Transcript The STAT family

The STAT family
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Class IIB(3)(b)
latent cytoplasmic factors
These families not present in fungi or plants, hinting
at an important evolutionary divergence leading to animals.
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STATs - a signal responsive TF family
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STATs: Signal Transducers and Activators
of Transcription
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1. Transducers for signals from many
cytokines
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two functions given in the name
Broad spectrum of biological effects
2. Transcriptional activators
characteristic activation mechanism
 activation at the cell membrane, response in the nucleus
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Rapid signal response
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The activation/deactivation cycle of STAT molecules is quite
short, about 15 min for an individual molecule.
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Simple signalling pathway
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The JAK-STAT signalling pathway
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Function: regulation of gene expression in
response to cytokines
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1. cytokines bind and aggregate the cytokine receptors in the cell membrane
2. associated JAK-type tyrosine kinases are activated by aggregation and
tyrosine-phosphorylates neighbouring-JAK (transphosphorylation) as well as
the C-terminal tail of the receptor (multiple sites)
3. Tyr-phosphates recruit inactive STAT-factors in the cytoplasm which are
bound through their SH2-domains
4. STATs become tyrosine-phosphorylated by JAK
5. phosphorylated STATs dissociate, dimerize (homo-/hetero-) and migrate to
the nucleus
6. STAT-dimers bind DNA and activates target genes
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Canonical JAK–STAT pathway
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Sequential tyrosine
phosphorylations
Receptor dimerization allows
transphosphorylation and
activation of Janus kinases
(JAKs).
 This is followed by
phosphorylation of receptor tails
and the recruitment of the STAT
proteins through their SH-2
domains. STAT tyrosine
phosphorylation then occurs.
 Dimerization of activated
(tyrosine phosphorylated) STAT
is followed by nuclear entry.
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IFN-response: two variants
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signalling pathway first discovered in studies of
interferon-response (IFN)
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IFN/
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IFN/  activation of Jak1+Tyk2  DNA-binding complexes (trimer:
STAT1+STAT2+p48, together designated ISGF3)  activation of target genes
with ISRE (IFN-stimulated response element)
IFN
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IFN  activation of Jak1+Jak2  DNA-binding complex (dimer: 2x STAT1)
 activation of target genes having GAS elements (IFN activated sequence)
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IFN-response:
two variants
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STAT-family members
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STAT1 - involved in IFN/- and IFN-response
STAT2 - involved in IFN/-response
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Mainly acting as partner for STAT1/p48
STAT3 - involved in response to several cytokines including IL6. It activates
several genes involved in acute phase response
Important in growth regulation, embryonic development & organogenesis
 Activation of STAT3 correlated with cell growth, link to cancer, bind c-Jun
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STAT4 - involved in IL12-response
STAT5a & 5b - involved in response to several cytokines including
prolactin, IL-2, and regulates expression of milk proteins in breast tissue in
response to prolactin
STAT6 - involved in IL4-response
non-mammalian family members (e.g. Drosophila)
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STAT-members
SH2
Y
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STAT-STAT interaction occurs through
reciprocal phospho-Tyr - SH2 interactions
SH2-domain
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SH2 = “Src-homology domain 2”
function: phospho-tyrosine binding
Three important functions in STATs:
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important for recruitment of STAT to receptor
important for interaction with the JAK kinase
important for dimerization of STATs to an active DNA-binding form
Tyr-701
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conserved key Tyr residue located just C-terminal to SH2
essensiell for dimerdannelse to an active DNA-binding form
function: TyrP bindingssted for SH2 in partner
P
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+
Y
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P
Y
Y
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dimerization via SH2-TyrP
TyrP from the left monomer
SH2 from the right monomer
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STAT-members
SH2
Y
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STATs - structure and function
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dimerization
Reciprocal SH2- TyrP interaction
 Homodimers
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Heterodimers
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(STAT1)2
STAT1-STAT2
STAT1-STAT3
GAS= TTN5-6AA
ISRE= AGTTTN3TTTCC
DNA-binding domain
DBD located in the middle of the protein
 Unique motif - se next slide
 All DBDs bind similar motifs in DNA
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symmetric inverted half sites
Only difference to STATs: preference for central nucleotide
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STAT-DBD structure
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Known structures
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[STAT1]2-DNA and [STAT3]2-DNA, as well as an
N-terminal of STAT4
Characteristic feature of DBD
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Symmetry-axis through DNA, each monomer
contacts a separate half site
structure resembles NFkB and p53
(immunoglobuline fold). The dimer forms a Cshaped ”clamp” around DNA.
