The STAT family

1. The STAT family

2. Class IIB(3)(b)latent cytoplasmic factors These families not present in fungi or plants, hinting at an important evolutionary divergence leading to animals.

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 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.

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 SH2 Y

11. Y 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

12. dimerization via SH2-TyrP TyrP from the left monomer SH2 from the right monomer

13. STAT-members SH2 Y

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 GAS= TTN5-6AA ISRE= AGTTTN3TTTCC

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 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/ISGF3g, 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.

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 INFg-signalling INFa-signalling

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 INFg-signalling INFa-signalling

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. Receptor tyrosinee kinase P P P Y Y Y Y MAPK 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

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. Effector SMADs (R-SMADs) Co-SMADs Repressor SMADs 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