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Signal transduction for mathematicians:
examples from the JAK-STAT
and Ras-Raf-Mek-Erk pathways.
Terry Speed, WEHI
BioInfoSummer 2006
1
Background and motivation
In multicellular organisms, cells must communicate with
each other and work together. Messages from outside
cells have to be conveyed to headquarters - the nucleus in order to be acted upon through transcription. This talk is
about that: intracellular signalling.
This subject is important for cancer, immunology and
development, and so of interest in biology generally.
(Herceptin is a humanized monoclonal antibody to ErbB2,
which in some breast cancers has a mutation which makes
its tyrosine kinase is constitutively active.)
This is often seen as an area where systems biology may
contribute, and that’s one reason why I’ve become involved
in it. Our examples are active in most mammalian tissues. 2
Intracellular signalling
(aka signal transduction)
These are the processes by which a cell converts one
kind of signal or stimulus to another. They often involve a
sequence of biochemical reactions inside the cell, which
are carried out by enzymes and linked through second
messengers (particular molecules).
Such processes take place in as little time as a
millisecond or as long as a few seconds. Slower
processes are rarely referred to as signal transduction.
(Wikipedia)
3
JAK-STAT signalling pathway
http://www.genome.ad.jp/kegg/pathway/hsa/hsa04630.html
4
MAPK signalling pathway
http://www.genome.ad.jp/dbget-bin/show_pathway?hsa04010+5594
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MAPK signalling pathway (blow-up of a bit)
http://www.genome.ad.jp/dbget-bin/show_pathway?hsa04010+5594
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Cartoon indicating some steps and players
JAK
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Figure 5.1 The Biology of Cancer (© Garland Science 2007)
My plan in these two talks
In the first, to introduce the key notions and to some extent
explain the nature of the Jak-Stat and Ras-Raf-Mek-Erk
pathways, as background to the topic of computational
models for them and similar pathways,
and then
In the second lecture, to describe some of the computational
modelling involving these pathways with which I’ve been
involved.
8
Growth factor
a protein that is able to stimulate the growth and/or
proliferation of a cell by binding to a cell surface receptor
displayed by that cell.
Examples include the Epidermal Growth Factor (EGF),
Platelet Derived Growth Factor (PDGF), the Fibroblast
Growth Factor (FGF), the Granulocyte-colony stimulating
factor (G-CSF), and the Granulocyte-macrophage colony
stimulating factor (GM-CSF).
9
Cytokines
are secreted proteins that regulate a variety of biological
functions by associating with transmembrane receptors at the
cell’s surface to activate signal transduction pathways.
Examples include the
Interleukins: literally, between leukocytes (white blood cells);
abbreviated IL-, e.g. IL1, etc; and the
Interferons, proteins found to interfere with reproduction of
viruses in cells, abbreviated IFN-, e.g. IFNγ, IFNα.
Another famous cytokine is erythropoietin (EPO).
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Receptors
A receptor is a protein found on the plasma membrane or
within a cell that is capable of specifically binding a signaling
molecule (its ligand). Most types of receptors emit signals,
such as those inducing cell proliferation in response to such
binding.
Examples include the estrogen receptor (ER), the PDGF
receptor (PDGF-R) and the Epidermal Growth Factor
receptor (EGF-R),
The EGF-R family of receptors consists of four distinct
proteins ErbB1 (EGF-R), ErbB2 (HER2, Neu), ErbB3 (HER3),
and ErbB4 (HER4). They often bind ligands as heterodimeric
receptors.
11
Phosphorylation
is the covalent attachment of a
phosphate group (PO3) to a
substrate, usually a protein. This
typically results in a functional
change in the target protein by
changing enzyme activity, cellular
location or interaction with other
proteins.
A protein can be phosphorylated at
a number of residues including
serine (S), threonine (T) and
tyrosine (Y). Many proteins have Phosphorylated serine
multiple phosphorylation sites.