The dimer is kept together by reciprocal SH2- TyrP
interactions between the SH2 domain in one
monomer and the phosphorylated Tyr in the other.
The SH2 domain in each monomer is closely linked
to the core DBD and is itself close to DNA, and is
assumed also to contribute to DNA-binding.
N-terminal coiled-coil region not close to DNA,
probably involved in prot-prot interaction with
flexible position
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3D
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STAT
domain
structure
and protein
binding
sites.
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Promoter recognition and selectivity
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Mechanisms to achieve
specific trx responses.
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Inverted repeat TTN5–6AA motif
common. Binding specificity to
individual elements based on evolved
preferences for specific positions.
In the ISGF3 heterotrimeric complex,
STAT1–STAT2 heterodimers bind to a
third protein, p48/ISGF3, a TF that
recognizes the ISRE sequence.
STAT N-domains mediate dimer–dimer
interactions allowing high-avidity
binding to tandemly arranged lowaffinity GAS elements.
Adjacent response elements bind to
other TFs. Cooperativity and synergy.
STAT directly recruit co-activators that
alter chromatin dynamics.
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TAD
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transactivation domain
C-terminal part of the protein, less conserved
 variants generated by alternative splicing + proteolysis
 STAT1 lacking the last 38aa has all functions retained except transactivation
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Regulation through TAD-modification
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Activity of TAD is regulated through Ser phosphorylation (LPMSP-motif)
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Ser727 in STAT1
Kinase not identified - candidates: p38, ERK, JNK
 A role in recruitment of GTF/coactivator
 Proteins identified that bind TAD in a Ser-dependent manner
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MCM5
BRCA1
TAD in STAT2 binds C/H-rich region of CBP
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STAT2 carries the principal TAD of the ISGF3-complex
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Other functional domains
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The N-domain is important for stabilizing
interactions between STAT dimers, bound
to tandemly arranged response elements
Tyr kinases
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The JAK-family of tyrosine kinases
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Family members
JAK1 (135 kDa)
 JAK2 (130 kDa)
 JAK3 (120 kDa)
 Tyk2 (140 kDa)
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Common feature
C-terminal kinase + pseudokinase
 ≠ RTK by lacking transmembrane domains and SH2, SH3, PTB, PH
 several regions homologous between JAK-members
 Associated with cytokine receptors (type in and II)
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Function
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Associated with cytokine receptors in non-stimulated cells in an inactive form
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The role of the kinases in the signalling
pathway
INF-signalling
INF-signalling
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The cytokine-receptor superfamily
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A receptor-family that mediates response to more than 30
different cytokines
Common feature: conserved extracellular ligand-binding
domain
Are associated with tyrosine-kinases in the JAK-family
Ligand-binding  Receptor dimerization or oligomerization
leads to JAK apposition  associated JAK Tyr kinases are
activated  transphosphorylation of neighbour-JAKs 
tyrosine-phosphorylation of C-terminal tail of receptors on
multiple sites  several cellular substrate-proteins associate
(including STATs)  multiple signalling pathways are activated
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The role of the kinases in the signalling
pathway
INF-signalling
INF-signalling
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Specificity in response
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Specific cytokines activate distinct STATs and lead to a specific
response - what mediate specificity?
each cytokine activates a subgroup STAT
 some cytokines activate only one specific STAT
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One contribution: the SH2 - receptor interaction specific for certain
combinations
swaps-experiments of SH2 between STATs change specificity
 affinity of the SH2-receptor interaction is affected by the sequence context of the Tyr
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Another contribution: different STAT-dimers bind different response
elements in the genome and turn on different genes
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STAT1 knock-out mice illustrate biological specificity
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STAT1-/- phenotype: total lack of IFN-response  highly sensitive to virus-infection
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Several signalling pathways linked
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STATs may also be Tyr-phosphorylated and hence activated by other
receptor families
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receptor tyrosine kinases (RTKs) such as EGF-receptor may phosphorylate STATs
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EGF stimulation  activation of STAT1, STAT3
non-receptor tyrosine kinases such as Src and Abl may also phosphorylate STATs (?)
