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1. The STAT family
2. Class IIB(3)(b)latent cytoplasmic factors
3. 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.
4. Simple signalling pathway
5. 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-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
6. 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.
7. IFN-response: two variants signalling pathway first discovered in studies of interferon-response (IFN)
IFN?/?
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?
IFN? ? activation of Jak1+Jak2 ? DNA-binding complex (dimer: 2x STAT1) ? activation of target genes having GAS elements (IFN? activated sequence)
8. IFN-response: two variants
9. 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)
10. STAT-members
11. 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
12. dimerization via SH2-TyrP
13. STAT-members
14. 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
15. STAT-DBD structure Known structures
[STAT1]2-DNA and [STAT3b]2-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
16. 3D STAT domain structure and protein binding sites.
17. 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.
18. 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
19. Other functional domains The N-domain is important for stabilizing interactions between STAT dimers, bound to tandemly arranged response elements
20. Tyr kinases
21. 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
22. The role of the kinases in the signalling pathway
23. 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
24. The role of the kinases in the signalling pathway
25. 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
26. 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
27. 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
28. Variations in mechanisms of STAT activation
29. SMAD family
30. 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
31. 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.
32. 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
33. SMAD-signalling pathway
34. 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.
35. SMAD-signalling pathway
36. 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.
37. The Smad-factors activate their target genes in combination with other TFs