12
Kinases
Are enzymes that covalently attaches phosphate groups
to substrate molecules, often proteins, i.e. phosphorylate
the substrate. Up to 30% of proteins may be modified by
kinases, and the human genome contains about 500
kinases.
Kinases act by removing a phosphate group (PO3) from
ATP and covalently attaching it to one of the 3 amino
acids (S, T, Y) that have a free hydroxyl group.
Examples include the JAKs (see later), RAF (see later),
AKT, ERK (also called MAPK, see later).
Receptor tyrosine kinases (RTKs) have a
transmembrane receptor with a tyrosine kinase domain
protruding into the cytoplasm. Example: the EGFR.
13
Phosphatases
enzymes that catalyse the removal of phosphate groups
from phosphorylated substrates. More than 100
phosphotyrosine phosphatases have been found in the
human genome, which remove the phosphate groups of
the 90 tyrosine kinases present in our cells.
Examples include alkaline phosphatase, PTEN, PP1,
PP2A and the SHPs.
14
Relevance of the foregoing to
intracellular signalling
The presence or absence of the phosphate group on
proteins, especially enzymes, is known to play a
regulatory role in many biochemical and signal
transduction pathways.
Hence together, specialized kinases and phosphatases
regulate enzymatic activity.
15
SH2 domains
Src (“sarc”) Homology 2 (SH2) domains are protein modules of
about 100 amino acids in size which are found in a large number
of proteins involved in signal transduction. The function of SH2
domains is to specifically recognize the phosphorylated state of
tyrosine residues, thereby allowing SH2 domain-containing
proteins to localize to tyrosine-phosphorylated sites.
It was established several years ago that specificity is conferred
by the sequence context of the phosphotyrosine within the
tyrosine-phosphorylated site, more specifically, by the three
residues immediately C-terminal to the phosphotyrosine.
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Attraction of signal-transducing proteins
by phosphorylated receptors
A constellation of
phosphotyrosines
can be formed
after the EGFR
binds to its ligand.
Listed to the right
are the different
cytoplasmic
proteins that can
bind to these via
their SH2 domain.
17
Figure 6.9b The Biology of Cancer (© Garland Science 2007)
Receptor dimerization & transphosphorylation
following ligand binding (EGFR works this way)
18
Figure 5.15 The Biology of Cancer (© Garland Science 2007)
SH3 domains
Src homology 3 domains bind specifically to certain
proline-rich sequence domains in partner proteins.
These proline-rich sequences thus serve as the ligands
of the SH3 domains.
An example of a protein with SH3 domains is the Growth
factor receptor binding protein 2 (Grb2 “grab two”), which
has two.
19
Adaptors
As we will see later, certain proteins play a key role in
signalling pathways by interacting with phosphorylated
receptors or derived molecules. The do so by forming
physical bridges between growth factor receptors and
other key molecules, e.g Son of sevenless (Sos, see
later).
Such proteins are known as adaptors. Examples include the
Src homology and collagen domain protein (Shc, “shick”)
and Grb2 . Both contain SH2 domains, and as just noted,
Grb2 contains two SH3 domains.
20
Transcription factors
Proteins that are involved in regulating the transcription of
genes, often by associating with sequences in the
promoter region of the gene. Examples below include
AP1, SP1, STAT1, MYC, JUN, FOS, ERG.
These are the proteins that make things happen (growth,
differentiation, migration, death, etc) though there are
several other ways in which gene expression is regulated.
21
Quick general review before we turn to specifics
JAK
22
Figure 5.1 The Biology of Cancer (© Garland Science 2007)
The JAK-STAT pathway
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The main players
Initially JAK meant “Just Another Kinase” but these days the
more sedate name “JAnus-like (tyrosine) Kinases” is given
as the explanation of the acronym. There are 4 Jak genes:
Jak1, Jak2, Jak3, Tyk2. They associate non-covalently with
certain receptors, including those for interferon (IFN),
erythropoietin (EPO) and thrombopoietin (TPO).