 G-protein coupled 7TMS receptors such as angiotensine receptor (?)
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STAT may also be modified by Ser-phosphorylation
DNA-binding reduced (STAT3)
 Transactivationdomain Ser-phosphorylated (important for transactivation in STAT1 and STAT3)
 Responsible kinases not identified - MAPkinases candidates, probably also others
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JAKs may activate other signalling pathways than STATs
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TyrP will recruit several protein-substrates and lead to phosphorylation and activation of other
signalling pathways
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e.g. JAK activation  activation of MAP-kinases
e.g. substrates: IRS-1, SHC, Grb2, HCP, Syp, Vav
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Crosstalk
Alternative inputs
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Alternative outputs
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JAK may phosphorylate
other targets and thus
activate signal transduction
pathways other than
through STATs
Receptor
tyrosinee
kinase
Cytokine
receptor
P
P
P
JAK
P
P
P
P
P
P
Y
P
MAPK
Y
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STATs may be Tyrphosphorylated by RTKs
Y
P
Y
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SH2
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Variations in mechanisms of STAT
activation
SMAD family
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SMAD-family - a logic
resembling the STAT-family
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The Smad-factors mediate response to
TGF-related growth- and
differentiation factors
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STAT-related logic
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Membrane-bound receptors (such as the TGFß-receptor) are
activated by binding of ligand (TGF). The receptors here are
transmembrane serine/threonine-kinases
Activated kinases phosphorylate specific Smad-factors
phosphorylated Smad-factors associate with a common
Smad-factor (Smad4)
The generated heteromeric complexes migrate to the nucleus
as transcription factors
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TGF effectors
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Latent cytoplasmic TFs
activated by serine
phosphorylation at their
cognate receptors
This family transduces
signals from the
transforming growth
factor- (TGF-)
superfamily of ligands.
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Classification
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Smad-factors - design and classification
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Nine different Smad-factors identified in vertebrates
common conserved domains: N-terminalt MH1-domain (DBD) + C-terminalt MH2domain
Can be divided into three groups
1. Receptor-activated Smad-factors - become phosphorylated by activated receptors in
their C-terminal (SSXS)
 2. common Smad-factors associated with activated Smad-factors and participate in several
signalling pathways
 3. Inhibitoriske Smad-factors
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SMAD-signalling pathway
Effector SMADs
(R-SMADs)
Co-SMADs
Repressor
SMADs
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Three groups of SMADs
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First group: The effector SMADs (also called the R-SMADs)
become serine-phosphorylated in the C-terminal domain by
the activated receptor.
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Smad1, Smad5, Smad8, and Smad9 become phosphorylated in response to bone
morphogenetic morphogenetic protein (BMP) and growth and differentiation factor
(GDF), and Smad2 and Smad3 become phosphorylated in response to the activin/nodal
branch of the TGF- pathway.
Second group: regulatory or co-SMADs (common SMADs).
There are two regulatory SMADs: Smad4 and Smad4 (also called Smad10).
 Smad4 binds to, and is essential for, the function of Smad1 and Smad2. The regulatory
Smad4 binds to all effector SMADs in the formation of transcriptional complexes, but it
does not appear to be required for nuclear translocation of the effector molecules.
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Third group: two inhibitory SMADs, Smad6 and Smad7.
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provide negative regulation of the pathway by blocking Smad4 binding.
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SMAD-signalling pathway
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Final steps - target gene activation
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Once an activated, serine-phosphorylated effector
SMAD binds Smad4 and escapes the negative
influences of Smad6 and Smad7, nuclear
accumulation and regu-lation of specific target
genes can occur.
In most cases, SMADs require partner transcription factors with
strong DNA binding capacity that determine the gene to be activated.
The DNA binding is then strengthened by association with SMADs
that on their own bind weakly to adjacent DNA sites. The SMADs
furnish transcriptional activation capacity.
 The specificity of response among different ligands can be partially
explained by the choice of DNA binding partner proteins. For
example, activin activation of SMADs results in combinations with
FAST1 and a particular set of genes is activated. Signaling by BMP
ligands results in association of activated SMADs with a DNA
binding protein called OAZ.
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The Smad-factors activate their target
genes in combination with other TFs