The STAT family

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The STAT family
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Class IIB(3)(b)latent cytoplasmic factors
These familys 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

• STATs Signal Transducers and Activators of
  Transcription
• two functions given in the name
• 1. Transducers for signals from many cytokines
• Broad spectrum of biological effects
• 2. Transcriptional activators
• characteristic activation mechanism
• activation at the cell membrane, response in the
  nucleus
• Rapid signal response
• 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

• Function regulation of gene expression in
  response to cytokines
• 1. cytokines bind and aggregate the cytokine
  receptors in the cell membrane
• 2. associated JAK-type tyrosine kinases are
  activated by aggregation and tyrosine-phosphorylat
  es 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 JAKSTAT pathway

• 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

• signalling pathway first discovered in studies of
  interferon-response (IFN)
• IFN?/?
• IFN?/? ? activation of Jak1Tyk2 ? DNA-binding
  complexes (trimer STAT1STAT2p48, together
  designated ISGF3) ? activation of target genes
  with ISRE (IFN-stimulated response element)
• IFN?
• IFN? ? activation of Jak1Jak2 ? 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

• STAT1 - involved in IFN?/?- and IFN?-response
• STAT2 - involved in IFN?/?-response
• 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
• 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
• SH2 Src-homology domain 2
• function phospho-tyrosine binding
• Three important functions in STATs
• 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
• 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

Y
Y
P
P
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

• dimerization
• Reciprocal SH2- TyrP interaction
• Homodimers
• (STAT1)2
• Heterodimers
• STAT1-STAT2
• STAT1-STAT3
• DNA-binding domain
• DBD located in the middle of the protein
• Unique motif - se next slide
• All DBDs bind similar motifs in DNA
• symmetric inverted half sites
• Only difference to STATs preference for central
  nucleotide

GAS TTN5-6AA
ISRE AGTTTN3TTTCC
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STAT-DBD structure

• Known structures
• STAT12-DNA and STAT3b2-DNA, as well as an
  N-terminal of STAT4
• Characteristic feature of DBD
• Symmetry-axis through DNA, each monomer contacts
  a separate half site
• structure resembles NFkB and p53 (immunoglobuline
  fold). The dimer forms a C-shaped 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

• STAT domain structure and protein binding sites.

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Promoter recognition and selectivity

• Mechanisms to achieve specific trx responses.
• Inverted repeat TTN56AA motif common. Binding
  specificity to individual elements based on
  evolved preferences for specific positions.
• In the ISGF3 heterotrimeric complex, STAT1STAT2
  heterodimers bind to a third protein, p48/ISGF3g,
  a TF that recognizes the ISRE sequence.
• STAT N-domains mediate dimerdimer interactions
  allowing high-avidity binding to tandemly
  arranged low-affinity 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

• 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
• Regulation through TAD-modification
• Activity of TAD is regulated through Ser
  phosphorylation (LPMSP-motif)
• 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
• MCM5
• BRCA1
• TAD in STAT2 binds C/H-rich region of CBP
• STAT2 carries the principal TAD of the
  ISGF3-complex

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Other functional domains

• The N-domain is important for stabilizing
  interactions between STAT dimers, bound to
  tandemly arranged response elements

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Tyr kinases
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The JAK-family of tyrosine kinases

• Family members
• JAK1 (135 kDa)
• JAK2 (130 kDa)
• JAK3 (120 kDa)
• Tyk2 (140 kDa)
• 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)
• Function
• 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
INFg-signalling
INFa-signalling
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The cytokine-receptor superfamily

• 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
INFg-signalling
INFa-signalling
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Specificity in response

• 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
• 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
• Another contribution different STAT-dimers bind
  different response elements in the genome and
  turn on different genes
• STAT1 knock-out mice illustrate biological
  specificity
• STAT1-/- phenotype total lack of IFN-response ?
  highly sensitive to virus-infection

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Several signalling pathways linked

• STATs may also be Tyr-phosphorylated and hence
  activated by other receptor families
• receptor tyrosine kinases (RTKs) such as
  EGF-receptor may phosphorylate STATs
• 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 (?)
• 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
• JAKs may activate other signalling pathways than
  STATs
• TyrP will recruit several protein-substrates and
  lead to phosphorylation and activation of other
  signalling pathways
• 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
• STATs may be Tyr-phosphorylated by RTKs
• Alternative outputs
• JAK may phosphorylate other targets and thus
  activate signal transduction pathways other than
  through STATs

Cytokine receptor
P
P
P
P
JAK
P
P
SH2
P
P
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Variations in mechanisms of STAT activation
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SMAD family
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SMAD-family - a logic resembling the STAT-family

• The Smad-factors mediate response to TGFb-related
  growth- and differentiation factors
• STAT-related logic
• Membrane-bound receptors (such as the
  TGFß-receptor) are activated by binding of ligand
  (TGFb). 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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TGFb effectors

• Latent cytoplasmic TFs activated by serine
  phosphorylation at their cognate receptors
• This family transduces signals from the
  transforming growth factor-b (TGF-b) superfamily
  of ligands.

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Classification

• Smad-factors - design and classification
• Nine different Smad-factors identified in
  vertebrates
• common conserved domains N-terminalt MH1-domain
  (DBD) C-terminalt MH2-domain
• 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
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Three groups of SMADs

• First group The effector SMADs (also called the
  R-SMADs) become serine-phosphorylated in the
  C-terminal domain by the activated receptor.
• 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-b pathway.
• Second group regulatory or co-SMADs (common
  SMADs).
• There are two regulatory SMADs Smad4 and Smad4b
  (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.
• Third group two inhibitory SMADs, Smad6 and
  Smad7.
• 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

• 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