six Classes of enzyme-linked receptors identified to date
1. Receptor tyrosine kinases – phosphorylate specific tyrosines on
specific intracellular signaling proteins. (EGFR)
2. Tyrosine-kinase-associated receptors – these enzymes associate
with intracellular proteins that have tyrosine kinase activity.
(Cytokine/Jak-Stat)
3. Receptor-like tyrosine phosphatases – remove phosphate groups
from tyrosines of specific intracellular proteins.
4. Receptor serine/threonine kinases – phosphorylate specific serines
or threonines on associated gene regulatory proteins. (TGF-b/Smad)
5. Receptor guanylyl cyclases – directly catalyze the production of
cyclic GMP in the cytosol. (Natriuretic peptides receptor)
6. Histidine-kinase-associated receptors- activate a 2-component
signaling pathway whereby the kinase phosphorylates itself on
histidine (autophosphorylation) and then immediately transfers the
phosphate to a second intracellular protein. Only occurs in yeasts and
plants involved in chemotaxis.
Like G-Protein Coupled Receptors, Enzyme-linked receptors are transmembrane
proteins with the ligand-binding domain on the outer surface and a cytosolic domain
that either has intrinsic enzymatic activity or associates with an enzyme.
1. Receptor Tyrosine Kinases
Activation of enzyme-linked receptors occurs by oligomerization of the
receptor. This causes reorientation of the receptor chains,
autophosphorylation, stimulating the kinase activity and creating docking
sites for intracellular proteins.
Three ways signaling proteins can cross-link receptors:
(A) PDGF is a dimer and cross-links two receptors together.
(B) FGF is a monomer and can bind in clusters to proteoglycans enabling
the ligands to cross-link the FGF receptor
(C) Ephrins are membrane-bound ligands and cluster prior to binding thus
causing receptor cross-linkng.
Phosphorylated Tyrosines Serve as Docking
Sites for Proteins with SH2 domains
Some examples:
Phospholipase C-g (enzyme)
Src (relay signaling protein)
PI-3’-kinase
All share a highly conserved
phosphotyrosine-binding domain,
either SH2 or SH3 domains (Src
homology region) or PTB
(phosphotyrosine bindng)
domains
These proteins act as adaptor
proteins to broadcast signals
along multiple pathways.
Hypothetical Signaling Pathway Using Modular Domains
Binding of SH2-Containing intracellular signaling
proteins to an activated PDGF receptor
X-ray crystal structure of an SH2 domain that acts as a
plug-in molecule recognizing phosphotyrosines and
adjacent amino acid side chains.
Ras proteins belong to the large Ras superfamily of
monomeric GTPases
-Ras proteins contain a covalently linked lipid group that anchors the protein
to the cytoplasmic face of the plasma membrane
-There are multiple Ras proteins, each acting in distinct cell types
-Ras functions as a switch, cycling between 2 conformational states
-mutations of the ras gene promote the development of cancer, 30% of
human tumors have a ras activation mutation.
Guanine nucleotide
exchange factor
(GEF) activates Ras
GTPase-activating
proteins (GAP) inactivates
Ras
Activation of Ras by an activated receptor tyrosine kinase
Grb-2 is an adaptor protein linking receptor tyrosine kinase to Ras via
the GEF called Sos. This is only one way of activating Ras.
Activation of ras is short-lived because it is inactivated by phosphatases and GAPS.
Once activated, Ras activates other signaling proteins to relay the signal
downstream along several pathways.
One of the pathways activated by Ras is the MAP Kinase Pathway
MAPK pathway consists of a kinase
cascade of no fewer than three enzymes:
Raf
MEK
MAP kinase (ERK) is
phosphorylated by MEK
on both a threonine and a
tyrosine. ERK then
travels to the nucleus
phosphorylating proteins
and activating immediate
early genes, G1 cyclins
are among them.
The Mitogen-activated Protein Kinase (MAPK) Cascades
SIGMA-ALDRICH
At least 5 parallel MAP kinase modules can operate in a mammalian cell. These
modules are activated by different kinds of cell stress such as UV irradiation, heat
shock, osmotic stress and stimulation by inflammatory cytokines.
The MAP kinase pathway
stimulates cell division
through the action of
mitogens. To prolliferate,
growth factors stimulate
proliferation. PI-3-kinase
activation mainly stimulates
cell growth. Ras can also
help to activate PI-3’-kinase.
Ras Effectors and Downstream Pathways
Macaluso et al., 2002, J Cell Physiol., 192:125-130
EGF Receptor Signal Transduction Pathway
SIGMA-ALDRICH
Strategy to prevent cross-talk between MAPK
Signaling Pathways:
Separate pathways are organized by scaffold proteins in budding yeast
and in mammalian cells
Phosphatidylinostitol 3-kinase (PI-3’-Kinase) Phosphorylates
Inositol Phospholipids Rather than Proteins
PI-3’-Kinase catalyzes the phosphorylation of inositol phospholipids
at the 3 position of the inositol ring
The 2 lipids in orange
can serve as docking
sites for signaling
proteins with Plecstrin
Homology (PH) domains
Inositol phospholipid
phosphatases (PTEN)
remove the 3 position
phosphate. PTEN is a tumor
suppressor gene, mutations
result in prolonged growth
signaling. These mutations
are found in various cancers
including endometrial, some
ovarian and some breast
cancers.