STAT abbreviates: Signal Transduction and Activator of
Transcription. These are latent transcription factors with
SH2 domains that are activated by receptor associated
JAK-mediated phosphorylation. The family includes STAT1,
STAT2, and STAT 3 (and several others).
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The JAK-STAT pathway
25
Figure 6.22 The Biology of Cancer (© Garland Science 2007)
Caption to preceding figure
Activation of signalling occurs when a cytokine binds to its
receptor at the cell surface, leading to dimerisation of the
receptor and activation of receptor-associated JAKs. Activated
JAK proteins transphosphorylate one another as well as the
C-terminal tails of the receptor. The resulting phosphotyrosines
attract STAT proteins, which bind via their SH2 domains and
become phosphorylated by the JAKs. Once phosphorylated,
STAT proteins can dimerise, each using its SH2 domain to bind
to the phosphotyrosine of its partner, and, upon transport into
the nucleus, operate as transcription factors to induce the
transcription of many cytokine-regulated genes.
Here there are no adaptors and no signalling cascades.
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Some other players
SOCS: suppressor of cytokine signalling; SOCS1, SOCS2, …
PIAS: protein inhibitors of activated STATs; PIAS1, etc
SUMO: small ubiquitin-like modifier
SHPs = SH2 containing phosphatases
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Beyond transcription: negative feedback
A)
B)
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Caption to right half of preceding figure
JAK-STAT signal transduction is repressed by three distinct
mechanisms.
SHP-1 is constitutively expressed, and can dephosphorylate
activated JAKs or receptors.
PIAS proteins, also constitutively expressed, ligate SUMOs
to STATs, thus inhibiting transcriptional activation.
SOCS proteins are induced in response to cytokine
signalling, and can inhibit JAK activity, or target signalling
components for ubiquitination and subsequent proteolysis.
29
Interferons and their receptors
β
τ
Type II
γ γ
ω
IFNγ-R1
IFNγ-R2
IFNα-R1
IFNα-R2
α
Type I
δ ε κ
ISGF-3
STAT1
STAT2
STAT1 P
STAT2 P
IRF-9
STAT1
STAT1 P
30
JAK-STAT signalling pathway
http://www.genome.ad.jp/kegg/pathway/hsa/hsa04630.html
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The Ras-Raf-Mek-Erk pathway
32
RAS
Ras is the name of a protein, the gene that encodes it, and
the family and superfamily of proteins to which it belongs.
Proteins in the Ras family are very important molecular
switches for a wide variety of signalling pathways.
Ras is a G-protein: a regulatory GTP hydrolase that cycles
between two conformations - an activated or inactivated form,
RAS-GTP and RAS-GDP. It is activated by guanine
exchange factors (GEFs, e.g. Sos), which are themselves
activated by mitogenic signals and through feedback from
Ras itself. It is inactivated by GTPase-activating proteins
(GAPs, the most common being RasGAP), which increase
the rate of GTP hydrolysis, returning RAS to its GDP-bound
form.
Ras is attached to the cell membrane by prenylation.
33
The Ras signalling sycle
34
Figure 5.30 The Biology of Cancer (© Garland Science 2007)
Sos
As just noted, Sos is a guanine nucleotide exchange
protein capable of activating Ras. Important to our story is
that it contains two distinct proline rich regions which can
bind to an SH3 domain.
Grb2 has two such domains, and serves as an adaptor
linking phosphorylated EGFR to Ras as in the next figure.
35
How the Receptor-Sos-Ras cascade operates
36
Figure 6.12 The Biology of Cancer (© Garland Science 2007)
RAF
(also called c-RAF or Raf-1) is a protein kinase functioning in
the MAPK/ERK pathway.
RAF activation is a multi-step process that involves the
dephosphorylation of inhibitory sites by Protein Phosphatase
2A (PP2A) as well as the phosphorylation of activating sites
by PAK (p21rac/cdc42-activated kinase), Src-family and yet
unknown kinases.
We assume RAF phosphorylation is caused directly by a free
Ras-GTP molecule.
Activated RAF has an intrinsic kinase activity and it
phosphorylates the MAPK/ERK Kinase, MEK.
