The STAT family

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Class IIB(3)(b)latent cytoplasmic factors

These families not present in fungi or plants, hintingat 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-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

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 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)

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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

YP

YP

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

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 [STAT1]2-DNA and [STAT3]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 NFB 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 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 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

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

INF-signalling INF-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

INF-signalling INF-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

Y

PP

PP

P

P

P

P

JAK

SH2YY

YCytokinereceptor

P

P

P

Receptortyrosineekinase

MAPK

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

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Variations in mechanisms of STAT activation

SMAD family

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SMAD-family - a logic resembling the STAT-family

The Smad-factors mediate response to TGF-related growth- and differentiation factors

STAT-related logic 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

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

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

Effector SMADs(R-SMADs)

Co-SMADs Repressor SMADs

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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- 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.

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