PI 3-K stimulates cell survival and growth by activating
protein kinase B (PKB or Akt)
One example of transmission
of the proliferation signal:
1.
2.
3.
4.
A survival signal activates a
RTK, with recruits and
activates PI 3’Kinase.
PI 3’K produces the
phosphorylated inositol
phospholipids which serve as
docking sites for 2
serine/threonine kinases PKB
and PDK1
PKB is then further
phosphorylated and activated
by PDK1.
Activated PKB dissociated
from the membrane, and
phosphorylates BAD,
releasing a death inhibitory
protein resulting in inhibition
of apoptosis.
The result is continued proliferation.
Five parallel intracellular signaling pathways activated
by G-protein-linked receptors, receptor tyrosine
kinases, or both
2. Tyrosine-Kinase-associated Receptors Depend
on Cytoplasmic Tyrosine Kinases for Their Activity
•These receptors lack intrinsic tyrosine kinase activity and
rely on cytoplasmic tyrosine kinases.
•The largest family of cytoplasmic TK receptors is the Src
family (Src, Yes, Fgr, Fyn, Lck, Lyn, Hck, Blk).
•These proteins contain SH2 and SH3 domains and are
located on the cytoplasmic side of the plasma membrane.
•Integrin receptors interact with focal adhesion kinase (FAK)
that is a cytoplasmic TK.
•Cytokine receptors are stably associated with a class of
cytoplasmic TKs called Jaks.
The Jak-STAT signaling pathway activated by a-interferon
Cytokines are secreted by cells in response to a viral infection and bind to
neighboring cells to induce proteins that increase resistance to virus. All
cytokine receptors are associated with one or more Jaks (4 types)
Janus kinases (Jaks)
Signal transducers and
activators of transcription
(STATs). 7 known STATs
3. Some Protein Tyrosine Phosphatases Act as
Cell-Surface Receptors
Protein tyrosine phosphatases (PTPs) remove selected
phosphotyrosines on a subset of tyrosine-phosphorylated
proteins. Exhibit high degree of substrate selectivity.
These enzyme ensure that the tyrosine phosphorylations
are short-lived and are responsible for regulating the
intensity of the signal.
There are about 30 known PTPs and occur as both
transmembrane and cytoplasmic forms.
Protein Tyrosine Phosphatases
The role of receptor-like
tyrosine phosphatases is not
yet clearly understood.
They are thought to act as
receptors, but their ligands
have not been identified.
Some have been shown to
display features of celladhesion.
Some can activate receptors
on neighboring cells.
4. Receptor Serine/Threonine Protein Kinase
Smad-dependent signaling pathway activated by TGF-b
Transforming growth factor b
(TGFb) consists of a large number
of structurally related, secreted,
dimeric proteins. They mediate a
wide range of biological functions in
animals: proliferation,
differentiation, ECM production, cell
death, tissue repair and immune
regulation.
They act through receptor
serine/threonine kinases type I and
type II
Smad family members are directly
phosphorylated by the type I
receptor, and moves to the nucleus
to direct gene transcription
The size and location of protein kinases
5. Receptor Guanylyl Cyclases
•Single pass transmembrane proteins with an extracellular
binding site for a signal molecule and intracellular guanylyl
cyclase catalytic domain.
•Binding of ligand activates the cyclase domain to produce
cyclic GMP, which in turn binds to and activates a cyclic
GMP-dependent protein kinase (PKG).
•PKG phosphorylates specific proteins on serine or
threonine.
•Some signaling molecules that use these receptor types
include natriuretic peptides (NPs) (regulate salt and water
balance and dilate blood vessels).
6. Histadine-Kinase Associated Receptors
Involved in Bacterial Chemotaxis
•Dimeric transmembrane proteins that bind specific attractants and
repellants
•The cytoplasmic tails of the receptors are stably associated with an
adapter protein CheW and a histidine kinase, CheA – couples the receptor
to the flagellar motor.
•Binding of a repellent activate CheA causing autophosphorylation on a
histidine, and immediate transfer of the phosphate to an aspartic acid on a
messenger protein, CheY.
•CheY dissociates from the receptor, diffuses through the cytosol, and
causes the flagellar motor to rotate clockwise and the bacterium tumbles.
•This type of signaling is used in yeast and plants, but not found in
animals.
What are the opportunities for therapeutic applications?
Modes of EGFR Inhibition
A. Levitzki. 2003. Lung Cancer, 41: S9-S14
Iressa/Gefitinib (EGFR Inhibitor)
• Used to treat non-small-cell lung cancer
(NSCLC) – 85% of all lung cancers
• Works in 25% of cases in Japan, but only
effective in 10% of U.S. patients.