37
MEK
MAPK/ERK Kinase (MEK) is another mitogen activated
protein kinase in the EGFR-ERK pathway. In its doubly
phosphorlyated form, it can phosphorylate ERK.
The requirement of double phosphorylation here and with
ERK seems to be a device which permits activation to be
switch-like.
Although RAF is the predominant MEK activator, MEK
can also be phosphorylated by other kinases, including
MEKK. The interaction between Raf-1 and MEK is also
regulated by RKIP (Raf kinase inhibitor protein), a protein
that binds to Raf and MEK preventing their interaction.
38
ERK (extracellular signal-regulated kinase)
= MAPK (mitogen-activated kinase)
As just noted, doubly phosphorylated MEK phosphorylates ERK.
In turn, ERK needs to be doubly phosphorylated to be activated.
Activated ERK has many substrates in the cytosol, e.g.
cytoskeletal proteins, phospholipase A2, and signalling proteins
including tyrosine kinase receptors, estrogen receptors, Sos,
STATs and others.
ERK can also enter the nucleus to control gene expression by
phosphorylating transcription factors such as Elk-1 and other
Ets-family proteins, c-myc, c-Fos and c-Jun, the last two
dimerising to form AP-1 (activation protein 1).
There is a negative feedback loop from doubly phosphorylated
ERK to Sos which causes the dissociation of Grb2-Sos from
39
the receptor complex.
Simplified view of the pathway
W. Kolch et al. / FEBS Letters 579 (2005) 1891–1895
40
Present animation here
http://www.bio.davidson.edu/courses/Immunology/Flash/MAPK.html
41
MAPK signalling pathway
http://www.genome.ad.jp/dbget-bin/show_pathway?hsa04010+5594
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MAPK Pathway II
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MAPK Pathway III
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MAPK Pathway IV
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MAPK Pathway V
Nat Rev Mol Cell Biol, 2005
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Cautionary notes (after RA Weinberg)
1. Although
already complicated, this was the simplified view.
Omitting details helps initially, but details usually matter. E.g.
many of the players in this pathway have more than one
alternative form (isozymes), and many are part of small
families each slightly different from the others (splice variants).
“In most cases. we are ignorant of the functional differences
between the superficially similar proteins operating…”
2. Complex positive- and negative-feedback loops serve to
amplify or dampen the signals fluxing through each of these
pathways. Each component is a complex signal processing
device that amplifies, attenuates and/or integrates the signals
it receives from upstream in a pathway before passing them
on to downstream targets. What is known is likely to be the tip
of a much larger iceberg.
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Cautionary notes (cont.)
3. The ability of proteins to transduce signals to one another
is affected by their intracellular concentrations,
postranslational modifications (such as phosphorylation)
and intracellular localization. We have only begun to plumb
the depths of this complexity.
4. None of these intracellular pathways operates in isolation.
Instead, each is influenced by cross connections with other
pathways, see next slide.
5. Even if these extensive cross connections did not exist, the
endpoints of signaling - specific changes in cell phenotype
- are the results of combinatorial interactions between
multiple converging signalling pathways. The dimension of
this complexity is baffling.
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Two-dimensional signaling maps
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Figure 6.32 The Biology of Cancer (© Garland Science 2007)
Cautionary notes (end)
6. Our depiction of how signals are transmitted through a
cell is likely to be fundamentally flawed. Each signaling
cascade is likely to operate in a finely tuned dynamic
equilibrium, where positive and negative regulators
continuously counterbalance on another. Signalling input,
e.g a mitogenic stimulus may operate like the plucking of
a fibre in one part of a spider web, which results in small
reverberations at distant parts throughout the web.
Neither our language nor our mathematical
representations suffice to clarify our understanding.
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Acknowledgements
WEHI
Sam Wormald & many others
UCB
Sach Mukherjee
ICGP project at LBNL
Rich Neve, Paul Spellman & many others
Robert A Weinberg’s fantastic book the biology of Cancer
Garland Science, 2007
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