• Suspected a genetic factor in efficacy
• Has variable effects based on a single gene
Science. 2004 Jun 4;304(5676):1458-61
EGFR Mutations in Lung Cancer:
Correlation with Clinical
Response to Gefitinib Therapy
J.Guillermo Paez,1,2 *Pasi A.Ja ¨nne,1,2 *Jeffrey C.Lee,1,3 *
Sean Tracy,1 Heidi Greulich,1,2 Stacey Gabriel,4 Paula Herman,1
Frederic J.Kaye,5 Neal Lindeman,6 Titus J.Boggon,1,3
Katsuhiko Naoki,1 Hidefumi Sasaki,7 Yoshitaka Fujii,7
Michael J.Eck,1,3 William R.Sellers,1,2,4 †
Bruce E.Johnson,1,2 † Matthew Meyerson 1,3,4 †
SCIENCE VOL 304 4 JUNE 2004
Convergence of Results With
Different Approaches
Lynch et al.:
• Sequenced the entire
coding region of the
EGFR from 9 patients
treated with gefitinib with
clinical response.
• 8/9 mutations found in
responders; 0/7 in
nonresponders
Paez et al.:
• Sequenced the exons of 47
TK activation loop domain
58 lung cancers from
Japanese patients and 61
from U.S. patients.
• Narrowed search to EGFR
in 119 cases of NSCLC
• Mutation found in 5/5
responders; 0/4 nonresponders
Both Studies Reveal Mutations in the EGRF Tyrosine Kinase Domain
-either small in frame deletions or amino acied substitutions
-Cluster around the ATP binding pocket
-activating mutations
Table 1. Protein kinase inhibitors in clinical trials
Genentech.com
Genentech.com
Data from Phase III clinical trials of Herceptin
Genentech.com
Table 2. Antibody protein kinase inhibitors in clinical trials
Overview of the PI3K/Akt Pathway
Compounds that Directly Inhibit the PI3K/Akt Pathway
Signaling Pathways that Depend on
Regulated Proteolysis:
-Notch
-Wnt
-NF-kB
The Notch Receptor Protein is Widely Used in
Animal Development
Notch genes encode heterodimeric transmembrane
receptors that regulate differentiation, proliferation and
apoptosis.
Mammals have 4 known Notch genes and 2 families of
Notch ligands: Delta and Jagged.
Both Notch and Delta are single-pass transmembrane
proteins that require proteolytic processing to function.
Well known for its role in nerve cell production in
Drosophila
Deregulated expression of Notch receptors, ligands and
downstream targets is described in many cancers
Activation of Notch by Proteolytic Cleavage
1. Full-length Notch is cleaved in the trans-golgi by a furin and is transported to the plasma
membrane. 2. Notch binds delta that is displayed on an adjacent cell triggers the next 2
proteolytic cleavages. 3. extracellular cleavage mediated by TACE leaving 12 amino acids.
4. Cleavage mediated by presenilin that releases the Notch tail to migrate to the nucleus.
Notch binds to the transcriptional repressor
CSL and activates gene transcription.
Wnt Proteins Bind to Frizzled Receptors and Inhibits b-Catenin
Degradation
Wnt proteins are secreted
glycoproteins that act as mediators to
control many aspects of development.
Wnt binds to the Frizzled receptor
(family of 7 transmembrane proteins
resembling G-proteins.
Signaling is mediated by the
cytoplasmic protein dishevelled.
Dishevelled acts by regulating the
proteolysis of a multifunctional protein,
b-catenin, which functions in cell-cell
adhesion and as a latent gene
regulatory protein.
In the absence of a wnt signal, bcatenin is phosphorylated in a
degradation complex and is degraded
in the proteosome.
In the presence of Wnt, in
association with a co-receptor
protein LRP and leads to inhibition of
b-catenin phosphorylation and
degradation.
As a result, the unphosphorylated bcatenin accumulates in the nucleus
and displaces the repressor protein
Groucho.
Wnt target genes are expressed
including c-myc , a powerful
stimulator of cell growth and
proliferation
APC (adenamatous polyposis coli)
gene mutations occur in 80% of
benign colon polyps that lead to
cancer.
Wnt ligand overexpression is
documented in many tumors
including breast.
Multiple Stress and Proinflammatory Stimuli Act
Through an NF-kB-Dependent Signaling Pathway
Two cytokines that are important in inducing
the inflammatory response are TNF-a and
interleukin-1 (IL-1). These cytokines bind to
cell-surface receptors and activate NF-kB.
There are 5 NF-kB proteins in mammals
(RelA, RelB, c-Rel, NF-kB1, NF-kB2). These
form a variety of homo- and heterodimers
each activating a specific set of genes.
Inhibitory proteins IkB, bind tightly to the
dimers and hold them in an inactive state.
TNF-a signals lead to recruitment of several
intracellular signaling proteins (RIP, TRADD,
TRAF2) that in turn activate a specific
serine/threonine kinase, IkB kinase. This
releases the IkB exposing the nuclear
localization signal of NF-kB and it
translocates to the nucleus.